CZ20003732A3 - Auxiliary preparation - Google Patents

Abstract

The solution deals with the aid that it contains a surfactant of formula HO (CH 2 CH 2 O) n -A-R and pharmaceutically acceptable vehicle. The solution also includes vaccination a composition which is composed of an adjuvant and an antigen or an antigenic preparation and which is intended to treat infections, Tumors or allergies in mammals.

Description

Technical field

The invention relates to an adjuvant comprising a polyoxyethylene ether or polyoxyethylene ester and a pharmaceutically acceptable vehicle, and a vaccine formulation comprising the adjuvant and an antigen. The invention further relates to the use of polyoxyethylene ethers and polyoxyethylene esters in the manufacture of adjuvants and vaccines and their use as medicaments.

BACKGROUND OF THE INVENTION

Vaccination at the mucosa, such as the nose or mouth, is an easy and more convenient method of vaccination than traditional systemic injection. Injection of the vaccine is associated with a number of unpleasant symptoms such as injection site pain and irritation. These manifestations may provoke a “fear of the needle” which leads to poor patient co-operation on the vaccination regimen. Systemic injection can also be a source of infection at the injection site.

Mucosal vaccination has become interesting since it has been shown in animals that administration of antigens to the mucosa is much more effective than systemic administration of antigens by injection in inducing an immune response on the surface of the mucosa that is the gateway to many pathogens. It is believed that vaccination on mucous membranes, such as the nose, can induce mucosal immunity not only to the nasal mucosa, but also to distant mucous membranes, such as the genital mucosa (Mestecky, 1987, Journal of Clinical Immunology, 7, 265). • · · · · · · · · · · · ·

- 276; McGhee and Kiyono, Infectious Agents and Disease, 1993, 2, 55, 73).

In order for mucosal immunization to replace or be an alternative immunization, it must be possible to produce a systemic immune response at least as effectively as by injection by administering the mucosal vaccine. Although certain mucosal antigens have been reported to be capable of inducing a systemic response (Cahill et al., 1993, FEMS Microbiology Letters, 107, 211-216), most soluble antigens administered alone on the nasal mucosa produce no or only a small immune response. .

Effective excipients have been discovered by many authors to help overcome this problem in various ways, such as the encapsulation of antigens (e.g., liposomes and microparticles); direct interaction with target cells with subsequent release of immunostimulatory cytokines (eg, cholera toxin or thermolabile toxin E. coli) ', increasing the absorption of antigens by the epithelium (eg, cholera toxin).

In this patent application, polyoxyethylene ethers and polyoxyethylene esters are shown as highly effective adjuvants for vaccine compositions. The adjuvants of the invention are safe, easy to sterilize, and administered. The advantage of these compositions is that they are capable of eliciting a sufficient systemic immune response when administered to the mucosa, at least the same as that of a conventional vaccine formulation by systemic injection.

Polyoxyethylene ethers, such as polyoxyethylene lauryl ether, are described in the Merck Index (12th Edition, 7717), their therapeutic use herein including: local anesthesia, anti-itching and sclerosing agents. Polyoxyethylene ethers and / or polyoxyethylene esters belong to the group of non-ionic surfactants.

Increased uptake of insulin in the nasal cavity has been reported following administration of polyoxyethylene ethers and polyoxyethylene esters to the nasal mucosa (Hirai et al., 1981, International Journal of Pharmaceutics, 9, 165-172; Hirai et al., 1981, International Journal of Pharmaceutics, 9, 173 - 184).

Other nonionic surfactants are also used in vaccine formulations. Vaccines containing a combination of polyoxyethylene castor oil or capillary / capric acid glycerides with polyoxyethylene monoesters of sorbitan and an antigen induce a systemic immune response upon topical administration to the mucous membrane (patent application WO 9417827). This patent application discloses that a combination of TWEEN 20 ™ (polyoxyethylene monoester sorbitan) and Imwitor 742 ™ (capillary / capric acid glycerides) or a combination of TWEEN 20 ™ and polyoxyethylene castor oil enhances the systemic immune response following intranasal immunization. Details of the effect of this agent on enhancing the immune response by administering antigens to the nasal mucosa are also reported in the literature (Gizurason et al., 1996, Vaccine Research, 5, 69-75; Aggerbeck et al., 1997, Vaccine, 15, 307- 316).

Surfactants may also take the form of nonionic surfactant vesicles (commonly known as neosomes, patent application WO 95/09651). These vesicles form lipid vesicles in the presence of cholesterol

Novasomes (US Patent Application No. 5,147,725) are paucilamenary vesicular structures containing polyoxyethylene ethers and cholesterol encapsulating antigen, which upon systemic administration enhance immune responses to the antigen.

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a bilayer that binds the antigen to the internal water phase or directly to the bilayer.

SUMMARY OF THE INVENTION

The invention is that polyoxyethylene ethers or polyoxyethylene esters administered together with antigens significantly enhance the systemic immune response. When these adjuvants are used in mucosal vaccines, their potentiating effect results in the organism's immunological response to the antigen being equal to or even higher than that of conventional antigen administration by systemic injection. These molecules represent a group of adjuvants suitable for administration to humans, both in conventional systemic vaccination and in the replacement of systemic vaccination with mucosal vaccination.

The invention is a potent adjuvant in vaccine formulations and, unlike many other available adjuvants that act by encapsulating the antigen, is in the form of a non-vesicular solution or suspension. The invention therefore relates to an excipient comprising a surfactant of formula (I) in the form of a vesicle-free solution or suspension. The invention further provides a vaccine adjuvant comprising a surfactant of formula (I) formulated in the absence of cholesterol.

The vaccine compositions and adjuvants of the invention comprise molecules of formula (I):

HO (CH ₂ CH ₂ O) _n -AR, wherein n is from 1 to 50, A is a bond or -C (O) -, R is an alkyl of 1 to 50 carbons or a phenylalkyl of 1 to 50 carbons.

- 5 - ··· * ·· · ·· ·· ·· • · · · · · · · · · · · ·· · ····· · · · · ······ • · · · ························ Invention se deals vaccinating preparation containing

a polyoxyethylene ether of formula (I) wherein n is a number between 1 and 50, preferably between 4 and 24, most preferably 9; R is an alkyl group of 1 to 50 carbons, preferably 4 to 24 carbons, most preferably 12 carbons; and A is a bond. The concentration of polyoxyethylene ether is in the range of 0.1-20 g / l, preferably 0.1-10 g / l, most preferably in the range of 0.1-1 g / l. Suitable polyoxyethylene ethers belong to the following group: polyoxyethylene-9 lauryl ether, polyoxyethylene-9-stearyl ether, polyoxyethylene-8 stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35 lauryl ether and polyoxyethylene-23-lauryl ether.

The invention further relates to a vaccine composition comprising a polyoxyethylene ester of formula (I), wherein n is a number between 1 and 50, preferably between 4 and 24, preferably 9; R is an alkyl group of 1 to 50 carbons, preferably 4 to 20 carbons, most preferably 12 carbons; and A is -C (O) -. The concentration of the polyoxyethylene ester is in the range of 0.1-20 g / l, preferably 0.1-10 g / l, most preferably in the range 0.1-1 g / l. Suitable polyoxyethylene esters include: polyoxyethylene 9-lauryl esters, polyoxyethylene 9-stearyl esters, polyoxyethylene-8 stearyl esters, polyoxyethylene-4-lauryl esters, polyoxyethylene-35 lauryl esters, and polyoxyethylene-23-lauryl esters.

The invention further relates to a vaccine composition comprising the polyoxyphenyl ethers of formula (I), wherein n is a number between 1 and 50, preferably between 4 and 24, preferably 9; R is a phenylalkyl group of 1 to 50 carbons, preferably 4 to 24 carbons, most preferably 12 carbons; and A is a bond. The concentration of polyoxyethylene phenyl ethers is preferably between 0.1 and 10 g / l, most preferably in the range 0.25 - 1 g / l.

The vaccine compositions of the present invention are used to protect mammals from infection or to treat mammals already ill by administering a vaccine vaccine. 9 9 9 9 9 9

9 · 9 9 9 9 ···················, oral, buccal, intestinal, vaginal, rectal or nasal. The formulations to be administered are in the form of drops, spray or powder. Also included are nebulized or aerosol formulations. The formulations of the invention further take the form of gastroresistant capsules, oral granules or suppositories for rectal or vaginal administration. The compositions of the invention also enhance the immunogenicity of antigens administered to the skin in the form of transdermal or transcutaneous systems. The compositions of the invention are also administered parenterally as intramuscular or subcutaneous injections, wherein the compositions are characterized in that they are not administered in vesicular form.

In particular, the invention relates to adjuvants for mucosal vaccine formulations. Such adjuvants are well tolerated in humans and the immune responses induced by them are strong. The adjuvants of the invention are in the form of solutions or suspensions without the presence of vesicles, thus avoiding the problems associated with the production of particulate adjuvant systems, including maintaining stability, uniformity and quality control. Such formulations are potent adjuvants, at the same time exhibit low reactogenicity and are well tolerated by patients.

The polyoxyethylene ethers of the invention have hemolytic effects. The haemolytic activity of polyoxyethylene ethers is measured by the following in vitro assay, where haemolytic activity is expressed as the highest concentration of detergent that does not cause lysis of red blood cells:

1. Fresh guinea pig blood is washed 3 times in phosphate buffered saline (PBS) in a table centrifuge.

2. Add 50 µl of PBS containing double dilutions of detergent. of this blood suspension.

· 4 4 4 4 4 4 4 4 4 4 4 4

4 4 4 4 4 4 4 4 4 ·· 444445

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3. Hemolysis is determined after 8 hours optically or by measuring the optical density of the supernatant. The presence of a red-colored light-absorbing supernatant at 570 min indicates hemolysis.

4. Results are expressed as the concentration of the first detergent dilution at which haemolysis no longer occurs.

Within the experimental variability of these bioassays, the polyoxyethylene ethers or surfactants of formula (I) of the invention have a hemolytic activity of between 0.5 and 0.0001 g / l, preferably between 0.05 and 0.0001 g / l, more preferably 0.005 and 0.0001 g / L, preferably between 0.003 and 0.0004 g / L. Preferably, the polyoxyethylene ethers or polyoxyethylene esters have a hemolytic activity similar (i.e. within a ten-fold difference) to polyoxyethylene-9-lauryl ether or polyoxyethylene-8-stearyl ether.

The ratio of the length of the polyoxyethylene moiety to the length of the alkyl chain in the surfactant (i.e., the ratio n: length of the alkyl chain) affects the aqueous solubility of this detergent group. The adjuvants of the invention are in the form of a solution or form particulate structures such as micelles. The adjuvants of the present invention are clean, non-turbid or opaque, stable, easy to sterilize by filtration on a 220 nm pore membrane, and processed in a simple and controlled manner due to their natural vesicle-free structure.

The vaccine compositions of the invention are in the form of a non-vesicular solution or suspension of the polyoxyethylene ether or polyoxyethylene ester of formula (I) in a pharmaceutically acceptable vehicle, such as PBS or water, with an antigen or antigenic preparation. These vaccines are administered to the mammalian mucosal surface either as a primary immunization or as a boost. Alternatively, the compositions of the invention may be administered systemically, e.g., transdermally, subcutaneously, or intramuscularly.

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Other adjuvants enhancing both mucosal and systemic immune responses are bacterial enterotoxins derived from Vibrio cholerae (cholera toxin (CT) and Escherichia coli (thermolabile enterotoxin (LT). CT and LT are heterodimers consisting of a pentameric ring of a subunit that fits a toxic A subunit The structure and biological activity of these toxins have been described in their articles by Clements and Finklestein, 1979, Infection and Immunity, 24, 760-769; Clements et al., 1980, Infection and Immunity, 24, 91-97. a derivative of LT that lacks the proteolytic site necessary to convert the non-toxic form of LT upon release from the cell into a toxic form, and due to the amino acid substitution arginine for glycine at position 192, this form of LT (designated mLT (R192G)) is non-susceptible to proteolytic cleavage. thus, it is significantly reduced while maintaining its strong side effect The process for producing mLT (R192G) is described in detail in patent application WO 96/06627. Other mutant forms of LT include active site mutants, such as mLT (A69G), in which glycine is replaced with alanine at the 69th site. WO 96/06627 describes the use of mLT (R192G) as a mucosal vaccine. These adjuvants may be combined with the nonionic surfactants of the present invention.

The invention includes combinations of a polyoxyethylene ether or polyoxyethylene ester with other adjuvants or immunostimulatory agents including cholera toxin and its B subunit, monophosphoryl lipid A and its non-toxic 3-de-O-acylated monophosphoryl lipid A derivative (described in UK patent application GB 2,220,211), saponins such as Quil A (obtained from the bark of the South American tree Quillaja Saponaria Molina) and its fractions, including QS21 and QS17 (US 5,057,540; Kensil, CR, 1996, Crit Rev Ther Drug Carrier System, 12 (1-2), 1). 55, EP 0 362 279 B1, Kensil et al., 1991, J. Immunology, 146, 431-437; WO 99/100008) and an oligonucleotide helper system.

An unmethylated CpG dinucleotide (described in patent application WO 96/02555). A particularly preferred immunostimulatory agent used in combination with POE is a CpG immunostimulatory oligonucleotide. Combined formulations with this agent have a strong inducing and reminder effect on immune responses in larger animals. Suitable oligonucleotides comprise the following sequences: all internucleotide linkages of these sequences are modified with phosphorothioate.

OLIGO 1: TCG ATG ACG TTC

OLIGO 2: TCT CCC AGG GTG CGC CAT

OLIGO 3: ACC GAT GAC GTC GGT GAC GGC ACC ACG

The CpG oligonucleotides used in the invention are synthesized by any method known in the art (for example, as described in patent application EP468520). Synthesizing on an automatic synthesizer is a convenient way of making these oligonucleotides.

Polyoxyethylene ethers and polyoxyethylene esters are combined with vaccine vehicles such as chitosan or other polycationic polymers, polylactide or polylactidoglycolide particles, particles composed of polysaccharides or chemically modified polysaccharides, cholesterol-free liposomes or lipid-water-based emulsions, WO 95 oil / emulsion 17210), particles composed of glycerol monoesters and others. The polyoxyethylene ethers and polyoxyethylene esters, when mixed with powdered vehicles such as lactose-containing antigen, are administered in the form of a dry powder.

The adjuvants of the invention contain surfactants with polyoxyethylene ethers or polyoxyethylene esters which are not vesicles. The invention therefore relates to the use of the polyoxyethylene ethers and the polyoxyethylene esters of formula (I) in the preparation of adjuvants and vaccines in which the surfactant of formula (I) is not present in the form of vesicles.

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The vaccine compositions of the invention comprise an antigen or antigenic preparation that elicits an immune response against a human pathogen. The antigen or antigenic preparation is derived from HIV-1 (such as tat, nef, gp120 or gp160), human herpes viruses (such as gD or derivatives thereof, Immediate Early protein such as ICP27 from HSV1 or HSV2), cytomegalovirus ((particularly human) (such as gB or derivatives thereof), rotaviruses (including attenuated viruses), Epstein-Barr virus (such as gp350 or derivatives thereof), varicella zoster virus (such as gpl, II and IE63) or hepatitis viruses such as virus hepatitis B (hepatitis B surface antigen or derivative thereof), hepatitis A virus, hepatitis C virus or hepatitis E virus, or other viral pathogens such as paramyxoviruses: respiratory syncytial virus (F and G proteins or derivatives thereof), parainfluenza virus, measles and mumps virus, human papillomaviruses (eg HPV6, 11, 16, 18, ...), flaviviruses (eg yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus) or influenza virus (whole live or inactivated virus, split influenza virus, grown on eggs, MDCK cells or Vero cells or whole influenza virosomes (described by Gluck R., 1992, Vaccine, 10, 915-920) or purified or recombinant proteins thereof, such as HA, NP, NA or M protein or combinations thereof) or antigens and antigenic preparations derived from bacterial pathogens such as Neisseria spp, including N. gonorrhea and N. meningitidis (for example capsular polysaccharides and their conjugates, transferrin binding proteins, lactoferrin binding proteins , PilC, adhesins); Streptococcus pyogenes (e.g., M proteins or portions thereof, protease C5A, lipoteichic acid), S. agalactiae, S. mutans', Haemophillus ducrer, Moraxella spp, including M. catarrhalis, also known as Branhamella catarrhalis (high and low molecular weight adhesives and ivazins) ; Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin or derivatives thereof, filamentous hemagglutinin, adenylate cyclase, fimbria), B. parapertussis and B. bronchiseptica ', Mycobacterium spp, including M. tuberculosis (for example ESAT6, antigen 85A, B or C), M. bovis, M. leprae, M. avium, M.

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4 44 4 4 · ·. II. 4444 4444 4444 44 · 4 paratuberculosis, M. smegmatis ', Legionella spp, including L. pneumophila', Escherichia spp, including enterotoxic E. coli (for example colonization factors, thermolabile toxin or its derivatives, thermostable toxin or its derivatives), enterohemorrhagic E. coli, enteropathogenic E. coli (e.g., shiga-like toxin or derivatives thereof); Vibrio spp, including V. cholerae (e.g. cholera toxin or derivatives thereof); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerr, Yersinia spp, including Y. enterocolitica (e.g. Yop protein), Y. pestis, Y. pseudotuberculosis', Campylobacter spp, including C. jejuni (e.g. toxins, adhesins) and invasins) and C. co //; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp, including L. monocytogenes; Helicobacter spp, including H. pylori (e.g. urease, catalase, vacuolization toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus spp, including S. aureus, S. epidermidis, Enterococcus spp, including E. faecalis, E. faeciunr, Clostridium spp, including C. tetani (for example, tetanus toxin and derivatives thereof), C. botulinum (for example, botulinum toxin and derivatives thereof) C. difficile (e.g., Clostridial toxins A, B and derivatives thereof); Bacillus spp, including B. anthracis; Corynebacterium spp, including C. diphteriae (for example diphtheria toxin and derivatives thereof); Borrelia spp, including B. burgdorferi (e.g. OspA, OspC, DbpA, DbpB), B. garinii (e.g. OspA, OspC, DbpA, DbpB), B. afzelii (e.g. OspA, OspC, DbpA, DbpB) B. andersonii (e.g. OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia spp, including E. equi and agents that cause human granulocyte ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp, including C. trachomatis (e.g., MOMP, heparin binding proteins), C. pneumoniae (e.g., MOMP, heparin binding proteins), C. psittaci, Leptospira spp, including L. interrogans; Treponema spp, including T. pallidum (for example, rare outer membrane proteins), T. denticola, T. hyodysenteriae, or antigens or antigenic preparations derived from parasites such as Plasmodium spp, including P. falciparum; Toxoplasma spp, including T. gondii (e.g., SAG2, SAG3, Tg34); Entamoeba spp, including E. histolytica, Babesia spp, including B. microti; Trypanosoma spp, including T. cruzi; Giardia spp, including G. lamblia; Leishmania spp, including L. major, Pneumocystis spp, including P. carinii;

· · · · · «« «· · · · · · · · · · · · · · · · · · · · · ···· · ··· ··· · ··· ♦

Trichomonas spp, including 7th vaginalis; Schistosoma spp, including S. mansoni, or antigen and yeast-derived antigenic preparations such as Candida spp, including C. albicans; Cryptococcus spp, including C. neoformans.

Preferred antibacterial vaccines include antigens derived from Streptococcus spp, including S. pneumoniae (e.g. capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline binding proteins, and protein antigen pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al. , Microbial Pathogenesis, 25, 337-342) and mutant detoxified derivatives thereof (WO 90/06951; WO 99/03884) Other preferred vaccine compositions include antigens derived from Haemophillus spp, including H. influenzae type B (e.g. PRP and its non-typeable H. influenzae (e.g., OMP26, high molecular weight adhesives, P5, P6, D protein and D lipoprotein, fimbrin and fimbrin-derived peptides (US 5,843,464) or multiple copy variants, or fusion proteins thereof). antigens derived from Moraxella catarrhalis (including its outer membrane vesicles and OMP106) (WO 97/41731)) and from Neisseria meningitidis B (including its outer membrane vesicles and NspA (WO 96/29412)).

Hepatitis B surface antigen derivatives are well known in the art and include, but are not limited to, the PreS1 and PreS2 S antigens described in European Patent Applications EP-A-414,374; EP-A-0304 578 and EP 198-474. One preferred vaccine composition of the invention comprises the HIV-1 gp120 antigen, especially when expressed in CHO cells. The vaccine composition of the invention also comprises the gD2t described herein above.

The vaccine compositions of the invention comprising the claimed adjuvant also comprise an antigen derived from human papillomavirus

- 13 • ·

(HPV), which causes genital warts (HPV 6, HPV 11 and others) and cervical cancer (HPV 16, HPV 18 and others).

A suitable prophylactic or therapeutic genital wart vaccine comprises L1 particles or capsomers and fusion proteins with one or more antigens - proteins E6, E7, L1 and L2 from HPV 6 and HPV 11.

The most suitable forms of fusion proteins include L2E7 described in patent application WO 96/26277 and protein D (1/3) - E7 described in patent application GB 9717953.5 (PCT / EP98 / 05285).

The vaccine for the prophylaxis and treatment of cervical infection or cancer caused by HPV contains HPV 16 or 18 antigens. These are L1 or L2 monomers in the form of monomers, or L1 and L2 antigens together in a virus-like particle (VLP) or L1 antigen alone in the VLP or in capsomer. These antigen, virus-like particles and capsomer are known and are described, for example, in patent applications WO94 / 00152, WO94 / 20137, WO94 / 05792 and WO93 / 02184.

These vaccine formulations also include early proteins, for example E7, E2 or E5, alone or in the form of fusion proteins. The most preferred form is a VLP comprising L1E7 fusion proteins (WO96 / 11272).

Suitable HPV 16 antigens are early E6 or E7 proteins in fusion with a D protein carrier, thereby forming a D protein-E6 or E7 fusion, or combinations thereof; or a combination of early E6 or E7 proteins with an L2 antigen (WO96 / 26277).

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The early E6 or E7 proteins from HPV 16 or 18 are also present separately, preferably in the D protein-E6 / E7 fusion. Such vaccine compositions optionally comprise one or both early proteins from HPV 18, preferably in the form of a D protein-E6 or D protein-E7 or D protein-E6 / E7 fusion.

The vaccine compositions of the invention additionally comprise antigens from other HPV strains, preferably from HPV strains 6, 11, 31, 33 or 45.

The vaccine compositions of the invention also contain antigens from malaria-causing parasites. Suitable antigens from, for example, Plasmodium falciparum are RTS, S and TRAP. RTS is a hybrid protein containing the entire C-terminal portion of a P. falciparum circumsporozoite protein linked by four amino acids from the preS2 portion of hepatitis B surface (S) antigens to hepatitis B virus surface antigen. The full structure of these antigens is described in International Patent Application No. PCT / EP92 / 02591, published under WO 93/10152, which claims priority from UK Patent Application No. 9124390.7. In yeast, RTS is expressed as a lipoprotein particle, coexpressing with the S antigen from HBV produces a mixed particle known as RTS, S. TRAP antigens are described in International Patent Application No. PCT / GB89 / 00895, published as WO90 / 01496. The malaria vaccine composition of the invention comprises an antigenic composition comprising a combination of RTS, S and TRAP antigens. Other plasmodial antigens suitable for a multi-stage malaria vaccine are MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMPI, Pf332, LSA1, LSA3, STARP, SALSA, PfEXPI, Pfs25, Pfs28, Pfs27 / 25, Pfs16, Pfs48 / 45, Pfs230 from Plasmodium falciparum and their analogs from Plasmodium spp.

The compositions of the invention also contain an anti-tumor antigen and are used in immunotherapy of tumors. Auxiliary agent according to the invention 9 9 9 9 999999

It contains, for example, tumor rejection antigens from prostate, breast, colon and rectum cancer, lung, pancreas, kidney or melanoma. Examples are MAGE 1, MAGE 3 or other MAGE antigens for the treatment of melanoma, as well as PRAME, BAGE or GAGE (Robbins and Kawakami, 1996, Current Opinions in Immunology, 8, 628-636; Van den Eynde et al., 1997, International Journal of Clinical and Laboratory Research, Correale et al., 1997, Journal of the National Cancer Institute,

89, 293). These antigens are expressed by many different tumors, such as melanoma, lung cancer, bladder cancer, and bladder sarcoma. Other specific tumor antigens, such as specific prostate antigen (PSA) or Her-2 / neu, KSA (GA733), MUC-1, carcinoembryonic antigen (CEA) and others, are used in conjunction with the adjuvant of the invention. Accordingly, the invention provides a vaccine composition comprising an adjuvant of the invention and a tumor rejection antigen.

The antigen in the vaccine composition of the invention used to treat many tumors or to immunocastrate is also the body's own peptide hormone, such as whole gonadotropin releasing hormone (GnRH, WO 95/20600), a short 10 amino acid peptide.

The adjuvants of the invention are suitable for the preparation of a vaccine composition with an antigen derived from Borrelia spp. Such an antigen is nucleic acid, pathogen-derived antigens or antigenic preparations, recombinantly produced proteins or peptides, and chimeric fusion proteins. For example, the antigen is OspA. OspA is a fully mature protein in a lipidated form obtained from a host cell (E. coli) called lipo-OspA or a non-lipidated derivative thereof. Non-lipidated derivatives include the non-lipidated NS1-OspA fusion protein, which contains the first 81 amino acids with the N-terminus of the non-structural influenza virus (NS1) protein and the entire OspA protein, and MDP-OspA, the non-lipidated form of OspA N - at the end.

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The vaccine compositions of the invention are used for the prophylaxis and treatment of allergy. These vaccines comprise allergen-specific antigens (e.g. Der p1) and non-allergen-specific antigens (e.g. human IgE-derived peptides, stanworth decapeptide (patent application EP 0 477 231 B1) and others).

The amount of protein in the vaccine dose is determined as the amount that induces an immunoprotective response without significant side effects. This amount varies according to the specific immunogen, the method of its processing and the form in which it is present. It is generally contemplated that each dose will contain 1-1000Dg of protein, preferably 1500Dg, more preferably 1-100dg, most preferably 1-50Dg. The optimal amount of protein in a particular vaccine formulation is determined based on the results of standard subject immune response studies. The initial vaccine dose may be followed by one or more booster doses at appropriate intervals.

The adjuvants of the invention are part of vaccines containing antigens from many different sources. These antigens include human, bacterial or viral nucleic acids, pathogen-derived antigens or antigenic preparations, tumor-derived antigens or antigenic preparations, host-derived antigens, including GnRH and IgE peptides, recombinantly produced proteins or peptides, and chimeric fusion proteins.

The vaccine compositions of the invention are administered by mouth. Pharmaceutically acceptable vehicles for such vaccine compositions are, but are not limited to, alkaline buffers, intestinal capsules, or microgranules. The vaccine compositions of the invention are administered vaginally. Pharmaceutically acceptable vehicles of these vaccine formulations include, but are not limited to, emulsifying agents, polymers such as CARBOPOL R, and other known vaginal stabilizers. · ······. 17 - ·· ·· ···· ^L '· ··· ···· ···· ·· ·· creams and suppositories. The vaccine compositions of the invention are administered rectally.

The vehicles of these vaccine compositions are waxes and polymers used to make rectal suppositories known in the art.

The adjuvants of the invention are used for both prophylaxis and treatment. The invention relates to a method of treating a mammal at risk of or suffering from an infectious disease, a cancer, an allergy or an autoimmune disease. Part of the invention is a vaccine composition for medical purposes described herein. A general method of making a vaccine formulation is described by Voller et al., 1978, New Trends and Development in Vaccines, University Park Press, Baltimore, Maryland, U.S.A.

The invention relates to the use of the polyoxyethylethers and polyoxyethylene esters of formula (I) in the manufacture of an excipient comprising a surfactant of formula (I) and a pharmaceutically acceptable vehicle. The invention relates to the use of the polyoxyethylene ethers and polyoxyethylene esters of formula (I) in the manufacture of a vaccine composition comprising a surfactant of formula (I), a pharmaceutically acceptable vehicle and an antigen. The invention relates to the use of the polyoxyethylene ethers and the polyoxyethylene esters of formula (I) in the manufacture of the adjuvant or vaccine composition described above, wherein the adjuvant is cholesterol-free. The invention relates to the use of the polyoxyethylene ethers and polyoxyethylene esters of formula (I) in the manufacture of the adjuvant or vaccine composition described above, wherein the adjuvant is in the form of a non-vesicular solution or suspension.

Pharmaceutically acceptable vehicles include water, phosphate buffered saline, and isotonic buffer solution.

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An alternative designation for polyoxyethylene lauryl ether is entered in the CAS registry. The polyoxyethylene lauryl ether registration number is CAS REGISTRY NUMBER: 9002-92-0.

The invention is illustrated by the following examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Techniques for measuring antibody (Ab) responses to a specific antigen

Measurement of OspA specific serum IgG antibodies - ELISA

Maxisorp Nunc immuno plates were coated overnight at 4 ° C with OspA antigen (1 µg / ml, 50 µΙ per well) or purified goat anti-mouse Ig antibody (Boerhinger, 5 µg / ml, 50 µΙ per well) in PBS , A series. Platelet free spots are blocked for 1 hour at 37 ° C with saturation buffer (PBS containing 1g / l BSA, polyoxyethylene monolaurate sorbitan (TWEEN 20) at 0.1g / l and normal bovine serum (NBS, at a concentration of Then add a series of two-fold dilutions (in saturation buffer, 50 μΙ per well) of a mixture of isotype IgG diluted in saturation buffer (50 μΙ per well) as a standard curve (mix of mouse monoclonal antibodies IgG1, IgG2a and IgG2b (Sigma)). starting at 200 ng / ml, row A) and serum samples (beginning at 1: 100 dilution, row B to H) are incubated at 37 ° C for 1 hour 30 minutes, then washed three times with wash buffer (PBS, polyoxyethylene mono). sorbitan lureate (TWEEN 20) at a concentration of 0.1 g / L) Biotinylated goat anti-mouse IgG (Amersham) diluted 1: 5000 in saturation buffer was incubated (50DI per well) for 1 hour 30 minutes at 37 ° C. After three washes followed by the addition of streptavidin-horseradish peroxidase conjugate (Amersham), the plates are washed five times and incubated for 20 minutes at room temperature with revolution buffer (50 μί / well, OPDA at 0.4 mg / ml (Sigma) and hydrogen peroxide in concentration of 0.03g / l in 50mM citrate buffer pH 4.5). Revolution is terminated by adding 50 μί 2N sulfuric acid to each well. The Biorad 3550 is measured at 492 and 630 nm for optical density. Antibody titer is calculated by a 4 parameter mathematical method using SoftMaxPro software.

Anti - TT, anti - FHA and anti - influenza IgG titers are measured by the same technique, replacing the OspA antigen with TT, FHA or whole influenza antigen. The TT antigen is supplied from a commercially available source (Behring). The FHA antigen is produced and purified as described in patent application EP 0 427 462 B. All influenza β-propiolactone inactivated virus (BPL) is obtained from SSD GmBH (Dresden, Germany).

Measurement of mouse serum polysaccharide-specific IgG antibodies (PS14 and PS19) of S. pneumoniae - ELISA

Maxisorp Nunc immuno plates are coated with PS14 antigen (5 μΙ / ml, 100 μΙ per well) or PS19 antigen (20 μΙ / ml, 100 μΙ per well) diluted in PBS for 2 hours at 37 ° C. The plates are then washed three times with wash buffer (PBS, polyoxyethylene monolaurate sorbitan (TWEEN 20) at a concentration of 0.1 g / l). Then add a series of two-fold dilutions (in PBS, TWEEN 20, 100 μί per well) of IgG14 monoclonal antibodies (mAbs) specific for PS14 or PS19 as a standard curve (starting at PS14 concentration 785 ng / ml, PS19 2040 ng / ml, series). A) and serum samples (starting at 1:20 dilution, series B to H) are incubated for 30 minutes at 20 ° C with shaking. To avoid non-specific reactions, both standard mAbs and serum samples are incubated at 37 ° C for 1 hour before addition and dilution on the plate.

- 20 with conventional polysaccharide (CPS). The plates are then washed three times with wash buffer (PBS, TWEEN 20). Thereafter, peroxidase-conjugated goat anti-mouse IgG (Jackson) diluted 1: 5000 in PBS with TWEEN 20 is incubated for 30 minutes with continuous shaking. After three washes, the plates are incubated for 15 minutes at room temperature with reversion buffer (100 μΙ per well, OPDA). at a concentration of 0.4 mg / ml (Sigma) and hydrogen peroxide at a concentration of 0.03 g / l in 50 mM citrate buffer pH 4.5). Revelation is terminated by the addition of 1N HCl (50 μΙ per well). The Biorad 3550 was read at 492 and 630 nm to determine the optical density. The antibody titer is calculated by a 4 parameter mathematical method using SoftMaxPro software.

Measurement of OspA specific serum Ig in monkeys - ELISA

Maxisorp Nunc immuno-plates are coated overnight at 4 ° C with OspA antigen (1 µg / ml, 50 μΙ per well) diluted in PBS. Plates are blocked for 1 hour at 37 ° C with saturation buffer (PBS containing 1g / L BSA, polyoxyethylene monolaurate sorbitan (TWEEN 20) at 0.1g / L). Then add a series of double dilutions (in saturation buffer, 50 μΙ per well) of the reference serum as a standard curve (the serum has a mean titer of 60,000 ELISA units / ml, starting at 12 EU / ml, series A) and serum samples (starting at dilution 1). (100, series B to H) are incubated at 37 ° C for 1 hour 30 minutes. The plates are then washed three times with wash buffer (PBS, polyoxyethylene monooleate sorbitan (TWEEN 20) at a concentration of 0.1 g / l). Biotinylated goat anti-human Ig antibodies (Amersham) diluted 1: 3000 in saturation buffer are incubated (50 μΙ per well) for 1 hour 30 minutes at 37 ° C. After three washes followed by the addition of streptavidin-horseradish peroxidase conjugate (Amersham), the plates are washed five times and incubated at room temperature for 20 minutes with Revolution Buffer (50 μΙ per well, OPDA at 0.4 mg / ml (Sigma) and hydrogen peroxide in concentration of 0.03g / l in 50mM citrate buffer pH 4.5). Revolution is terminated by adding 50 μΙ 2N

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Sulfuric acid was added to each well. Biorad 3550 immunoassay at 492 a

630 nm measures the optical density. The antibody titer is calculated by a 4 parameter mathematical method using SoftMaxPro software.

Anti-influenza antibody titers are measured by a similar technique except that the OspA antigen is replaced with whole influenza inactivated by β-propiolactone (BPL) obtained from SSD GmBH (Dresden, Germany).

Measurement of OspA specific monkey nasal IgA antibodies - ELISA

Maxisorp Nunc immuno-plates are coated overnight at 4 ° C with OspA antigen (1 µg / ml, 50 μΙ per well) diluted in PBS (series B to H). Platelets are blocked for 1 hour at 37 ° C with saturation buffer (PBS containing 1g / l BSA, polyoxyethylene monolaurate sorbitan (TWEEN 20) at 0.1g / l and normal bovine serum (NBS) at 4g / l ). Then add a series of double dilutions (in saturation buffer, 50 μ 50 per well) of the reference secretion as a standard curve (secretion has a mean titer of 3000 ELISA units / ml, starting at 30 EU / ml, series A) and nasal swabs (starting at dilution 1). (5, rows B to H) were incubated at 22 ° C for 2 hours. The plates are then washed three times with wash buffer (PBS, polyoxyethylene monooleate sorbitan (TWEEN 20) at a concentration of 0.1 g / l). Biotinylated goat anti-human IgA antibodies (ICN, 0.2 μΙ / ml) in saturation buffer are incubated (50 μΙ per well) for 1 hour 30 minutes at 37 ° C. After three washes followed by the addition of streptavidin-horseradish peroxidase conjugate (Amersham), the plates are washed five times and incubated for 10 minutes at room temperature with reversion buffer (50 μΙ per well, TMB, Biorad). of each well. The Biorad 3550 is measured at 450 and 630 nm for optical density. The antibody titer is calculated by a 4 parameter mathematical method using

9 · 9 · 1

- ZZ ”SoftMaxPro software. Samples are considered positive if the IgA titer exceeds the end of the assay (0.3 EU / ml).

Measurement of serum LA2 - like antibody titres to the Lipo antigen - OspA inhibition assay

Antibody titers in vaccines are studied for their LA2-like specificity. LA2 is a murine monoclonal antibody that recognizes a conformational OspA epitope on the bacterial surface and that is capable of killing B. burgdorferi in vitro and protecting mice in encountering laboratory-grown spirochetes (Schaible UE et al., 1990, Proc Natl Acad Sci USA, 87, 3768-3772). In addition, the monoclonal antibody LA-2 correlates with bactericidal antibodies. In human serum studies, LA-2 titers correlate with total anti-OspA IgG titers (measured by ELISA).

Maxisorp Nunc immuno-plates were coated overnight at 4 ° C with Lsp OspA antigen (at a concentration of 0.5 µg / ml, 50 µΙ per well) diluted in PBS. Free sites are blocked for 1 hour at 37 ° C with saturation buffer (PBS, BSA at 1 g / l, TWEEN 20 at 0.1 g / l, NBS at 4 g / l). A series of two-fold dilutions of LA2 monoclonal antibody (mAb) (starting at 4Dg / ml) is diluted in saturation buffer (50 μΙ per well) to produce a standard curve. Dilutions of serum samples from the vaccine preparations (starting at 1:10 dilution) are also added and the plates incubated for 2 hours at 37 ° C. After incubation, the plates are washed three times with wash buffer (PBS, TWEEN 20 at 0.1 g / l). 50 μΙ of peroxidase conjugate with monoclonal antibody LA2 (1: 10,000) diluted in saturation buffer is added to each well and incubated for 1 hour. at 37 ° C. After five washes, the plates are incubated for 20 minutes at room temperature (in the dark) with revellation buffer (50 μΙ per well, OPDA at 0.4 mg / ml (Sigma) and hydrogen peroxide at 0.03g / l in 50mM citrate buffer. pH 4.5). The reaction and coloring is terminated by addition of 2N sulfuric acid. The Biorad 3550 is read optical at 492 and 630 nm

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494 4 9 94 4 • 9 4 4 4 4 4

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9444944 44 44 density. LA2 - like antibody titers are calculated by a 4 parameter mathematical method using SoftMaxPro software. LA2-like antibody titers are determined by comparison with a standard curve.

Example 2

Intranasal stimulation of mice with OspA antigen

- weekly female Balb / c strain (groups of 8 animals) are immunized by intramuscular injection of 1 µg lipo-OspA antigens on 50 µg alum. After three months, mice under general anesthesia receive an intranasal boost of - 10 µl of solution (5 µl into one nostril by dropwise pipette) containing A: 5 µg of lipo-OspA antigens; B: 5 µg lipo-OspA antigens in tween 20 at 36 g / l, Imwitor 742 at 10 g / l; C: 5 µg lipo-OspA antigens in tween 20 at a concentration of 36 g / L; D: 5 µg of Lipo OspA antigens in polyoxyethylene-9-lauryl ether D at 18 g / L.

days after the booster dose, serum anti-lipo-OspA IgG antibodies and anti-OspA-LA2 antibodies were detected in the sera by ELISA (see Example 1). The results (Figure 1) show that intranasally administered lipo-OspA antigen is able to induce a systemic increase in titer of lipo-OspA-specific IgG antibodies. However, this increase is only marginal in the presence of tween 20 and Imwin 742 or tween 20 alone. However, an increase in antibody titer is very significant with the presence of polyoxyethylene-9-lauryl ether. A similar situation occurs with the LA2 antibody response (see Figure 2).

Example 3

Intranasal stimulation of mice with amspigen OspA ·· ··· ♦

Groups of mice are primarily immunized as described in Example 2 with 5 µg of lipo-OspA antigen alone (groups A and C) or in the presence of B: taurocholic acid at a concentration of 1 g / L; D: dodecyl maltoside at a concentration of 1g / L; E: tween 20 at 36 g / l; or F: polyoxyethylene-9-lauryl ether at 18 g / l. Since the test with groups A and B runs at a different time than the test with groups C, D, E and F, they are separated in the figures (see Figure 3). Sodium taurocholate at a concentration of 1 g / l does not significantly increase the response to the boost. Dodecylmaltoside at a concentration of 1 g / l and tween 20 at a concentration of 36 g / l have only a weak enhancing effect, but polyoxyethylene-9-lauryl ether potentiates the IgG antibody response very significantly. A similar effect is observed in the LA2 antibody response (see Figure 4).

Example 4

Intranasal stimulation of mice - dose range study

To determine the concentration of polyoxyethylene-9-lauryl ether required to achieve the adjuvant effect observed in the previous examples, a dose range test is performed. As other polyoxyethylene ethers also have auxiliary effects, the test is also performed with polyoxyethylene - 23 - lauryl ether. Mice primarily immunized as in Example 1 are intranasally administered a 10 μΙ boost with 5 μg lipo-OspA antigen in A: PBS; B: polyoxyethylene-9 lauryl ether at a concentration of 1g / L; C: polyoxyethylene-9-lauryl ether at a concentration of 2 g / l; D: polyoxyethylene-9-lauryl ether at a concentration of 5 g / l; E: polyoxyethylene-23-lauryl ether at a concentration of 1 g / l; F: polyoxyethylene-23-lauryl ether at a concentration of 10 g / l. 14 days after the boost, the sera were analyzed in the same manner as in Example 2.

- 25 * · * 9 9 9 9 99 ·· · 9 9 9

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

It can be seen from Figures 5 and 6 below that even a concentration of polyoxyethylene-9-lauryl ether as low as 1 g / l will result in a very significant enhancement of the immune response. Likewise, polyoxyethylene - 23 - lauryl ether significantly increases the immune response to an intranasally administered booster dose.

Example 5

Combined vaccine - intranasal boost

This assay verifies the ability of polyoxyethylene ethers to enhance the systemic immune response following intranasal administration. Female balb / c mice are immunized primarily by intramuscular injection of a commercial vaccine DTPa (diphtheria, tetanus, cell-free pertussis vaccine: INFARIX ™ Smith Kline Beecham, Belgium). Mice are immunized with one of two 50 μΙ intramuscular injections corresponding to 20% of the human dose. After three months, mice receive an intranasal boost (as in Example 2) with tetanus toxoid (TT: 5 µg) or filamentous hemagglutinin (FHA: 5dg) in A: PBS; B: polyoxyethylene-9-lauryl ether at a concentration of 1 g / l; or C: intramuscular injection of DTPa only (twice 50 μΙ). After 14 days, serum specific TT and FHA specific IgGs were determined. Antibody titers are shown in Figures 7 and 8.

TT itself does not induce a significant boost in the immune response, but polyoxyethylene - 9 - lauryl ether is able to significantly enhance the immune response. Surprisingly, the enhancement of the immune response following intranasal administration of the antigen in the presence of this adjuvant is greater than the enhancement following intramuscular administration of the immunization. Administration of FHA induces an immune response by itself, and by adding polyoxyethylene-9 lauryl ether adjuvant, this response is further enhanced.

Example 6 • ·

9 9 • · ·

9 9 • 9 9

Intranasal stimulation in monkeys AGMs

Many adjuvants are effective in small rodents, but their administration has no effect in larger animals. In this example, the ability of polyoxyethylene ethers to potently enhance the immune response after intranasal administration in larger animals is demonstrated. Monkeys of the Green Vervet (AGM) strain are divided into groups of four and primarily immunized by intramuscular injection of 10 µg lipo - OspA antigen per 500 µg alum. After 10 months, animals under general anesthesia were dosed intranasally with a 200 μΙ (100 μΙ per nostril) dual dose spray device from Pfeiffer GmBH, Germany, containing 60 µg lipo-OspA antigen in A: PBS; or B: polyoxyethylene-9-lauryl ether at a concentration of 1 g / l. After 14 days, anti - OspA and LA2 immunoglobulin titers are measured in serum. Geometric mean titers for each group are depicted in Figures 9 and 10. For group C, consisting of 10 monkeys and receiving both primary and boosting lipo-OspA antigen on intramuscular injection, anti-OspA immunoglobulin titers (geometric mean titers) were determined. are shown only for LA2 titers, see Figure 10).

The lipo - OspA antigen is capable of enhancing the immune response when administered intranasally to monkeys, but this enhancement is significantly greater when polyoxyethylene - 9 - lauryl ether is administered concomitantly at a concentration of 1 g / l. Again, the antibody titers found after intranasal boost with polyoxyethylene-9-lauryl ether are higher than those found after intramuscular injection (group C).

Example 7

Primary and Remaining Intranasal Immunization of AGMs 444 «• ··············

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4 4 4 4 4

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In the previous examples, it is shown that polyoxyethylene ethers assist in intranasal administration of a booster dose to enhance the systemic immune response. In this example, the possibility of administering both primary and booster immunization to animals intranasally is established in order to induce a sufficient systemic immune response. In order to investigate the possibility of administering this adjuvant to larger animals, the test is carried out on monkey monkeys (AGMs).

Monkeys of the Green Vervet (AGM) strain (groups of three animals) are both intranasally administered a primary and booster immunization dose of 200 μΙ (100 μί into one nostril, given by a two-dose spray device from Pfeiffer GmBH, Germany) containing 60 μρ lime-OspA v A: PBS; or B: polyoxyethylene-9-lauryl ether at a concentration of 1 g / l. After 14 days, OspA specific immunoglobulin is measured in serum. Figure 11 shows that there is no systemic immune response following intranasal administration of both primary and booster immunization doses of antigen without adjuvant. After administration of the antigen with polyoxyethylene-9auryl ether as adjuvant, significant anti-OspA antibody titers are shown in the diagram.

Example 8

Auxiliary effect of CpG in intranasally administered vaccine on induction of systemic and nasal humoral immune response to lipo-OspA antigen in primates

In this model, the adjuvant effect of polyoxyethylene-9 lauryl ether (POE-9LE) is verified without and in conjunction with other immunostimulatory agents in the primary and mock immunization of primates. Serum and nasal immunoglobulins are determined. The immunostimulant used herein is CpG 1001 described in Example 9.

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9

9 * 9

999 * 99

Methodology

Green monkey monkeys are given intranasal primary and booster immunizations on day 0 (pl and day 14) .The vaccine is administered by a Pfeiffer two-dose spray device (100 μΙ into each nostril, under general anesthesia). :

group antigen auxiliary agent n = administration 1 lipoOspA (60 pg) None 2 i.n. 2 lipoOspA (60pg) CpG (100 μ9) 3 i.n. 3 lipoOspA (60 pg) CpG (100 µ9), POE - 9 LE (0.25 g / l) 3 i.n. 4 lipoOspA (60 pg) POE - 9 LE (0.25 g / L) 4 i.n. 5 lipoOspA (60 pg) POE - 9 LE (0.25 g / L) 4 i.n.

On day 14 after pI, serum samples were taken and lipo-OspA antibody titers were determined. Nasal antigen-specific IgA is measured by very sensitive ELISA on nasal swabs taken at the same time. Animals are considered positive if their IgA titers reach a predetermined level that is significantly higher than baseline.

Results ·· · * · »

Serum OspA specific immunoglobulin

The serum levels of anti - lipo - OspA immunoglobulins measured on day 14 after piling are shown in Figure 12. Lipo - OspA antigen administered alone as a primary and booster immunization did not induce any detectable increase in serum immunoglobulins. In the presence of CpG, the situation is the same. Co-administration of POE-9LE at a concentration of 0.25 g / l or 0.5 g / l induces a greater immune response than administration of CpG, with POE-9 LE concentration of 0.5 g / l being most effective. When both CpG and POE - 9 LE are administered at 0.25 g / l, the antibody response is similar to that of POE - 9 LE at 0.5 g / l. This suggests a synergy between the CpG and POE components.

Nasal OspA specific IgA

Vaccines containing lipo-OspA antigen alone or in combination with CpG do not induce any increase in captured IgA antibodies in the nose (Figure 13 summarizes all antibody responses in the nose). When administering a combination of lipo-OspA antigens with polyoxyethylene lauryl ether at a concentration of 0.25 g / l, only 25% of animals are positive for 'nasal IgA' (as opposed to 50% positive animals when co-administered with POE-9 LE at 0.5 g / l). l). When the combination of lipo - OspA and POE - 9 LE at 0.25 g / l is supplemented with CpG, 100% of the animals develop an IgA antibody response. Thus, CpG and polyoxyethylene lauryl ether are also synergistic in inducing mucosal antibodies.

Thus, CpG and polyoxyethylene lauryl ether show synergy in inducing antigen-specific serum immunoglobulins and nasal IgA in monkeys.

Example 9 • φ φφφφφφφφφφφφφφφφφ φφφφφφφφφφφφφφφφφφφφφφφφφφ

ΦΦΦΦΦΦ · ·· ··

Auxiliary effect of intranasally administered CpG on enhancing systemic humoral response to lipo-OspA antigen

In this example, the effect of additional immunostimulants added to the polyoxyethylene ether adjuvant (POE-9 LE) in mice is demonstrated. CpG is a known immunomodulatory oligonucleotide described in PCT Patent Application WO 96/02555. The immune responses boosted by CpG vaccines are at least as high as those induced by conventional intramuscular boosting. These preparations are further compared with the known intranasal adjuvant, thermolabile enterotoxin from E. coli (mLT).

In this assay, the CpG 1001 (TCC ATG AGC TTC CTG AGC TT) and CpG 1002 (TCT CCC AGC GTG CGC CAT) and negative control sequences of non-immunostimulatory CpG 1005 (TCC ATG AGC TTC CTG AGC TT) are used.

Methodology

Balb / c mice are primarily immunized on day 0 by intramuscular administration of 100 µl of a vaccine containing 1 µg lipo-OspA antigen adsorbed to 50 µg aluminum hydroxide. On day 107, animals under general anesthesia are given an intranasal boost immunization with 10 µl (5 µl per nostril) of nasal drops with a micropipette. Groups of 6 mice receive a boost of the following vaccines intranasally (i.n) or intramuscularly (i.m.):

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

9999 9 999

Group antigen auxiliary agent Filed 1 LipoOspa (5pg) AIOH ₂ (50pig) i.m. 2 LipoOspa (5pg) CpG 1005 (20µg), POE -9 LE (2g / l) i.n. 3 lipoOspa (δμρ) CpG 1002 (2µμα), POE - 9 LE (1g / l) i.n. 4 LipoOspa (5pg) CpG 1001 (20μ), POE 9 LE (1 g / l) i.n. 5 lipoOspa ^ g) CpG 1005 (ig) i.n. 6 lipoOspa (4μ $) CpG 1002 (ig) i.n. 7 ipipoOspa (5μ ^) CpG 1001 (g) i.n. 8 LipoOspa (5pg) POE-9 LE (1g / l) i.n. 9 lipoOspa ^ g) mLT (5 mg) i.n. 10 lipoOspa ^ g) None i.n. 11 neimunizováno

Blood samples are taken on the day of administration of the boost and 14 days thereafter (dran). OspA titers of specific IgG and LA2 antibodies are determined by ELISA.

Results

From Figure 14 (OspA specific serum IgG determined by antigen specific ELISA) and 15 (bactericidal LA2 titers in serum), administration of CpG alone does not improve the serum antibody response to OspA antigen. Vaccine containing OspA s

- 32 ···························· Polyoxyethylene leuryl ether increases the resulting IgG and LA2 titers with polyoxyethylene leuryl ether. The best response is achieved when the lipo - OspA antigen is administered concurrently with both polyoxyethylene lauryl ether and CpG.

Example 10

Dose study

In Example 4, it is described that even low concentrations of polyxyethylene-9 lauryl ether, such as 1 g / l, greatly enhance the immune response. In this example, a dose range test with lower concentrations of adjuvant is shown to determine the concentration of polyoxyethylene-9-lauryl ether needed to achieve efficacy of the adjuvant in nasal mucosal vaccines.

Balb / c mice are primarily immunized as described in Example 2. A booster dose of 10 μΙ is administered to the animals intranasally. The dose contains 5 µg lipo-OspA antigen in A: PBS; B: polyoxyethylene-9-lauryl ether at a concentration of 1 g / l; C: polyoxyethylene-9-lauryl ether at a concentration of 0.5 g / l; D: polyoxyethylene-9-lauryl ether at a concentration of 0.25 g / l or a boost is given intramuscularly by injection of E; 1 μg lipo OspA antigen adsorbed on 50 pg alum. 14 days after the boost, the serum titers are determined as described in the example

1.

Results

It can be seen from Figures 16 and 17 that polyoxyethylene-9-lauryl ether at a concentration of 0.25 g / l significantly enhances the immune response. This adjuvant already achieves a low antibody-like concentration.

the response obtained with parenteral administration of the vaccine.

Example 11

Vaccination of monkeys against influenza

In Example 11, polyoxyethylene-9-lauryl ether was shown to increase influenza virus immunogenicity in mice. In this example, it is verified whether this surfactant also has a similar adjuvant effect in larger species. Green monkey monkeys (AGMs, 2 animals per group, blood collection in 2 animals per day) are immunized with primary and booster intranasally administered (as in Example 6) with a dose of 50 µg HA equivalent from inactivated whole A / Beijing / 262/95 virus in 200 μΙ A: PBS; B: polyoxyethylene-9 lauryl ether at a concentration of 0.5 g / l. On days 2, 7, and 14 after administration of the boost, immunoglobulins specific for A / Beijing / 262/95 virus are determined in the sera of the animals. The figure clearly shows that when polyoxyethylene 9-lauryl ether is used as an adjuvant, the immune response to influenza antigen is better.

Example 13

Vaccination against polysaccharide antigens

In the previous examples, the ability of polyoxyethylene-9 lauryl ether to enhance the immune response to a protein antigen is shown. In this example, the ability of this adjuvant to enhance the immune response to an intranasally administered boost of the polysaccharide antigen after previous parenteral primary immunization in mice is tested. Mice are primarily immunized with a single subcutaneous 100 μΙ injection containing PS14 and PS19 polysaccharides (1 µg each) from • mice.

Streptococcus pneumoniae conjugated to carrier D protein. After two months, mice under general anesthesia receive an intranasal dose of 40 μΙ of solution (at 0 10 μΙ into each nostril, 30 minutes later again 10 μΙ into each nostril, by dropwise pipette) containing 1 pg of PS14 and 1 Dg conjugate. PS19 conjugate in A: 150 mM NaCl pH 6.1; B; polyoxyethylene - 9 - lauryl ether at a concentration of 1 g / l. 14 days after the boost, serum antibodies specific for PS14 and PS19 are determined in serum.

Results

It can be seen from Figures 20 and 21 that separate administration of PS14 and PS19 elicits an immune response which is further enhanced by the administration of polyoxyethylene 9-lauryl ether as adjuvant.

Example 14

Polyoxyethylene - 8 - stearyl ether

In this example, it is shown that other polyoxyethylene ethers, such as polyoxyethylene-8-stearyl ether, also have an adjuvant effect on the immune response.

Balb / c mice primarily immunized as in Example 2 received an intranasal boost of 10 μΙ containing 5 µg of lipo-OspA antigen in A: PBS; B; polyoxyethylene - 9 - lauryl ether at a concentration of 1 g / l; C; polyoxyethylene-8-stearyl ether at a concentration of 1 g / L; or dose D: intramuscular injection of 1 µg lipo-OspA antigen adsorbed on 50 µg alum. 14 days after the boost, the sera were analyzed in the same manner as in Example 1.

9

- 35 Results

Figures 22 and 23 show that polyoxyethylene-8-stearyl ether enhances the immune response to the antigen as significantly as polyoxyethylene-9 lauryl ether. After administration of both polyoxyethylene ethers, antibody titers increase as well as after administration of the vaccine parenterally.

Claims (41)

PATENT CLAIMS An auxiliary agent, characterized by a surfactant of the formula (I) ΗΟ (ΟΗ ₂ ΟΗ ₂ Ο) _η - A - R, 50, A is a bond or a -C (O) - group, R is an alkyl or phenylalkyl group of 1 to 50 carbons; and a pharmaceutically acceptable vehicle, wherein the surfactant of formula (I) is in the form of an aqueous solution or micelles. An excipient comprising a surfactant of formula (I) HO (CH ₂ CH ₂ O) _n - A - R, wherein n is a number from 1 to 50, A is a bond or a -C (O) -, R group is an alkyl or phenylalkyl group of 1 to 50 carbons; and a pharmaceutically acceptable vehicle, wherein the surfactant of formula (I) is not in the form of vesicles and the excipient does not comprise an acrylic acid polymer. Adjuvant according to claim 1 or characterized in that it does not contain an oil-in-water or water-in-oil emulsion. Adjuvant according to any one of claims 1 to 3, characterized in that the surfactant of formula (I) has hemolytic effects. An excipient comprising a surfactant of formula (I) HO (CH ₂ CH ₂ O) _n - A - R, wherein n is a number from 1 to 50, A is a bond or a -C (O) - group R is an alkyl or phenylalkyl group of 1 to 50 carbons; and a pharmaceutically acceptable vehicle, wherein the surfactant of formula (I) is not in the form of vesicles and the degree of haemolytic activity in the guinea pig blood hemolysis assay is in the range of 0.05-0.0001%. • · · · - 37 Adjuvant according to claims 4 or 5, characterized in that the haemolytic activity of the surfactant of formula (I) in the guinea pig blood hemolysis assay does not differ more than ten times from the haemolytic activity of polyoxyethylene-9-lauryl ether and polyoxyethylene-8 stearyl ether. Adjuvant according to any one of claims 1 to 6, characterized in that it contains a surfactant of formula (I), wherein n is a number from 4 to 24. An adjuvant according to claims 1 to 7 comprising a surfactant of formula (I) wherein R is an alkyl or phenylalkyl group of 8 to 20 carbons. An excipient comprising a surfactant of formula (I) HO (CH ₂ CH ₂ O) _n - A - R, wherein n is a number 9, A is a bond or a -C (O) -, R is alkyl or a phenylalkyl group of 1 to 50 carbons; and a pharmaceutically acceptable vehicle, wherein the surfactant of formula (I) is not in the form of vesicles. The adjuvant according to claim 8 or 9, wherein R is an alkyl group of 12 carbons. An excipient comprising a surfactant of formula (I) HO (CH ₂ CH ₂ O) _n - A - R, wherein n is a number 8, A is a bond or a -C (O) -, R is an alkyl or a phenylalkyl group of 1 to 50 carbons; and a pharmaceutically acceptable vehicle, wherein the surfactant of formula (I) is not in the form of vesicles. • · · · · · · · · · · · · · · · · · · · · · · - 38 12. An adjuvant according to claim 1 wherein R is an alkyl group of 18 carbons. An adjuvant according to any one of claims 1 to 12, characterized in that it comprises a surfactant of formula (I), wherein A is a bond and thereby forms an ether. An adjuvant according to any one of claims 1 to 12, characterized in that it contains a surfactant of formula (I), wherein A is -C (O) - and thereby forms an ester. An adjuvant according to claim 1, characterized in that the polyoxyethylene ether or polyoxyethylene ester of formula (I) belongs to the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene - 9 - lauryl ester, polyoxyethylene - 9 - stearyl ether, polyoxyethylene 8 - stearyl ether, polyoxyethylene - 4 - lauryl ether, polyoxyethylene - 35 - lauryl ether, polyoxyethylene - 23 - lauryl ether. The adjuvant according to any one of claims 1 to 10, characterized in that the surfactant concentration is in the range of 0.1-10 g / l. The adjuvant according to claim 16, wherein the surfactant concentration is in the range of 0.25 - 1 g / l. An adjuvant combination comprising an adjuvant comprising a surfactant of formula (I) HO (CH ₂ CH ₂ O) _n - A - R, wherein n is a number from 1 to 50, A is a bond or a group -C (O) -, R is an alkyl or phenylalkyl group of 1 to 50 carbons and a pharmaceutically acceptable vehicle, wherein: The surfactant of formula (I) is not in the form of vesicles; and at least one immunostimulatory agent selected from the group of: LT, CT, 3D-MPL, CpG, and QS21. An adjuvant combination comprising an adjuvant according to any one of claims 1 to 17 in combination with at least one immunostimulatory agent. The adjuvant combination of claim 19, wherein the at least one immunostimulatory agent belongs to the group: LT, CT, 3D-MPL, CpG, and QS21. 21. An adjuvant according to claim 1, wherein the CpG immunostimulant comprises the following sequences: TCC ATG ACG TTC CTG ACG TT (SEQ ID NO. 1). 22. Auxiliary combination comprising polyoxyethylene ether, either polyoxyethylene-9 lauryl ether or polyoxyethylene-8-stearyl ether, and 3D-MPL. The adjuvant of any one of claims 1 to further comprising a vehicle belonging to the group: chitosan and other polycationic polymers, polylactide and polylactidoglycolide particles, particles composed of polysaccharides or chemically modified polysaccharides, or particles composed of glycerol monoesters. 24. An excipient comprising polyoxyethylene-9-lauryl ether. - 40 A vaccine composition comprising an adjuvant according to any one of claims 1 to 24, further comprising an antigen or antigenic preparation. • · ···· 26. The vaccine composition of claim 1, wherein the antigen or antigenic preparation is derived from the following pathogens: human immunodeficiency virus, varicella zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalovirus, virus dengue fever, hepatitis A, B, C or E viruses, respiratory syncital virus, human papilloma virus, infectious virus, Hib, meningitis virus, salmonella, neisseria, borrelia, chlamydia, bordetela, streptococcus, mycoplasma, mycobacterium, haemophilia or plasmoplasm, plasmus IgE peptides such as stanworth decapeptide; or from tumor-associated antigens (TMA): MAGE, BAGE, GAGE, MUC-1, Her-2-neu, LnRH, CEA, PSA, KSA or PRAME. 27. A vaccine composition comprising polyoxyethylene-9-lauryl ether and an infusion virus antigen. A vaccine composition according to any one of claims 25 to 27, in the form of an aerosol or a spray. Use of a surfactant of formula (I) HO (CH ₂ CH ₂ O) _n - A - R, wherein n is an integer from 1 to 50, A is a bond or -C (O) -, R is an alkyl or phenylalkyl group of 1 up to 50 carbons, wherein the surfactant of formula (I) is not in the form of vesicles, in the manufacture of a medicament for application to the mucosal surface or to the skin of a patient. Use of an adjuvant according to any one of claims 1 to 24 in the manufacture of a medicament for application to the mucosal surface or skin of a patient. ··· “H1 - · · · ··· 9 999 9999 99 99 Use of a surfactant of formula (I) according to claim 29, characterized in that the medicament does not contain an acrylic acid polymer. Use of a surfactant of the general formula (I) according to claim 1, characterized in that the medicament does not contain an oil-in-water or water-in-oil emulsion. Use of polyoxyethylene-9-lauryl ether or polyoxyethylene-8-stearyl ether in the manufacture of a vaccine for application to the mucosal surface or skin of a patient. A spray device, in particular a two-dose spray device, with a vaccine characterized in that the vaccine composition comprises (a) a surfactant of formula (I) HO (CH 2 CH 2 O) _n -A-R, wherein n is a number from 1 to 50, A is a bond or -C (O) -, R is an alkyl or phenylalkyl group of 1 to 50 carbons; (b) a pharmaceutically acceptable vehicle; and (c) an antigen or antigenic preparation. Use of a vaccine composition according to any one of claims 25 to 28 * in the manufacture of a vaccine composition for the treatment of viral, bacterial and parasitic infections, allergies or tumors. A method of treating a mammal at risk or already ill with an infection, tumor, or allergy, comprising administering to the mammal a safe and effective amount of a vaccine composition according to any one of claims 25 to 28. · 4 4 4>> «4> 4 4 4 4 4 4 4 4 4 · · · · ΊΊ · «· ·« - τ · 2 ~ · · · ··· · 9999 9 999 9999 ·· ·· 37. A method of treating a mammal at risk or already ill with an infection, tumor, or allergy, comprising administering to the mammal a safe and effective amount of a vaccine composition according to any one of claims 25 to 28. 38. A method of treating a mammal at risk or already ill with an infection, tumor, or allergy, comprising administering to a mammal intranasally a safe and effective amount of a vaccine composition according to any one of claims 25 to 28. A method for producing a vaccine composition according to any one of claims 25 to 28, comprising (a) an adjuvant according to claims 1 to 24, (b) a pharmaceutically acceptable vehicle, and (c) an antigen or antigenic preparation. . A vaccine composition or adjuvant according to any one of claims 1 to 28, characterized in that it is used as a medicament. A vaccine composition comprising an adjuvant, a pharmaceutically acceptable vehicle and an antigen or antigenic preparation, wherein the adjuvant is a surfactant of formula (I) HO (CH ₂ CH ₂ O) _n - A - R, wherein n is a number from 1 to 50, A is a bond or -C (O) -, R is an alkyl or phenylalkyl group of 1 to 50 carbons.