CZ20003732A3 - Aids - Google Patents

Abstract

The solution deals with an adjuvant comprising a surfactant of the formula HO(CH2CH2O)n - A - R and a pharmaceutically acceptable vehicle. The solution also includes a vaccine comprising the adjuvant and an antigen * or antigen preparation and intended for the treatment of 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 to a vaccine 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.

State of the art

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

Mucosal vaccination has become of interest since it was demonstrated in animals that mucosal administration of antigens is much more effective than systemic administration of antigens by injection in inducing an immune response at the mucosal surface, which is the entry point for many pathogens. It is assumed that vaccination at a mucosal site, such as the nose, can induce mucosal immunity not only in the nasal mucosa but also in distant mucosal sites, 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).

For mucosal immunization to replace or be an alternative to injection immunization, it must be possible to induce a systemic immune response by mucosal administration of the vaccine at least as effectively as by injection. Although it has been reported that certain antigens administered mucosally are capable of eliciting a systemic response (Cahill et al., 1993, FEMS Microbiology Letters, 107, 211-216), most soluble antigens administered alone to the nasal mucosa elicit no or only a small immune response.

Many authors have discovered effective adjuvants that help overcome this problem in various ways, such as antigen encapsulation (e.g. liposomes and microparticles); direct interaction with target cells with subsequent release of immunostimulatory cytokines (e.g. cholera toxin or E. coli thermolabile toxin); and increased absorption of antigens by the epithelium (e.g. cholera toxin).

In this patent application, polyoxyethylene ethers and polyoxyethylene esters are shown as very effective adjuvants for vaccine preparations. The adjuvants according to the invention are safe, easy to sterilize and administer. The advantage of these preparations is that when administered to the mucosa, they are able to induce a sufficient systemic immune response, at least the same as when the vaccine is conventionally administered by systemic injection.

Polyoxyethylene ethers, such as polyoxyethylene lauryl ether, are described in the Merck Index (12th edition, 7717), and their therapeutic uses listed therein include: local anesthetics, antipruritics, and sclerosing agents. Polyoxyethylene ethers and/or polyoxyethylene esters belong to the group of nonionic surfactants.

Increased insulin uptake in the nasal cavity has been reported after 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 vaccines. Vaccines containing a combination of polyoxyethylene castor oil or caprylic/capric glycerides with polyoxyethylene sorbitan monoesters and antigen elicit a systemic immune response after topical administration to the mucous membrane (patent application WO 9417827). This patent application describes that the combination of TWEEN 20™ (polyoxyethylene sorbitan monoester) and Imwitor 742™ (caprylic/capric glycerides) or the combination of TWEEN 20™ and polyoxyethylene castor oil enhances the systemic immune response after intranasal immunization. Details of the effect of this preparation on enhancing the immune response to antigens administered 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 can also take the form of non-ionic surfactant vesicles (commonly known as neosomes, patent application WO 95/09651). These vesicles form lipid-containing vesicles in the presence of cholesterol.

Novasomes (US patent application 5,147,725) are paucilamenar vesicular structures containing polyoxyethylene ethers and cholesterol encapsulating antigen, which upon systemic administration enhance immune responses to antigen.

• ·

bilayer, which binds antigen to the internal aqueous phase or directly to the bilayer.

The essence of the invention

The invention consists in the fact that polyoxyethylene ethers or polyoxyethylene esters administered together with antigens significantly enhance the systemic immune response. When these adjuvants are used in vaccines administered to the mucosa, their enhancing effect leads to the fact that the immunological response of the organism to the antigen is the same or even higher than when the antigen is conventionally administered by systemic injection. These molecules represent a group of adjuvants suitable for administration to humans, both in conventional systemic vaccination and in replacing systemic vaccination with mucosal vaccination.

The invention is a potent vaccine adjuvant and, unlike many other available adjuvants which act by encapsulating the antigen, is in the form of a non-vesicular solution or suspension. The invention therefore relates to an adjuvant which comprises a surfactant of formula (I) in the form of a vesicle-free solution or suspension. The invention further relates to a vaccine adjuvant which comprises a surfactant of formula (I) formulated without the presence of cholesterol.

The vaccines and adjuvants according to the invention contain molecules of general formula (I):

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

- 5 - ···*·· · ·· ·· ·· • · · ♦ · · · · » · · ·· · ····· • · · · · ······ • · · · ···· · ···· · ···· · ···· ···· ···· Invention with deals with vaccination a preparation containing

a polyoxyethylene ether of the general formula (I), where n is a number between 1 and 50, preferably between 4 and 24, most preferably 9; the component 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 the 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 preparation comprising a polyoxyethylene ester of the general formula (I), where 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 20 carbons, most preferably 12 carbons; and A is a -C(O)- group. The concentration of the polyoxyethylene ester is in the range of 0.1-20 g/l, preferably 0.1-10 g/l, most preferably 0.1-1 g/l. Suitable polyoxyethylene esters belong to this group: 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 preparation comprising polyoxyphenyl ethers of the general formula (I), where n is a number between 1 and 50, preferably between 4 and 24, most 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 of 0.25 - 1 g/l.

The vaccines according to the invention are used to protect mammals from infection or to treat mammals already sick, by administering the vaccine • · · · * · • · · · · · · · · · · · • · 9 · · · · · · • « · 9 9 9 9 9 9 9

9 · · 9 9 9 9 9 ···· ·

The invention is particularly concerned with adjuvants for mucosal vaccine formulations. Such adjuvants are well tolerated in humans and the immune responses they elicit are potent. The adjuvants of the invention are in the form of solutions or suspensions without the presence of vesicles and are therefore not burdened with the problems associated with the manufacture of adjuvant particulate systems, including maintaining stability, uniformity and quality control. Such formulations are potent adjuvants, exhibit low reactogenicity and are well tolerated by patients.

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

1. Fresh blood from laboratory guinea pigs is washed 3 times with phosphate buffered saline (PBS) in a benchtop centrifuge.

2. Add 50 µl of this blood suspension to 800 µl of PBS containing two-fold dilutions of detergent.

·· 4··· • · · 4 4 4 4 · · · · · · 4 4 4 4 4

4 4 4 4 4 4 4 4 4 4 ·· 444444

- 7 - 4444 4 444 4444 44 44

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

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

Within the experimental variability of these biological tests, the polyoxyethylene ethers or surfactants of general formula (I) according to the invention have a hemolytic activity between 0.5 and 0.0001 g/l, preferably between 0.05 and 0.0001 g/l, more preferably between 0.005 and 0.0001 g/l, most preferably between 0.003 and 0.0004 g/l. In the best case, the polyoxyethylene ethers or polyoxyethylene esters have a hemolytic activity similar (i.e. within a tenfold 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:alkyl chain length) affects the solubility of this detergent moiety in an aqueous environment. The auxiliaries of the invention are in the form of a solution or form particulate structures such as micelles. The auxiliaries of the invention are, due to their natural vesicle-free structure, clear, not cloudy or opaque, stable, easily sterilized by filtration on a membrane with pores of 220 nm in diameter and processed in a simple and controlled manner.

The vaccines of the invention are in the form of a non-vesicular solution or suspension of a polyoxyethyl ether or polyoxyethylene ester of the general formula (I) in a pharmaceutically acceptable vehicle such as PBS or water, with an antigen or antigen preparation. These vaccines are administered to the mucosal surface of a mammal either as a primary immunization or as a booster dose. Alternatively, the preparations of the invention may be administered systemically, for example transdermally, subcutaneously or intramuscularly.

- 8 • ·9 · ·

Other adjuvants that enhance both mucosal and systemic immune responses are bacterial enterotoxins obtained from Vibrio cholerae (cholera toxin (CT)) and Escherichia coli (thermolabile enterotoxin (LT). CT and LT are heterodimers consisting of a pentameric ring of subunits into which the toxic A subunit fits. The structure and biological activity of these toxins were 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. Recently, a non-toxic derivative of LT has been developed that lacks the proteolytic site necessary for the conversion of the non-toxic form of LT after its release from the cell to the toxic form. Due to the replacement of the amino acid arginine with glycine at position 192, this form of LT (designated mLT(R192G)) to the proteolytic cleavage. The toxicity of this form is thus significantly reduced while maintaining its strong adjuvant effect. mLT(R192G) is therefore called a proteolytic site mutant. The method 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 by alanine at position 69. Patent application WO 96/06627 describes the use of mLT(R192G) as a mucosal vaccine. These adjuvants can be combined with the nonionic surfactants of the present invention.

The invention includes combinations of polyoxyethylene ether or polyoxyethylene ester with other adjuvants or immunostimulants, including cholera toxin and its B subunit, monophosphoryl lipid A and its non-toxic derivative 3-de-O-acylated monophosphoryl lipid A (described in UK patent application number 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, C.R., 1996, Crit Rev Ther Drug Carrier Syst, 12 (1-2), 1-55; EP 0 362 279 B1; Kensil et al., 1991, J. Immunology, 146, 431-437; WO 99/100008) and oligonucleotide auxiliary system ·· ♦···

·· ·· * · · · • · « · • · · · · · · · · · · · · · containing 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. Combination compositions with this agent have a potent inductive and priming effect on immune responses in larger animals. Suitable oligonucleotides contain the following sequences: all internucleotide linkages of these sequences are modified with phosphorothioate.

OLIGO 1: TCC ATG ACG TTC CTG ACG TT

OLIGO 2: TCT CCC AGC GTG CGC CAT

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

The CpG oligonucleotides used in the present invention are synthesized by any method known in the art (for example, as described in patent application EP468520). A convenient way to produce these oligonucleotides is by synthesis on an automatic synthesizer.

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

The adjuvants according to the invention contain polyoxyethylene ether or polyoxyethylene ester surfactants which are not in the form of vesicles. The invention therefore concerns the use of polyoxyethylene ethers and polyoxyethylene esters of the general formula (I) in the production of adjuvants and vaccines in which the surfactant of the general formula (I) is not present in the form of vesicles.

·· ···· ··· · ·♦ ♦ · ·· • · · · · · « · · · » • · · · · · · · · · · · · · · · · ·

The vaccine preparations of the invention contain an antigen or antigenic preparation that will elicit an immune response against a human pathogen. The antigen or antigen 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 ((especially 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 gp1, II and IE63) or hepatitis viruses such as hepatitis B virus (hepatitis B surface antigen or derivative thereof), hepatitis A virus, hepatitis C virus or hepatitis E virus, or from other viral pathogens such as paramyxoviruses: respiratory syncytial virus (F and G proteins or derivatives thereof), parainfluenza virus, measles virus and mumps virus, human papillomaviruses (for example HPV6, 11, 16, 18, ...), flaviviruses (e.g. yellow fever virus, dengue fever 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 (as described by Gluck R., 1992, Vaccine, 10, 915-920) or their purified or recombinant proteins 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 (e.g. capsular polysaccharides and their conjugates, transferrin binding proteins, lactoferrin binding proteins, PilC, adhesins); Streptococcus pyogenes (e.g. M proteins or parts thereof, C5A protease, lipoteichoic acids), S. agalactiae, S. mutans', Haemophilus ducrei, Moraxella spp, including M. catarrhalis, also known as Branhamella catarrhalis (high and low molecular weight adhesins and ivasins); Bordetella spp, including B. pertussis (e.g. pertactin, pertussis toxin or its derivatives, filamentous haemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica', Mycobacterium spp, including M. tuberculosis (e.g. ESAT6, antigen 85A, B or C), M. bovis, M. leprae, M. avium, M.

···· ♦» 4 >

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4 * 4 4 4 4 · · F

4 44 4 4 · · . II . 4444 4 4444 4444 44 ·4 paratuberculosis, M. smegmatis', Legionella spp, including L. pneumophila', Escherichia spp, including enterotoxic E. coli (for example, colonization factors, heat-labile toxin or its derivatives, heat-stable toxin or its derivatives), enterohemorrhagic E. coli, enteropathogenic E. coli (for example, shiga-like toxin or its derivatives); Vibrio spp, including V. cholerae (for example, cholera toxin or its derivatives); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerr, Yersinia spp, including Y. enterocolitica (for example, Yop protein), Y. pestis, Y. pseudotuberculosis', Campylobacter spp, including C. jejuni (for example, 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, vacuolating 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 (e.g. tetanus toxin and its derivatives), C. botulinum (e.g. botulinum toxin and its derivatives), C. difficile (e.g. clostridial toxins A, B and their derivatives); Bacillus spp, including B. anthracis; Corynebacterium spp, including C. diphteríae (e.g. diphtheria toxin and its derivatives); 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 the causative agent of human granulocytic 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 (e.g. rare outer membrane proteins), T. denticola, T. hyodysenteriae, or antigens or antigen 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 7. vaginalis; Schistosoma spp, including S. mansoni, or antigen and antigen preparations derived from yeasts such as Candida spp, including C. albicans; Cryptococcus spp, including C. neoformans.

Preferred antibacterial vaccines contain antigens derived from Streptococcus spp, including S. pneumoniae (e.g. capsular polysaccharides and their conjugates, PsaA, PspA, streptolysin, choline binding proteins and the 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 vaccines contain antigens derived from Haemophilus spp, including H. influenzae type B (e.g. PRP and its conjugates), non-typeable H. influenzae (e.g. OMP26, high molecular weight adhesins, P5, P6, D protein and D lipoprotein, fimbrin and fimbrin-derived peptides (U.S. Pat. No. 5,843,464) or multiple copy variants or their fusion proteins). Other preferred antibacterial vaccines contain 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)).

Derivatives of the hepatitis B surface antigen are well known in the art and include, among others, the S antigens PreS1 and PreS2 described in European patent applications EP-A-414 374; EP-A-0304 578 and EP 198-474. One of the preferred vaccines of the invention comprises the HIV-1 gp120 antigen, particularly when expressed in CHO cells. The vaccine of the invention also comprises gD2t as described hereinabove.

Vaccine preparations according to the invention containing the claimed adjuvant also contain 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 vaccine against genital warts comprises L1 particles or capsomeres 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).

A 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 antigens in the form of monomers, or L1 and L2 antigens together in a virus-like particle (VLP) or L1 antigen alone in a VLP or in a capsomer. These antigens, 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 vaccines also include early proteins, such as E7, E2 or E5, either alone or in the form of fusion proteins. The most suitable form is VLP containing L1E7 fusion proteins (WO96/11272).

Suitable HPV 16 antigens are the early proteins E6 or E7 fused to a D protein carrier, thus forming D protein-E6 or E7 fusions, or combinations thereof; or combinations of the early proteins E6 or E7 with the L2 antigen (WO96/26277).

• ·

The early proteins E6 or E7 from HPV 16 or 18 are also present alone, preferably in a D protein-E6/E7 fusion. Such vaccines optionally contain 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 preparations according to the invention additionally contain antigens from other HPV strains, preferably from HPV strains 6, 11, 31, 33 or 45.

The vaccines of the invention also contain antigens from malaria parasites. Suitable antigens, for example from Plasmodium falciparum, are RTS,S and TRAP. RTS is a hybrid protein comprising the entire C-terminal portion of the P. falciparum circumsporozoite protein linked by four amino acids from the preS2 portion of the hepatitis B surface (S) antigen to the hepatitis B virus surface antigen. The full structure of this antigen is described in International Patent Application No. PCT/EP92/02591, published as WO 93/10152, which claims priority from UK Patent Application No. 9124390.7. In yeast, RTS is expressed as a lipoprotein particle, and when co-expressed with the HBV S antigen, a mixed particle known as RTS,S is formed. TRAP antigens are described in International Patent Application No. PCT/GB89/00895, published as WO90/01496. The malaria vaccine of the invention comprises an antigenic preparation comprising a combination of RTS,S and TRAP antigens. Other Plasmodium antigens suitable for use in 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 analogues from Plasmodium spp.

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

9 · · 9 9 · · 9 9 9 · · · · · · · contains, for example, tumor rejection antigens from prostate, breast, colorectal, lung, pancreatic, kidney or melanoma cancers. Examples are the antigens MAGE 1, MAGE 3 or other MAGE antigens for melanoma treatment, also PRÁME, 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 carcinoma, bladder carcinoma and bladder sarcoma. Other specific tumor antigens, such as prostate specific antigen (PSA) or Her-2/neu, KSA (GA733), MUC-1, carcinoembryonic antigen (CEA) and others, are used together with the adjuvant of the invention. The invention therefore includes a vaccine comprising the adjuvant of the invention and a tumor rejection antigen.

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

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

4 4 • · 4444 44·· • · 4 4*444 • · · 444444

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

The amount of protein in a vaccine dose is determined as the amount that will induce an immunoprotective response without significant side effects. This amount varies depending on the specific immunogen, its processing method and the form in which it is presented. It is generally expected 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 preparation is determined based on the results of standard studies of the immune responses of the subject. The initial vaccine dose may be followed by one or more booster doses at appropriate intervals.

The adjuvants of the invention are incorporated into vaccine compositions containing antigens from a variety of sources. These antigens include human, bacterial or viral nucleic acids, pathogen-derived antigens or antigen preparations, tumor-derived antigens or antigen preparations, host-derived antigens including GnRH peptides and IgE, recombinantly produced proteins or peptides, and chimeric fusion proteins.

The vaccines of the invention are administered orally. Pharmaceutically acceptable vehicles for these vaccines include, but are not limited to, alkaline buffers, enteric capsules or microgranules. The vaccines of the invention are administered vaginally. Pharmaceutically acceptable vehicles for these vaccines include, but are not limited to, emulsifiers, polymers such as CARBOPOL ^R , and other known stabilizers for vaginal creams and suppositories. The vaccines of the invention are administered rectally.

The vehicle for these vaccine preparations is waxes and polymers used to make rectal suppositories known in the art.

The adjuvants of the invention are used both for prophylaxis and for treatment. The invention relates to a method of treating mammals at risk of or suffering from an infectious disease, a tumor disease, an allergy or an autoimmune disease. The invention includes a vaccine for medical purposes as described herein. A general method for producing a vaccine 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 polyoxyethyl ethers and polyoxyethylene esters of the general formula (I) in the manufacture of an adjuvant comprising a surfactant of the formula (I) and a pharmaceutically acceptable vehicle. The invention relates to the use of polyoxyethylene ethers and polyoxyethylene esters of the general formula (I) in the manufacture of a vaccine preparation comprising a surfactant of the formula (I), a pharmaceutically acceptable vehicle and an antigen. The invention relates to the use of polyoxyethylene ethers and polyoxyethylene esters of the general formula (I) in the manufacture of an adjuvant or vaccine preparation as described above, wherein the adjuvant does not contain cholesterol. The invention relates to the use of polyoxyethylene ethers and polyoxyethylene esters of the general formula (I) in the manufacture of an adjuvant or vaccine preparation as described above, wherein the adjuvant is in the form of a non-vesicular solution or suspension.

Pharmaceutically acceptable vehicles include water, saturated phosphate-buffered saline, and isotonic buffered saline.

• · • · · ·

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

The invention is illustrated by the following examples.

Examples of embodiments 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 immunoplates are coated overnight at 4°C with OspA antigen (1 pg/ml, 50 μΙ per well) or purified goat anti-mouse Ig (Boerhinger, 5 pg/ml, 50 μΙ per well) in PBS, series A. The blank spots on the plate are blocked for 1 hour at 37°C with saturation buffer (PBS containing 1 g/l BSA, 0.1 g/l polyoxyethylene monolaurate sorbitan (TWEEN 20) and normal bovine serum (NBS, 4 g/l). Then a series of two-fold dilutions (in saturation buffer, 50 μΙ per well) of a mixture of isotype IgGs diluted in saturation buffer (50 μΙ per well) are added as a standard curve (mixture of mouse monoclonal antibodies IgG1, IgG2a and IgG2b (Sigma), starting at concentration of 200 ng/ml, row A) and serum samples (starting at a dilution of 1:100, rows B to H) are incubated for 1 hour 30 minutes at 37°C. The plates are then washed three times with washing buffer (PBS, polyoxyethylene monolureate sorbitan (TWEEN 20) at a concentration of 0.1 g/l). Biotinylated goat anti-mouse IgG antibodies (Amersham) diluted 1:5000 in saturation buffer are incubated (50 µL per well) for 1 hour 30 minutes at 37°C. After three washes and subsequent addition of streptavidin-horseradish peroxidase conjugate (Amersham), the plates are washed five times and incubated for 20 minutes at room temperature with development buffer (50 µL per well, OPDA at a concentration of 0.4 mg/ml (Sigma) and hydrogen peroxide at a concentration of 0.03 g/l in 50mM citrate buffer pH 4.5). Development is completed by adding 50 μί 2N sulfuric acid to each well. Optical density is measured at 492 and 630 nm with a Biorad 3550 immunoreader. Antibody titer is calculated by a 4-parameter mathematical method using SoftMaxPro software.

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

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

Maxisorp Nunc immunoplates 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, 0.1 g/l polyoxyethylene sorbitan monolaurate (TWEEN 20). Then, a series of two-fold dilutions (in PBS, TWEEN 20, 100 μί per well) of monoclonal antibodies (mAbs) lgG1 specific for PS14 or PS19 are added as a standard curve (starting at a concentration of PS14 785 ng/ml, PS19 2040 ng/ml, series A) and serum samples (starting at a dilution of 1:20, series B to H) are incubated at 20°C for 30 minutes with constant 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 common polysaccharide (CPS). The plates are then washed three times with washing buffer (PBS, TWEEN 20). Then, goat anti-mouse IgG conjugated with peroxidase (Jackson) diluted 1:5000 in PBS with TWEEN 20 is incubated for 30 minutes with constant shaking. After three washes, the plates are incubated for 15 minutes at room temperature with development 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). Development is completed by adding 1N HCl (50 μΙ to each well). The optical density is determined at 492 and 630 nm with a Biorad 3550 immunoreader. 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 immunoplates are coated overnight at 4°C with OspA antigen (1 pg/ml, 50 μΙ per well) diluted in PBS. The blank spots on the plate are blocked for 1 hour at 37°C with saturation buffer (PBS containing 1 g/l BSA, 0.1 g/l polyoxyethylene monolaurate sorbitan (TWEEN 20). Then a series of two-fold dilutions (in saturation buffer, 50 μΙ per well) of reference serum are added as a standard curve (serum has a mean titer of 60,000 ELISA units/ml, starting at 12 EU/ml, row A) and serum samples (starting at 1:100 dilution, rows B to H) are incubated for 1 hour 30 minutes at 37°C. The plates are then washed three times with washing buffer (PBS, polyoxyethylene monolaurate 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 and subsequent addition of streptavidin-horseradish peroxidase conjugate (Amersham), the plates are washed five times and incubated for 20 minutes at room temperature with development buffer (50 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). Development is terminated by the addition of 50 μΙ 2N

- 21 - · · · ·

4 4 4 « 4444444 sulfuric acid into each well. The Biorad 3550 immunoreader reads at 492 and

Optical density is measured at 630 nm. Antibody titer is calculated by a 4-parameter mathematical method using SoftMaxPro software.

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

Measurement of OspA-specific monkey nasal IgA antibodies - ELISA

Maxisorp Nunc immunoplates are coated overnight at 4°C with OspA antigen (1 pg/ml, 50 μΙ per well) diluted in PBS (rows B to H). The blank spots on the plate are blocked for 1 hour at 37°C with saturation buffer (PBS containing 1 g/l BSA, 0.1 g/l polyoxyethylene monolaurate sorbitan (TWEEN 20) and 4 g/l normal bovine serum (NBS). Then a two-fold dilution series (in saturation buffer, 50 μΙ per well) of the reference secretion is added as a standard curve (the secretion has a mean titer of 3000 ELISA units/ml, starting at 30 EU/ml, row A) and nasal swabs (starting at 1:5 dilution, rows B to H) are incubated for 2 hours at 22°C. The plates are then washed three times with washing buffer (PBS, polyoxyethylene monolaurate sorbitan (TWEEN 20) at a concentration of 0.1 g/l). Biotinylated goat anti-human IgA antibodies (ICN, at a concentration of 0.2 μΙ/ml) in saturation buffer are incubated (50 μΙ per well) for 1 hour 30 minutes at 37°C. After three washes and subsequent addition of streptavidin-horseradish peroxidase conjugate (Amersham), the plates are washed five times and incubated for 10 minutes at room temperature with development buffer (50 μΙ per well, TMB, Biorad). Development is completed by adding 50 μΙ of 0.4N sulfuric acid to each well. Optical density is measured at 450 and 630 nm with a Biorad 3550 immunoreader. The antibody titer is calculated by a 4-parameter mathematical method using

9 · 9 · 1

- ZZ ” · · · · •··· · ·«*···· of SoftMaxPro software. Samples are considered positive if the IgA titer exceeds the end of the test (0.3 EU/ml).

Measurement of serum LA2-like antibody titers to lipo-OspA antigen - inhibition assay

The titers of antibodies in vaccine preparations are studied with respect to their LA2-like specificity. LA2 is a murine monoclonal antibody that recognizes the conformational epitope of OspA on the surface of the bacterium and is capable of killing B. burgdorferi in vitro and protecting mice when challenged with laboratory-grown spirochetes (Schaible UE et al., 1990, Proc Nati Acad Sci USA, 87, 3768-3772). In addition, the LA-2 monoclonal antibody correlates with bactericidal antibodies. In studies with human sera, LA-2 titers correlate with total anti-OspA IgG antibody titers (measured by ELISA).

Maxisorp Nunc immunoplates are coated overnight at 4°C with lipo OspA antigen (0.5 pg/ml, 50 μΙ per well) diluted in PBS. The blanks are blocked for 1 hour at 37°C with saturation buffer (PBS, 1 g/l BSA, 0.1 g/l TWEEN 20, 4 g/l NBS). A two-fold dilution series of monoclonal antibody (mAb) LA2 (starting at 4 μΙ/ml) is diluted in saturation buffer (50 μΙ per well) to generate a standard curve. Serum sample dilutions from the vaccine preparations (starting at 1:10 dilution) are also added and the plates are incubated for 2 hours at 37°C. After incubation, the plates are washed three times with washing buffer (PBS, TWEEN 20 at a concentration of 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 development buffer (50 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). The reaction and staining are terminated by adding 2N sulfuric acid. The optical absorbance is measured at 492 and 630 nm with a Biorad 3550 immunoreader.

- 23 • · · 4· · 4

494 4 9 94 4 • 9 4 4 4 4 • 4 4 4 4 4 4

4 9 9 4 4

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

- one-week-old female Balb/c mice (groups of 8 animals) are immunized by intramuscular injection of 1 pg of lipo-OspA antigens in 50 pg of alum. After three months, mice under general anesthesia receive an intranasal booster dose of - 10 μl of a solution (5 μl into one nostril with a dropper pipette) containing A: 5 pg of lipo-OspA antigens; B: 5 pg of lipo-OspA antigens in tween 20 at a concentration of 36 g/l, Imwitor 742 at a concentration of 10 g/l; C: 5 pg of lipo-OspA antigens in tween 20 at a concentration of 36 g/l; D: 5 pg of lipo-OspA antigens in polyoxyethylene-9-lauryl etherD at a concentration of 18 g/l.

days after the administration of the booster dose, IgG antibodies against lipo-OspA antigens and LA2 antibodies against OspA antigens are 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 the titer of IgG antibodies specific for the lipo-OspA antigen. However, this increase is only slight in the presence of tween 20 and Imwitor 742 or tween 20 alone. However, the increase in the titer of antibodies is very significant in 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 the amtigen OspA ·· ···♦

- 24 Groups of mice are primed as described in Example 2 with 5 pg 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: dodecylmaltoside at a concentration of 1 g/l; E: tween 20 at a concentration of 36 g/l or F: polyoxyethylene-9-lauryl ether at a concentration of 18 g/l. Since the test with groups A and B is carried out 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 booster dose. 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 enhances 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

A dose range test was performed to determine the concentration of polyoxyethylene-9-lauryl ether required to achieve the adjuvant effect observed in the previous examples. Since other polyoxyethylene ethers also have adjuvant effects, the test was also performed with polyoxyethylene-23-lauryl ether. Mice primed as in Example 1 were intranasally administered a 10 μΙ booster dose containing 5 μg of 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 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 booster dose, sera are analyzed in the same manner as in Example 2.

- 25 *·*· 9 99 9 9 99 ·· · ♦ · 9 9 9

9 9 9 9 9 9 • 9 9 9 9 9 9 9 9 9 • 9 · · · 9 · • *··*··· 99 99

Figures 5 and 6 below show that even a low concentration of polyoxyethylene-9-lauryl ether, such as 1 g/l, causes a very significant enhancement of the immune response. Similarly, polyoxyethylene-23-lauryl ether significantly enhances the immune response to an intranasally administered booster dose.

Example 5

Combined vaccine - intranasal booster dose

This test tests the ability of polyoxyethylene ethers to enhance the systemic immune response after their intranasal administration. Female mice of the balb/c strain are primarily immunized by intramuscular injection of the commercial DTPa vaccine (diphtheria, tetanus, acellular pertussis vaccine: INFARIX™ Smith Kline Beecham, Belgium). The mice are immunized with one or two 50 μΙ intramuscular injections, which correspond to 20% of the human dose. After three months, the mice receive an intranasal booster dose (as in Example 2) with tetanus toxoid (TT: 5 pg) or fibrous hemagglutinin (FHA: 5dg) in A: PBS; B: polyoxyethylene-9-lauryl ether at a concentration of 1 g/l; or C: only intramuscular injection of DTPa vaccine (twice 50 μΙ). After 14 days, TT and FHA specific IgG are determined in the serum. Antibody titers are shown in Figures 7 and 8.

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

Example 6 • ·

9 9 • · ·

9 9 • 9 9

- 26 Intranasal stimulation in monkeys AGMs

Many adjuvants are effective in small rodents but have no effect in larger animals. This example demonstrates the ability of polyoxyethylene ethers to potentiate the immune response after intranasal administration in larger animals. Green monkeys (AGM) are divided into groups of four and primed with an intramuscular injection of 10 pg of lipo-OspA antigen in 500 pg of alum. Ten months later, the animals are given a booster dose of 200 μΙ (100 μΙ in one nostril) intranasally, under general anesthesia, using a two-dose spray device from Pfeiffer GmBH, Germany, containing 60 pg of lipo-OspA antigen in A: PBS; or B: polyoxyethylene-9-lauryl ether at a concentration of 1 g/l. After 14 days, serum titers of anti-OspA and LA2 immunoglobulins are measured. Geometric mean titers for each group are shown in Figures 9 and 10. Anti-OspA immunoglobulin titers are determined for Group C, consisting of 10 monkeys, which received both a primary and a booster dose of lipo-OspA antigen on alum by intramuscular injection (geometric mean titers are shown for LA2 titers only, see Figure 10).

The lipo-OspA antigen is able to enhance the immune response when administered intranasally to monkeys, but this enhancement is significantly greater when administered simultaneously with polyoxyethylene-9-lauryl ether at a concentration of 1 g/l. Antibody titers detected after an intranasal booster dose when administered simultaneously with polyoxyethylene-9-lauryl ether are again higher than titers detected after intramuscular injection (group C).

Example 7

Primary and booster intranasal immunization of AGMs ·· 444« • ·· ·· ·· • · · · · · · >

4 9 4 4 4 «9 4 4 4 4

4 4 4 4 4

4444444 44 44

In the previous examples, it has been demonstrated that polyoxyethylene ethers help to enhance the systemic immune response when administered intranasally as a booster. In this example, the possibility of administering both the primary and booster immunizations to animals intranasally to induce a sufficient systemic immune response is verified. As part of the investigation of the possibility of administering this adjuvant to larger animals, the test is performed on albino marmosets (AGMs).

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

Example 8

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

This model is used to verify the adjuvant effect of polyoxyethylene-9 lauryl ether (POE-9LE) alone and in combination with other immunostimulants in primary and booster immunization of primates. Serum and nasal immunoglobulins are determined. The immunostimulant used here is CpG 1001 described in Example 9.

- 28 • 9 9 9 9 9 • · 9 9 9 9 • 9 9 9 9

9 9 9 9 9

9 9 *9

999* 99 99

Methodology

Green monkeys are given a primary and booster dose intranasally on day 0 (pl) and day 14 (pil). The vaccine is administered using a two-dose spray device from Pfeiffer (100 μΙ into each nostril, under general anesthesia). The test products are:

group antigen aid n= submission 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 the injection, serum samples are collected and lipo-OspA antibody titers are determined. Antigen-specific nasal IgA is measured by a highly sensitive ELISA on nasal swabs collected at the same time. Animals are considered positive if their IgA titers reach a defined level that is significantly higher than the baseline level.

Results ·· ·*·»

Serum OspA specific immunoglobulin

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

Nasal OspA-specific IgA

Vaccines containing the lipo-OspA antigen alone or in combination with CpG do not induce any increase in captureable IgA antibodies in the nose (Figure 13 summarizes all antibody responses in the nose). When the combination of lipo-OspA antigens with polyoxyethylene lauryl ether at a concentration of 0.25 g/l is administered, only 25% of the animals are positive for “nasal IgA” (in contrast to 50% of the animals positive when POE-9 LE is simultaneously administered at a concentration of 0.5 g/l). When the combination of lipo-OspA and POE-9 LE antigens at a concentration of 0.25 g/l is supplemented with CpG, then 100% of the animals develop an IgA antibody response. This shows that 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 φφφφ φφφ φφφφ φφφφ φφφφ φ φ φφφφ φ φ φφφφφφ φ φφφφφ

ΦΦΦΦΦΦ· ·· ··

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

This example demonstrates the effect of additional immunostimulatory agents added to a polyoxyethylene ether (POE-9 LE) adjuvant in mice. 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 elicited by conventional intramuscular booster vaccination. These preparations are further compared with a known intranasal adjuvant, E. coli thermolabile enterotoxin (mLT).

The sequences used in this assay are CpG 1001 (TCC ATG AGC TTC CTG ACG TT) and CpG 1002 (TCT CCC AGC GTG CGC CAT) and a negative control sequence of non-immunostimulatory CpG 1005 (TCC ATG AGC TTC CTG AGC TT).

Methodology

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

·· ···· ·« «· ·· * · < *· · · 9 9 9 9 • 9 β ···«· * » · * ···»«· • · · · · · 9 9

9999 9 999 9999 99 99

Group antigen aid Submitted 1 lipoPospa (5pg) AIOH ₃ (50pig) i.m. 2 lipoPospa (5pg) CpG 1005(20pg), POE -9 LE(1g/l) i.n. 3 lipoOspa (δμρ) CpG 1002(2Όμα), POE - 9 LE (1g/I) i.n. 4 lipoPospa (5pg) CpG 1001(20μρ), POE - 9 LE (1 g/l) i.n. 5 lipoPospa ^g) CpG 1005^ig) i.n. 6 lipoOspa (5μ$) CpG 1002^ig) i.n. 7 íipoOspa (5μ^) CpG 1001^g) i.n. 8 lipoPospa (5pg) POE-9 LE(1g/l) i.n. 9 lipoPospa ^g) mL (5^mg) i.n. 10 lipoPospa ^g) None i.n. 11 not immunized

Blood samples are taken on the day of the booster dose and 14 days later (pill). In individual sera, titers of OspA-specific IgG antibodies and LA2 antibodies are determined by ELISA.

Results

Figures 14 (OspA-specific serum IgG determined by antigen-specific ELISA) and 15 (LAB2 bactericidal titers in serum) show that administration of CpG alone does not improve the serum antibody response to the OspA antigen. The vaccine containing OspA with

- 32 ·· ··*· · ·« «· ·· · · · · · · *·· · ····· • · « · ······ • · ·· ····« ·»·· · «*· «··· ·Φ ·· polyoxyethylene lauryl ether increases the resulting IgG and LA2 titers. The best responses are achieved when the lipo-OspA antigen is administered simultaneously with both polyoxyethylene lauryl ether and CpG.

Example 10

Dose study

Example 4 shows that even low concentrations of polyoxyethylene-9-lauryl ether, such as 1 g/l, significantly increase the immune response. In this example, a dose range test with lower concentrations of adjuvant is demonstrated to determine the concentration of polyoxyethylene-9-lauryl ether required to achieve efficacy as an adjuvant in nasal vaccine formulations.

Balb/c mice are primed as described in Example 2. A 10 μΙ booster dose is administered intranasally to the animals. The dose contains 5 pg of 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 the booster dose is administered intramuscularly by injection E; 1 μg of lipo-OspA antigen adsorbed on 50 pg of alum. 14 days after the booster dose, antibody titers are determined in the sera as described in Example

1.

Results

It is clear from Figures 16 and 17 that polyoxyethylene-9-lauryl ether at a concentration of 0.25 g/l significantly increases the immune response. This adjuvant achieves similar antibody response at low concentrations • · · · « · • ·

response, as achieved with parenteral administration of the vaccine.

Example 11

Vaccination of monkeys against influenza

In Example 11, polyoxyethylene-9-lauryl ether was shown to enhance the immunogenicity of influenza virus in mice. This example examines whether this surfactant has a similar adjuvant effect in larger species. Green monkeys (AGMs, 2 animals per group, blood sampling from 2 animals per day) are immunized with a primary and booster dose administered intranasally (as in Example 6) of 50 pg equivalent of HA 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 the booster dose, 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

The previous examples demonstrate the ability of polyoxyethylene-9 lauryl ether to enhance the immune response to a protein antigen. This example tests the ability of this adjuvant to enhance the immune response to an intranasally administered booster dose of a polysaccharide antigen following a previous parenteral primary immunization in mice. Mice are primed with a single subcutaneous 100 μΙ injection containing polysaccharides PS14 and PS19 (1 pg each) from • · · · · ·

Streptococcus pneumoniae conjugated with the D protein carrier. After two months, mice under general anesthesia receive an intranasal booster dose of 40 μΙ of a solution (at time 0 10 μΙ into each nostril, 30 minutes later again 10 μΙ into each nostril, administered dropwise with a pipette) containing 1 pg of PS14 conjugate and 1 Dg of 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 booster dose, serum IgG antibodies specific for PS14 and PS19 are determined.

Results

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

Example 14

Polyoxyethylene-8-stearyl ether

This example shows that other polyoxyethylene ethers, such as polyoxyethylene-8-stearyl ether, also have an adjuvant effect on the immune response.

Balb/c mice, primed as in Example 2, are given a 10 μΙ booster dose containing 5 pg 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 pg of lipo-OspA antigen adsorbed on 50 pg of alum. Sera are analyzed 14 days after the booster dose 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 in the same way as after parenteral administration of the vaccine.

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.