MXPA02003067A - Use of combination of polyoxyethylene sorbitan ester and octoxynol as adjuvant and its use in vaccines. - Google Patents

Abstract

The invention relates to a novel adjuvant system comprising a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol and vaccines comprising the adjuvant system together with an antigen. Further provided are methods of manufacturing the adjuvants and vaccines and the use of the adjuvants and vaccines in the prophylaxis or therapy of disease.

Description

USE OF COMBINATION OF POLYOXYETHYL ESTER SORBITAN AND OCTOXINOL AS AN ADJUVANT AND ITS USE IN VACCINES Field of the Invention The present invention relates to a novel adjuvant system comprising a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol. The present invention provides novel adjuvants, vaccines containing them and methods for their manufacture and for their formulation in vaccines. The use of the adjuvants or vaccines of the present invention in the prophylaxis or therapy of a disease is also provided. The adjuvants are particularly useful as mucosal adjuvants, although they are also effective systemically. The adjuvants are especially useful in the context of influenza vaccines. In addition to eliminating the requirement for painful injections and the associated negative effect with respect to compliance by the patient due to "fear of the needle", mucosal vaccination is attractive since it has been shown in animals that the mucosal administration of antigens has a greater efficacy of inducing protective responses on mucous surfaces, which are the entry route for many pathogens. Furthermore, it has been suggested that mucosal vaccination, such as intranasal vaccination, 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). In addition to much research in the field, safe and effective mucosal adjuvants that are suitable for use in humans, remain to be identified. The present invention provides a solution to this problem. 5 The medical uses of certain nonionic surfactants have been described. For example, intranasal administration of polyoxyethylene ethers and esters to increase insulin uptake in the nasal cavity has been described (Hirai et al., 1981, International Journal of Pharmaceuticals, 9, 165-172; Hirai et al., 1981, Journal International of Pharmaceuticals, 9, 173-184). Other nonionic surfactants have been used in vaccine formulations. For example, vaccine preparations comprising a mixture with additions of either polyoxyethylene castor oil or caprylic / capric acid glycerides, with sorbitan monoesters of Polyoxyethylene, and an antigen, have the ability to induce systemic immune responses after topical administration to a mucosal membrane (WO 94/17827). This patent application describes the combination of the nonionic surfactant TWEEN20 ™ (monoester sorbitan polyoxyethylene) and lmwitor742 ™ (glycerides of caprylic / capric acid), or a combination of TWEEN20 ™ and polyoxyethylene castor oil, have the ability to increase the systemic immune response after intranasal immunization. They have also been described in the literature (Gizurarson and associates, 1996, Vaccine Research, 5, 69-75, Aggerbeck and associates, 1997, Vaccine, 15, 307-316, Tebbey and associates, Viral Immunology, 1999; 12 (1): 41-5), details of the effect of this ^^ M ^ &j £ & i "," formulation in the increase of the immune response towards antigens administered in intranasal form. In the examples shown in WO94 / 1782, (in particular from Example 4) the concentration of TWEEN 20 ™ that is required to increase the immune response is very high (36%), while 28% even in the presence of a glyceride of caprylic-capric acid does not increase the emergence of the immune response. The nonionic surfactants have also been formulated in such a manner that they form vesicles of nonionic surfactants (commonly known as NISV, US Patent No. 5, 679, 355). Such formulations of nonionic surfactants, often in the presence of cholesterol, form bilayer vesicles of lipids that entrap the antigen within the internal aqueous phase or within the bilayer itself. Documents WO 96/36352 and US 5, 653, 987), describes a liquid pharmaceutical agent comprising at least two absorption and water enhancers, mainly for oral administration of insulin wherein the amount of each agent that increases absorption is present in a concentration of from 1 to 10. % w / w of the total formulation. Surfactants are commonly used in the formulation of oil emulsion adjuvants for systemic administration, and function to stabilize oil droplets. For example, polyoxyethylene sorbitan esters (TWEEN ™) and sorbitan fatty acid esters (SPAN ™) are used to stabilize the oil in water emulsions (EP 0399843 B, WO 95/17210).

In the past, influenza virus vaccines have been prepared using Triton X-100 or a mixture of TWEEN and ether with the fragmented influenza virus. A clinical comparison of the systemic immunogenicity of the two divisions shows that they are comparable (Gross et al. 1981, J. Clin Microbiol 14,534-8). Other surfactants have been investigated for their effect on the immunogenicity of the resulting fragmented vaccine. In a comparative study of parenteral administration, Mukhlis and associates (1984 Vaccine 2, 199-203) showed that the whole virus was more immunogenic than the virus interrupted by detergent, although such difference between the different detergents Triton X-100 and the bromine of Cetyl trimethylammonium (C ) produced immunogenic cleavages marginally superior to those of the detergent empyeon. The present application surprisingly discovered that polyoxyethylene sorbitan esters, in combination with an octoxynol, act together as a potent adjuvant for vaccines. Conveniently, such compositions can be administered systemically, although they are also potent in the induction of systemic immune responses when the vaccine compositions are administered to the mucosa. The immune responses induced by the mucosal administration of vaccines of the present invention can be at least as high as those observed after a systemic injection of a conventional vaccine.

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The present invention provides safe and potent adjuvants f which are easy to manufacture, and which can be administered either through mucosal or systemic routes. In a first aspect, the present invention provides an adjuvant comprising a polyoxyethylene sorbitan ester and an octoxynol. In another aspect, the present invention provides a vaccine comprising an adjuvant according to the same, together with an antigen. Particularly preferred is a vaccine composition comprising an adjuvant according to the present invention with the virus antigen for administration to a mucosal surface, in particular to the nasal mucosa. However, there are alternative routes of administration and other possible antigens to be used in a vaccine according to the present invention, which will be described later. The octoxinols and sorbitan esters of polyoxyethylene are described in the publication "Surfactant systems" editors: Adwon and Florence (1983, Chapman and Hall). The octoxynol series, which includes t-20 octylphenoxypolyethoxyethanol (TRITON X-100 ™) is also described in the Merck Index Entry 6858 (page 1162, 12th Edition, Merck and Co., Inc. Whitehouse Station, NJ, USA; 0911910-12-3). Polyoxyethylene sorbitan esters that include polyoxyethylene sorbitan monooleate (TWEEN 80 ™) are described in the Index Entry Merck 7742 (page, 1308, 12th edition, Merck and Co., Inc, i ^ a i á J - *. * »s. . ~ A. ^ - Whitehouse Station, N.J..EUA; ISBN 0911910-12-3.). Both can be manufactured using the methods described herein, or purchased from commercial sources, such as Sigma Inc. Preferred octoxins to be used in the adjuvants according to the present invention include other non-ionic surfactants of the Triton series, such as Triton X -45, Triton X-102, Triton X-114, Triton X-165, Triton X-205, Triton X-305, Triton N-57, Triton N-101 and Triton N-128, although t-octylphenoxypolyethoxyethanol is particularly preferred. (Triton X-100). The adjuvants of the present invention comprise a sorbitan ester of polyoxyethylene and an octoxynol. Preferably, octoxynol is t-octylphenoxypolyethoxyethanol (Triton X-100 ™). Preferably, the polyoxyethylene sorbitan ester is polyoxyethylene sorbitan monooleate (TWEEN 80 ™). The adjuvant according to the present invention may conveniently further comprise a bile salt or a cholic acid derivative. Thus, the adjuvant may comprise a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80), an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) and a bile salt or cholic acid derivative, such as sodium deoxycholate or taurodeoxycholate In a preferred embodiment, the present invention provides an adjuvant formulation comprising polyoxyethylene sorbitan monooleate (Tween 80), t-octylphenoxypolyethoxyethanol (Triton X-100) and sodium deoxycholate.

Preferably, the total concentration of nonionic surfactants Mr present in the adjuvant formulation is less than 40%, more preferably up to about 20%. A preferred range is between about 0.001% to 20%, more preferably from 0.01% to 10% and most preferably up to about 2% (p / v). The individual nonionic surfactants have the preferred concentrations in the composition of the final vaccine indicated below: octyl or nonylphenoxy polyethoxy ethanols such as Triton X-100 or other detergents of the Triton series: from 0.001% to 20%, preferably from 0.001% to 10%, more preferably from 0. 001% up to 1% and most preferably from 0.005% up to 0.1% (p / v); polyoxyethylene sorbitan esters such as Tween 80: from 0. 01 to 1%, most preferably approximately 0.0% (w / v). The particularly preferred ranges for the concentrations of the nonionic surfactants are: Tween 80 ™: from 0.01 to 1%, more preferably approximately 0.1% (w / v); Triton X-100 ™: from 0.001 to 0.1, more preferably 0.005 up to 0.02% (p / v). One aspect of the present invention is a vaccine formulation comprising a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol, wherein the antigen present in the vaccine is not trapped within a vesicle of surfactant not ionic. - ¿ÉÉf ^ £ &i * & * fc, .j iíi rt .1 Mam nifjfjt? Influenza virus antigens for use in the vaccine according to the present invention, can have any form of influenza antigens suitable for raising an immune response, including complete viruses, live or inactivated, fragmented viruses or subunit antigens prepared from complete viruses or through recombining media. The influenza virus for the production of the antigen, can be grown in embryonated eggs in a conventional process, or the virus can be grown in tissue culture. Suitable cell substrates for influenza tissue culture include, for example, dog kidney cells such as MDCK cells, cells of a MDCK clone, or MDCK-like cells, monkey kidney cells such as AGMK cells including cells. Vero, and any other types of cells suitable for the production of the influenza virus for the purposes of vaccination. Suitable cell substrates also include human cells, for example MRC-5 cells. Suitable cell substrates are not limited to cell lines, for example primary cells such as chicken embryo fibroblasts are also included. An antigen preparation of the influenza virus comprising the divided virus, which has undergone a series of purification steps, is preferred. Therefore, the preparation of antigens can be produced through a number of different commercially applicable processes, for example the fragmented flow process described in patent No. DD 300 833 and DD 211 444, incorporated herein by reference. t.i i. * .t á .: The fragmented influenza vaccine available on the market includes Fluarix ™ which is sold by SmithKine Beecham. Therefore, a preferred vaccine formulation according to the present invention comprises eggs or tissue culture derived from the influenza antigen, preferably the fragmented influenza antigen, together with a polyoxyethylene sorbitan ester and an octoxynol, further comprising Optional a bile salt or derivative of cholic acid. More preferably, the formulation comprises the fragmented influenza virus antigen, polyoxyethylene sorbitan monooleate, (Tween 80), t-octylphenoxypolyethoxyethanol (Triton X-100) and sodium deoxycholate. The influenza vaccine according to the present invention is preferably a multivalent influenza vaccine comprising two or more strains of influenza. More preferably it is a trivalent vaccine comprising three strains. Conventional influenza vaccines include three strains of influenza, two strains A and one strain B. However, monovalent vaccines that may be useful for example in a pandemic situation, are not excluded from the present invention. A monovalent pandemic flu vaccine will likely contain the influenza antigen from a single strain A. The vaccine preparations of the present invention can be used to protect or treat a mammal susceptible to, or suffering from, a disease by administering the vaccine. vaccine through a mucosal route, such as the oral / vocal / intestinal / vaginal / rectal or nasal route. Such jjja é? .. i? i i administration can take the form of drop, spray or dry powder. Formulations of nebulized or aerosolized vaccines also form part of the present invention. Enteric formulations such as gastro-resistant capsules and granules for oral administration, suppositories for rectal or vaginal administration are also part of the present invention. The present invention can also be used to increase the immunogenicity of antigens applied to the skin (dermal or transcutaneous administration). In addition, the adjuvants of the present invention can be administered parenterally, by example intramuscular or subcutaneous administration. When used for intranasal vaccination, the vaccines of the present invention have a preferably hemolytic nature. Depending on the route of administration, a variety of administration devices can be used. For example, for administration Intranasal, a spray apparatus such as that available commercially from Acuspray ™ (Becton Dickinson) can be used. The preferred spray devices for intranasal use are devices for which the performance of the device does not depend on the pressure applied by the user. These devices are known as pressure threshold devices. The liquid is released from the nozzle only when a threshold pressure is achieved. These devices make it easier to achieve a spray with a regular drop size. Pressure threshold apparatuses suitable for use with the present invention are known in the art and are described for example in the document WO 91/13281 and EP 311 863 B. Such apparatuses are commercially available from Pfeiffer GmbH. Preferred intranasal devices produce drops (measured using water as a liquid) within the range of 1 to 200μm, preferably 10 to 120μm. Below 10μm there is a risk of inhalation, therefore it is desirable to have drops of no more than 5% below 10μm. Drops above 120μm can not be spread as well as smaller drops, so it is desirable to have drops no greater than about 5% above 120μm. The administration of bidosis is a further preferred feature of an intranasal delivery apparatus for use with vaccines according to the present invention. The bidosis devices contain two sub-doses of a single dose of vaccine, one sub-dose for administration to each nostril. Generally, the two sub-doses are in a single chamber and the construction of the apparatus allows the efficient administration of a single sub-dose at the same time. The present invention provides in a further aspect, a kit comprising an intranasal delivery apparatus as described in the present invention, which contains a vaccine formulation according to the same. In a preferred embodiment of this aspect of the present invention, the intranasal delivery apparatus is filled with an influenza vaccine. For certain vaccine formulations, other vaccine components may be included in the formulation. As such, the adjuvant formulations of the present invention may also comprise a bile acid or cholic acid derivative. Preferably, the cholic acid derivative is a salt thereof, and more preferably a sodium salt thereof. Examples of bile acids and derivatives thereof include cholic acid, deoxycholic acid, taurodeoxycholate, cholic kenodeoxi acid, ursodeoxycholic acid, lithocholic acid, iodeoxycholic acid, and derivatives similar to amidopropyl-2-hydroxy-1-propanesulfonic derivatives, glyco, tauro, amidopropyl-1-propanesulfonic acid of the aforementioned bile acids, or deoxycolamide N, N-bis (3DGUuconoamidopropyl). A particular preferred example is sodium deoxycholate (NaDOC) which may be present in the final vaccine dose. Preferably, the adjuvant formulation of the present invention is convenient when it is in the form of an aqueous solution or a suspension of non-vesicular forms. Such formulations are easy to manufacture reproducibly, and also easy to sterilize (terminal filtration through a pore membrane of 450 or 220 nm) and are also easy to administer to the nasal mucosa in the form of a spray without degradation of the physical structure of the adjuvant complex. In a further aspect of the present invention, there is provided a method for preparing a vaccine, wherein the method comprises admixing an adjuvant according to the present invention with an antigen. In a still further aspect, there is provided a method for inducing or enhancing an immune response in a subject, which comprises mixing 1 * 4 »* Fc. with additions the antigen and the adjuvant of the present invention, and administering the mixture to the subject. Preferably, the route of administration to the subject is through a mucosal surface and more preferably through the nasal mucosa. When the vaccine is administered through the nasal mucosa, the vaccine is preferably administered in the form of a spray. In a preferred method for inducing or increasing an immune response, a systemic response is induced by nasal administration of the vaccine. Therefore, a mucosal vaccine according to the present invention preferably has the ability to induce a systemic immune response when administered through a mucosal route. The present invention further provides the use of a sorbitan ester of polyethylene and an octoxynol in the manufacture of an adjuvant formulation, in particular an adjuvant formulation for application to the mucosa of a patient. The present invention also relates to the use of a polyoxyethylene sorbitan ester, an octoxynol and an antigen, in the manufacture of a vaccine formulation, especially a vaccine formulation for application to the mucosa. Preferably, the antigen is the influenza virus antigen. Adjuvants and vaccines for administration to the nasal mucosa are particularly preferred. Preferably, administration of a vaccine according to the present invention comprises administration of a starting or boosting dose of the vaccine, such as a starting or boosting dose of the influenza vaccine comprising a preparation of the antigen of the vaccine. influenza. It is considered that the compositions of the present invention will be used to formulate vaccines containing antigens derived from a wide variety of sources. For example, the antigens may include antigenic or antigen preparations derived from human, bacteria, viral nucleic acid or pathogens, antigen derived from tumor or antigenic preparations, antigens derived from host, including GnRH and IgE peptides, proteins or peptides produced in recombinant form and chimeric fusion proteins. Preferably, the formulations of the present invention contain an antigen or antigenic composition having the ability to obtain an immune response against a human pathogen, wherein the antigen or antigenic composition is derived from HIV-1, (such as tat, nef, gp120 or gp160), human herpes viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from VSH1 or VSH2, cytomegalovirus (human) such as gB or derivatives thereof, Rotavirus (including viruses) live attenuated), Epstein Barr virus (such as gp350 or derivatives thereof), Varicella Zoster Virus (such as gpl, II and IE63), or a hepatitis virus such as hepatitis B virus (e.g. Hepatitis or a derivative thereof), hepatitis A virus, hepatitis C virus and hepatitis E virus, or other viral pathogens, such as paramyxoviruses: respiratory syncytial virus (such as proteins F and G or der ivados of them), parainfluenza virus, measles virus, mumps virus, human papillomavirus (for example VPH6, 11, I 16, 18, ..), flaviviruses (for example, Yellow Fever Virus, Dengue, Encephalitis virus by tick birth, Virus of Japanese Encephalitis) or Influenza virus (all live or inactivated virus, fragmented influenza virus grown in egg cells or MCDK cells, or Vero cells or all flu virosomes (as described by R. Glick, Vacuna 1992, 10, 915 -920) or purified or recombinant proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof) or derivatives of bacterial pathogens such as Neisseria spp, including N. gonorrhea, including N. meningitidis, (for example, capsular polysaccharides and conjugates thereof, transferrin binding proteins, lactoferin binding proteins, PilC, adhesins); S. pyogenes (for example M proteins or fragments thereof, protease C5A, lipoteichoic acids), S. agalactiae, S. mutans; H. ducreyi; Moraxella spp, Including M catarrhalis, also known as Branhamella catarrhalis (for example adhesins and invasins of high and low molecular weight); Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin or derivatives thereof, filamentous haemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium spp; including M. tuberculosis (eg ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli (for example colonization factors, heat-labile toxin derivatives thereof, heat-stable toxin or derivatives of the - JFI? AÁ itself), enterohemorrhagic E. coli, enteropathogenic E. coli (for example toxin similar to shiga toxin or derivatives thereof); Vibrio spp, including V. cholera (for example cholera toxin and derivatives thereof); Shigella spp, Including S. sonnei, S. dysenteriae, S. flexnerii; Yersina spp, including Y. Enterocolitica (for example a Yop protein), Y. pestis, Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (for example toxins, adhesins and invasins) and C. coli; Salmonella spp, including S. tiphi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp, including L. monocytogenes; Helicobacter spp, including H. pylori (e.g. urease, catalase, vacuolation toxin); Pseudomonas spp, including P. aeruginosa; Staphilococcus spp, including S. aureus, S. epidermidis; Enterococcus spp, including E. faecalis, E. faecium; Clostridium spp, including C. tetani (for example tetanus toxin and derivatives thereof), C. botulinum (for example botulinum toxin and derivatives thereof), C. difficile (for example clostridium toxin A or B and derivatives thereof); Bacillus spp, including B. anthracis (for example botulinum toxin and derivatives thereof); Corynebacterium spp, including C. diphtheriae (for example diphtheria toxin and derivatives thereof); Borrelia spp, including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB), B Anderson (for example OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia spp, including E. eand the agent of Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp, including C. trachomatis (for example MOMP, eparin binding proteins) C. pneumoniae (by IM.Í? -. A -iíl í-? you i. -t example MOMP, eparin binding proteins) C. psittaci; Leptospira spp, including T. interrogans; Treponema spp, including T. pallidum (for example, rare outer membrane proteins), T. denticola, T. hyodysenteriae; or parasite derivatives such as Plasmodium spp, including P. falciparum; Toxoplasma spp, including T. gondii (for example SAG2, SAG3, Tg34); Entamoeba spp, including histolytica; Babesia spp, including B. microti; Trypanosoma spp, including 7. Cruzi; Giardia spp, including G. lamblia; Leshmania spp, including L. major; Pneumocystis spp, including P. carinii; Trichomonas spp, including T. vaginalis; Schisostoma spp, including S. mansoni, or yeast derivatives such as Candida spp, including C. albicans; Cryptococcus spp, including C. neoformans. Preferred bacterial vaccines comprise antigens derived from Streptococcus spp, including S. pneumoniae (eg capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline binding proteins) and protein antigen Pneumolicin (Acta Biophysical Biochemistry , 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25, 337-342), and mutant toxified derivatives thereof (WO 90/06951, WO 99/03884). Other preferred bacterial vaccines comprise antigens derived from Haemophilus spp, including H. influenzae type B (for example PRP and conjugates thereof), non-typeable H. influenzae, for example OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrine and peptides derived from fimbrin (US Patent 5, 843, 464) or multiple copy variants or fusion proteins thereof. Other preferred bacterial vaccines comprise antigens derived from Morexella Catarrhalis (including outer membrane vesicles thereof, and OMP106 (WO 97/41731)) and from Neisseria mengitidis B (including outer membrane vesicles thereof, (WO 96/29412) The Hepatitis B Surface antigen derivatives are well known in the art and include, inter alia, those PreS1, PreS2 S antigens established and described in European Patent Applications No. EP-A-414 374; EP-A-0304 578, and EP 198-474. In a preferred aspect the vaccine formulation of the present invention comprises the HIV-1 antigen, gp120, especially when expressed in CHO cells. In a further embodiment, the vaccine formulation of the present invention comprises gD2t, as defined above. In a preferred embodiment of the present invention, vaccines containing the claimed adjuvant comprise antigen derived from Human Papilloma Virus (HPV) considered to be responsible for genital warts, (HPV 6 or HPV 11 and others), and HPV viruses responsible for cervical cancer (HPV16, HPV18 and others). Particularly preferred forms of prophylactic or therapeutic vaccines of genital warts comprise L1 particles or capsomeres and fusion proteins comprising one or more antigens selected from the HPV 6 and HPV 11 E6, E7, L1, and L2 proteins. The most preferred formulas of fusion protein are. L2E7 as described in WO 96/26277, and protein D (1/3) -E7 described in GB 97179535 (PCT / EP98 / 05285). l .i.

A vaccine composition for prophylaxis or therapeutics of infection or cervical cancer by preferred HPV may comprise HPV 16 or 18 antigens. For example, the antigen monomers L1 or L2 or the antigens L1 or L2 presented together as a virus-like particle ( VLP) or the L1 protein alone presented alone in a VLP or capsomere structure. Such antigens, virus-like particles and capsomeres are already known. For example, see documents WO94 / 00152, WO94 / 20137, WO94 / 05792, and WO93 / 02184. Additional early proteins may be included alone or as fusion proteins such as preferably E7, E2 or E5 for example; Particularly preferred embodiments of this include a VLP comprising L1E7 fusion proteins (WO 96/11272). Particularly preferred HPV 16 antigens comprise E6 or E7 early proteins in fusion with a protein D carrier to form Protein D-E6 or E7 from HPV 16 or combinations thereof; or combinations of E6 or E7 with L2 (WO 96/26277). Alternatively, E6 and E7 of early HPV proteins 16 or 18 can be presented in a single molecule, preferably a fusion of Proteins D-E6 / E7. Such a vaccine may optionally contain either or both of the E6 and E7 proteins from HPV 18, preferably in the form of a Protein D-E6 or Protein D-E7O fusion protein, a Protein D E6 / E7 fusion protein.

S ^^^ & ^^^^ 3é &m The vaccine of the present invention can traditionally comprise antigens from other strains of HPV, preferably from strains HPV 6, 11, 31, 33 or 45. The present invention further comprises antigens derived from parasites that cause Malaria. For example, preferred antigens from Plasmodia falciparum include RTS.S and TRAP. RTS is a hybrid protein that comprises substantially all of the C-terminal part of the circumsporozoite (CS) protein of P. falciparum linked through four amino acids from the preS2 part of the Hepatitis B surface antigen to the surface antigen (S) of the hepatitis B virus. Its entire structure is described in International Patent Application No. PCT / EP92 / 02591, published under number WO 93/10152 which claims the priority of United Kingdom patent application No. 9124390.7. When expressed in yeast RTS a lipoprotein particle is produced, and when coexpressed with the S antigen from VBH, a mixed particle known as RTS, S is produced. In the International Patent Application No. PCT / GB89 / 00895, published under the number WO 90/01496, TRAP antigens are described. One embodiment of the present invention is a Malaria vaccine wherein the antigenic preparation comprises a combination of the RTS.S and TRAP antigens. Other plasmodia antigens that are likely candidates for components of a multistage Malaria vaccine are P.faciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Secuestrim, PfEMPI, Pf332, LSA1, LSA3, STARP, SALSA, PfEXPI, Pfs25, Pfs28, PFS27 / 25, Pfs16, Pfs48 / 45, Pfs230 and their analogs in Plasmodium spp. The formulations may also contain an anti-thyroid antigen and be useful for cancers of immunotherapeutic treatment. For example, the adjuvant formulation has utility with tumor expulsion antigens such as prostate, breast, colorectal, lung, pancreatic, renal or melanoma cancers. Example antigens include MAGE 1 and MAGE 3 or other MAGE antigens for the treatment of melanoma, PRAME, BAGE or GAGE (Robbins and Kawakami, 1996, Normal Options in Immunology 8, pages 628 to 636: Van den Eynde and associates, Journal International Clinical and Laboratory Research (presented in 1997); Corréale et al. (1997), Journal of the National Cancer Institute 89, page 293. In fact, these antigens are expressed in a wide range of tumor types such as melanoma, carcinoma of tumor, carcinoma of sarcoma and bladder. Other tumor-specific antigens are suitable for use with the adjuvant of the present invention and include, but are not limited to, prostate specific antigen (PSA) or Her-2 / neu, KSA (GA733), MUC-1 and carcinoembryonic antigen ( CEA), Therefore, in one aspect of the present invention, there is provided a vaccine comprising an adjuvant composition according to the present invention and a tumor expulsion antigen. Further, the antigen may be a self-peptide hormone tai as a hormone releasing gonadotropin hormones of all length (GnRH, WO 95/20600), a long peptide of 10 ..t-jt. * k4..i short amino acids, in the treatment of many cancers or immunocastration. It is considered that the compositions of the present invention will be used to formulate vaccines containing antigens derived from Borrelia sp .. For example, the antigens may include nucleic acid, antigen derived from pathogen or antigenic preparations, proteins or peptides produced in recombinant form and proteins. of chimeric fusion. In particular. the antigen is OspA. AND! OspA can be a total mature protein in a Sipidated form by virtue of the host cell (E: Coli) termed (Lipo-OspA) or a non-lipidated derivative. Such non-lipid derivatives include a non-cleaved NS1-OspA fusion protein having the first 81 N-terminal amino acids of the non-structural protein (NS1) of the influenza virus and the complete OspA protein, and another, MDP-OspA, is a form non-lipidated OspA that carries 3 additional N-terminal amino acids. The vaccines of the present invention can be used for prophylaxis or allergy therapy. Such vaccines could comprise allergen-specific antigens (e.g. Dev p1) and non-allergen-specific antigens (e.g., human IgE derived polypeptides, including but not limited to Stanworth decapeptide (EP 0 477 231 B1)). The amount of protein in each vaccine dose is selected as an amount that induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending on the specific immunogen that is used and i-.t.j .j i .¿.¿. .. U. Ifi?? .ff ~, i ^ M. gj ^^ a? í of how it presents itself. Generally, it is expected that each dose will comprise from 1 to 1000 μg of protein, preferably from 1 to 500 μg, preferably from 1 to 100 μg, most preferably from 1 to 50 μg. An optimal amount of a particular vaccine can be determined by standard studies comprising observing an appropriate immune response in subjects. After an initial vaccination, the subjects may receive one or more booster immunizations spaced appropriately. The vaccines of the present invention can also be administered orally. In such cases the pharmaceutically acceptable excipient may also include antacid buffers, or enteric capsules or microgranules. The vaccines of the present invention can also be administered through the vaginal route !. In such cases, the pharmaceutically acceptable excipients may also include emulsifiers, polymers, such as CARBOPOL.0, and other known stabilizers of vaginal creams and suppositories. The vaccines of the present invention can also be administered rectally. In such cases the excipients may also include waxes and polymers known in the art to form rectal suppositories. The formulations of the present invention can also be used for both prophylactic and therapeutic purposes. Therefore, the present invention provides a method for treating a mammal susceptible to or suffering from an infectious disease or cancer, or allergy, or autoimmunological disease. In one aspect In addition to the present invention, a combination of adjuvant and a tai vaccine as described in the present invention is provided for use in medicine. In an alternative embodiment related to the present invention, the adjuvant of the present invention can be further combined with other adjuvants, including Cholera toxin and its subunit B, the lipid Monophosphoryl A and its non-toxic derivative of monophosphoryl lipid 3- de-O-acylated A, (as described in United Kingdom Patent No. GB 2220,211), immunologically active saponin fractions, eg, Qui A derived from the bark of the Quillaja Saponaria Mohna tree of America of the South and derivatives thereof (for example QS21, US Patent No. 5,057,540), and the CpG oligonucleotide adjuvant system (as described in WO 96/0255). especially 5'TCG TCG TTT TGT CGT TTT GTC GTT3 '(SEQ ID NO.1), The present invention is illustrated but not limited to the following examples. In the examples below, we use the complete flu virus grown with eggs, deactivated with formaldehyde, or fragmented ether-Tween virus or virus grown by fragmented NaDOC eggs supplemented with Triton X-100. The concentrations of Tween-80 and Triton X-100 are shown in the following examples. Example 1, Method that is used to measure the antibody (Ab) responses in ELISA sera for the measurement of Abs: g of influenza specific serum: t it * ^. faith I í.

Maxisorp Nuric immunoplates are coated overnight at a temperature of 4 ° C with 50 μl / 1 μg / ml HA reservoir of inactivated influenza virus p-propylactone (BPL) (supplied by the manufacturer SSD GmBH, Dresden, Germany) diluted in PBS The udder sites are blocked in the plates (1 hour at 37 * 0), using saturation buffer: PBS containing 1% BSA, 01% polyoxyethylene sorbitan monolaurate (TWEEN 20), then incubated for 1 hour. hour 30 minutes at a temperature of 37 ° C, serial dilutions of 2 bends (in saturation buffer, 50 μl per deposit) of a reference serum added in the form of a standard curve (serum having a mid-point titrant) expressed as ELISA Unit / ml and placed in wing A) and serum samples (beginning with a 1/100 dilution and placed on the B-to-H feces), then the plates are availed (x3) with buffer of avado (PBS, 0.1% sorbitan monolaurate of polyoxyethylene) eno (TWEEN 20)), Subsequently, incubate (50 μf / tank) for 1 hour 30 minutes at a temperature of 37 ° C. Biotinylated goat antihuman Ig (Amersham) diluted 1/3000 in saturation buffer. After 3 washes, and the subsequent addition of streptavidin-radish peroxidase conjugate (Amersham) the plates are spiked 5 times and incubated for 20 minutes at room temperature with 50 μi / reservoir of the revelation buffer (OPDA 04 mg / ml (Sigma) and H202 0.3% in 50mM pH 45 of citrate buffer) The detection is stopped by adding 50 μl / tank of H2S04 2N The optical densities are measured at 492 and 630 nm using a Biorad 3550 integrator.

? To such < ** - £ &jjéyd.jrl-a & Antibody are calculated by the 4-parameter mathematical method using SoftMaxPro software. Hemaqlutination Inhibition Activity (HAI) of flu-specific Abs serum in mice. The sera were first treated (25 ul) for 20 minutes at room temperature (RT) with 100 μl of 0.5 M borate buffer (pH 9) and 125 μl of kaolin purchased from Dade Behring. After centrifugation (30 minutes, 3000 RPM or 860 g), 100 μl of supernatant (corresponding to a 1/10 dilution of the serum) was taken and incubated for 1 hour at a temperature of 4 ° C with 0.5% of chicken red blood cells. The sobendant was collected after centrifugation for 10 minutes at 3200 RPM (970 g). Both operations were performed to eliminate the natural haemagglutination factors contained in the sera. Subsequently, 25 μl of treated sera are diluted in 25 μl of PBS (series of double-fold dilutions starting at 1/20) in 96-well Greiner plates. The complete virus deactivated by BPL (25ul / tank) was added in a concentration of 4 units of hemagglutination (for example in a dilution which is 4 folds lower than the last one that causes an agglutination of the red blood cells) during 30 minutes at RT under agitation. Subsequently, the cells of chicken red blood cells (25 μl / tank) were added for one hour at RT. Finally the plates were kept overnight at a temperature of 4 ° C before being read. The HAI titrator corresponds to the inverse of the last serum dilution inhibiting the hemagglutination induced by the virus.

Example 2, Effect of TWEEN 80 and Triton on the intranasal immunogenicity of all influenza virus inactivated in mice. Previously, preclinical evaluations of alternative influenza vaccines (eg, adjuvanted parenteral vaccines, DNA-based vaccines or mucous-delivered vaccines) had been carried out mainly in "naive" animals. In general, the promising results obtained from these studies were not confirmed in humans. This was probably due to the fact that most adults had been "immunologically prepared" by means of natural infections before vaccination, unlike the "naive" animals. Tfore, the best way to evaluate intranasal influenza vaccines in animal models could be to test their ability to reinforce previously established immune responses in animals prepared in nasal form. In the present example, the effect of Tween 80 and Triton X-100 on such responses was evaluated. The preparation was carried out in female Balb / c mice (8 weeks of age) on day 0, administering with a pipette (under anesthesia) in each nostril, 2.5 μg HA of influenza A / Beijin / 262/95 virus inactivated by BPL contained in 10ul of PBS. After 28 days, the mice (6 / animals / group) were reinforced in intranasal form (under anesthesia) with 20 μl of solution with (10 μl per nostril, administered in the form of drops by means of a pipette) containing 5 μg of HA of influenza A / Beijing / 262/95 virus inactivated by BPL in eitA: PBS; B: TWEEN80 (0.11%) plus Triton X-100 (0.074%); or by C: intramuscular injection of 1.5 μg HA of the influenza vaccine. The antigens were supplied by the manufacturer SSD GmBH (Dresden, Germany). HAI Ab responses were measured in the sera, as described in Example 1. As shown in Figure 1, when formed with TWEEN80 and Triton, the inactivated influenza virus delivered in intranasal form has the ability to reinforce systemic HAI Ab responses previously established as efficiently as the classic parenteral influenza vaccine. However, the same antigen provided in The intranasal form in the absence of TWEEN80 and Triton is significantly less immunogenic. Comparison of the immunogenicity of a fragmented intranasal influenza vaccine with TWEEN 80 and Triton X-100, with a conventional parenteral vaccine with license (Fluarix ™) in subjects healthy adults Formulations used in the study. Two formulations (A, B) of fragmented influenza antigens derived from eggs were evaluated. A is an intranasal formulation and B is Fluarix ™ / a-Risk R provided in intramuscular. The formulations contain 3 fragmented inactivated virgin antigens prepared from strains recommended by WHO of the 1998-1999 season. The apparatus used for intranasal administration is the Accuspray ™ intranasal syringe from Becton Dickinson. In each nostril, 100 μl of formulation A was sprayed. ÜIIIÜ- A-i¿i.ta. , A. t ti ^ ,,. ,, ^.

^ Composition of the Formulation The intranasal formulation (A) contained the following fragmented virions deactivated. 1.30μg HA A / Beijing / 262/95 (H1N1) 2.30μg HA A / Sydney / 5/97 (H3N2) 3.30μg HA of B / Harbin / 7/94 and phosphate buffered saline with pH 7.4 i 0.1, 0.1 % of TWEEN80. 0.015% Triton-X-100, 0.0045% sodium deoxycholate and thiomersal below 35 μg / ml. The volume of a dose was 200 μl (sub-dose of 10Oμl for each nostril). The Fluarix ™ / a-Rix R comparator is the commercially available trivalent fragmented influenza vaccine from SmithKIine Beecham Bíologícals. The 500 μl dose is administered intramuscularly. This dose contains; 15 μgHa of the three strains mentioned above, Tween 80 between 500 and 1,000 μg per ml (0.05% - 0.1%), Triton X-100 between 50 and 170 μg / ml (0.005% - 0.017%), of maximum sodium oxicolate 100 μg / ml, thiomersal 100 μg / ml and phosphate buffered saline with pH between 6.8 and 7.5. Immunogenicity Study. A randomized controlled open-label study evaluated the immunogenicity of a fragmented intranasal influenza vaccine formulated with Tween 80 and Triton X-100, compared with the vaccine can . i tqaJ, ,,. i. * ^ & conventional parenteral ai X (for example Fluarix ™). Twenty healthy adult subjects (aged 18 to 40 years) received a dose of Fluarix ™, and 10 subjects received a dose of intranasal influenza vaccine. The intranasal formulation (200ul) contained the following deactivated virions: 30 μg A / Beijing / 262/95 hemagglutinin (H1N1) 3.0 μg A / Sydney / 5/97 haemagglutinin (H3N2), 30 μg B / Harbin / 7/94 hemagglutinin with phosphate buffered saline (pH 7.4 +/- 0.1) Tween 80 (0.1%), Triton X-100 (0.015%), sodium deoxycholate (0.0045%) and thiomersal (<35μg / ml ). There was a 8-day follow-up period of local and general symptoms and both vaccines were well tolerated with respect to safety and reactogenicity. No serious adverse events related to vaccination were reported. The immunogenicity of the vaccines was examined by evaluating the serum hemagglutination inhibitor (Hl) titrators to determine the range of serum conversion (defined as the percentage of vaccines that have at least a 4-fold increase in serum Hl titers in the day 21, compared to day 0 for each vaccine strain), conversion factor (defined as the increase in folds in Geometric Average HL Titrators (GMTs) on day 21 compared to day 0 for each vaccine strain) and range of serum protection (defined as the percentage of vaccines with a titrant Hl of serum >40 after vaccination (for each strain of vaccine), this is accepted as indication protection. Generally, an influenza vaccine needs to have more than or equal to 40% of the serum conversion range, more than or equal to 70% of the range of serum protection and a conversion factor > to 2.5 for each strain, in order to comply with the requirements of international regulation. This applies to adults between 18 and 60 years old; and different criteria apply for the elderly. In addition, the mucosal IgA antibody response was evaluated by the Enzyme Linked Immunoabsorbent Assay (Elisa). Table 1 shows the ranges of serum positivity, serum conversion and serum Hl protection twenty-one days after a dose of Fluarix ™ or the intranasal formulation.

In all cases the conversion factor (increase in Hl GMTs of serum after vaccination) was greater than 2.5, the level required for a successful influenza vaccine.

The percentage of subjects with an increase of two folds or four folds in the proportion of specific / total mucosal IgA antibody between day 21 and day 0 (dose 1), can be seen in Table 2.

Summary The immunogenicity results tabulated above, show that the intranasal formulation produced levels of serum positivity, serum conversion and serum protection similar to those produced by the conventional parenteral vaccine (Fluarix ™), 21 days after a dose. The intranasal formulation produced a better mucosal IgA response after a dose than the conventional parenteral vaccine (Fluarix ™).

. To Me-j r? ..

Claims (18)

1. CLAIMS 1. The use of a combination of a sorbitan ester of polyoxyethylene and octoxynol in the preparation of an adjuvant for application to a mucosal surface of a patient.
2. 2. The use according to claim 1, wherein the polyoxyethylene sorbitan ester is polyoxyethylene sorbitan monooleate (Tween 80 ™).
3. 3. The use according to claim 1 or 2, wherein the octoxynol is t-octylphenoxypolyethoxyethanol (TRITON X-100 ™).
4. 4. The use according to any one of claims 1 to 3, further comprising a bile salt or a cholic acid derivative.
5. 5. The use of an adjuvant according to any of claims 1 to 4, together with an antigen, in the manufacture of a vaccine for mucosal administration. The use according to claim 5, wherein the antigen is selected from the group comprising: Human Immunodeficiency Virus, Varicella Zoster Virus, Herpes Simplex Virus type 1, Herpes Simplex Virus type 2, Human cytomegalovirus, Virus of Dengue, Hepatitis A, B, C, or E, Respiratory Syncytial Virus, Human Papilloma Virus, Influenza Virus, Hib, Meningitis Virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Streptococcus, Mycoplasma, Mycobacteria, Hemofilus, Plasmodium o Toxoplasma, stanworth or antigens associated with tumor (TMA), MAGE, BAGE, GAGE, MUC-1, Her-2 neu, LnRH, CEA, PSA, KSA, or PRAME.
6. É & .4 ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ bti
7. 7. The use according to claim 6, wherein the antigen is an antigen or antigenic preparation from the influenza virus.
8. 8. The use according to claim 7, wherein the antigenic preparation is a fragmented influenza virus preparation.
9. 9. The use according to claim 7 or claim 8 for the manufacture of a vaccine for prophylaxis against influenza.
10. 10. Use according to any of claims 5 to 9, for the manufacture of a vaccine for use in medicine.
11. 11. A method for producing a vaccine, wherein the method comprises mixing with additions (a) a polyoxyethylene sorbitan ester, (b) an octoxinol and (c) an antigen, and providing the vaccine in the form of a vaccine dose. for mucosal administration.
12. The method according to claim 11, wherein the vaccine is provided in an aerosol or intranasal spray apparatus.
13. 13. An aerosol or spray apparatus, more particularly a bidosis apparatus, filled with a vaccine comprising a polyoxyethylene sorbitan ester, an octoxynol and an antigen.
14. 14. The spray or spray apparatus according to claim 13, wherein the antigen is an influenza antigen or an antigenic preparation.

    9,
15. 15. The spray or aerosol apparatus according to claim 14, wherein the antigenic preparation is a fragmented influenza virus preparation.
16. 16. A method for treating a mammal suffering from or susceptible to a pathogenic infection, or cancer or allergy wherein the method comprises administering to the mucosa of the mammal, a safe and effective amount of a vaccine composition comprising a sorbitan ester of polyoxyethylene, an octoxynol and an antigen.
17. 17. The method according to claim 16, wherein the vaccine is administered in intranasal form.
18. 18. The method according to claim 16 or claim 17, wherein the vaccine is an influenza virus vaccine comprising an influenza antigen or antigenic preparation, such as a fragmented influenza virus preparation.