Title of Invention	A NOVEL VACCINE COMPOSITION
Abstract	The present invention relates to an aqueous vaccine composition comprising: hepatitis B surface antigen; an inactivated hepatitis A virus (HAV); and formol, in which there is 0.015 - 0.1 mg aluminium phosphate per µg hepatitis B surface antigen and 0.05 - 0.10 mg aluminium hydroxide per ml, for administration to adolescents or children in a 2 dose schedule.

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Vaccines for the prophylaxis of hepatitis A and hepatitis B infections are well known. For example the vaccine Engerix-B (Trade Mark) from SmithKline Beecham Biologicals is used to prevent Hepatitis B. This vaccine comprises hepatitis B surface antigen (specifically the 226 amino acid S- antigen described in Harford et al. in Postgraduate Medical Journal, 1987, 63 (Suppl. 2), 65-70) and is formulated using aluminium hydroxide as adjuvant. The vaccine Havrix (Trade Mark), also from SmithKline Beecham Biologicals can be used to prevent hepatitis A infections and is also formulated with aluminium hydroxide as adjuvant. This vaccine comprises an attenuated strain of the HM-175 Hepatitis A virus inactivated with formol (formaldehyde); see Andre et al [Prog Med. Virol. 1990, vol 37; p72-95]. The vaccine Twinrix (Trade Mark) which is a combination of the above hepatitis A and hepatitis B antigens may be used to protect against hepatitis A and hepatitis B simultaneously. European patent 0 339 667 (Chemo Sero) describes the general concept of combining a hepatitis A antigen and a hepatitis B antigen to make a combination vaccine. In that specification it is stated that the adjuvant which is used is not critical: it must only be capable of enhancing the immune activity to a desired extent and not cause any side-effects. It is stated that aluminium gel may be used, in particular aluminium hydroxide gel and aluminium phosphate gel. PCT application WO 93/24148 (SmithKline Beecham) describes the preparation of vaccines comprising hepatitis B surface antigen in which aluminium phosphate is used as adjuvant. Multivalent combination vaccines which may optionally contain a hepatitis A antigen, are described. The use of formol is not disclosed. European Patent Number 0 633 784 (SmithKline Beecham) describes novel vaccine formulations comprising a hepatitis B component, particularly hepatitis B surface antigen, in combination with aluminium phosphate, and 3 de-O-acylated monophosphoryl lipid A. It has now been surprisingly found that vaccines comprising hepatitis B and/or hepatitis A antigens give exceptionally good results if the vaccine is formulated in a specific manner. Using vaccine formulations according to the invention, it is possible to administer the vaccines in a 2 dose, rather than a 3 dose, regimen. In a first aspect of the invention, there is provided an aqueous vaccine composition comprising hepatitis B surface antigen which is formulated with aluminium phosphate as adjuvant, the concentration of aluminium phosphate being selected such that there is a ratio of 0.015-0.lmg aluminium phosphate per ug hepatitis B surface antigen. Preferably the ratio is in the range 0.02 to 0.08mg aluminium phosphate per ug HBsAg. In a further aspect of the invention an inactivated hepatitis A virus (HAV) may optionally be added to the formulation of the invention, providing a combined hepatitis A plus B vaccine which may be administered in a 2 dose schedule. The hepatitis A antigen is preferably the HM-175 strain used in the commercial product Havrix (SmithKline Beecham Biologicals). The concentration of hepatitis A antigen in the vaccine formulation of the invention is preferably about 720-2880 EU units per ml. For the definition of EU units see Andre etal(1990)loc cit. The compositions of the invention which comprise HAV may additionally comprise aluminium hydroxide, the total amount of aluminium hydroxide generally being 0.05-0.10 mg per ml. The total amount of aluminium salt per 0.5 or 1 ml dose is normally in the range 0.4-l.Omg. In the vaccine composition of the invention it is advantageous to add formol (formaldehyde) such that the formol concentration is 10-200ug per ml. Preferably the formol concentration is about 20-160 ug per ml. Normally the hepatitis B antigen will be hepatitis B surface antigen (HBsAg). The preparation of Hepatitis B surface antigen (HBsAg) is well documented. See for example, Harford et al in Develop. Biol, Standard 54, page 125 (1983), Gregg et al in Biotechnology, 5, page 479 (1987), EP-A- 0 226 846, EP-A-0 299 108 and references therein. As used herein the expression 'Hepatitis B surface antigen' or 'HBsAg' includes any HBsAg antigen or fragment thereof displaying the antigenicity of HBV surface antigen. It will be understood that in addition to the 226 amino acid sequence of the HBsAg S antigen (see Tiollais et al, Nature, 317, 489 (1985) and references therein) HBsAg as herein described may, if desired, contain all or part of a pre-S sequence as described in the above references and in EP-A- 0 278 940. HBsAg as herein described can also refer to variants, for example the 'escape mutant' described in WO 91/14703. In a further aspect the HBsAg may comprise a protein described as SL* in European Patent Application Number 0 414 374, that is to say a protein, the amino acid sequence of which consists of parts of the amino acid sequence of the hepatitis B virus large (L) protein (ad or ay subtype), characterised in that the amino acid sequence of the protein consists of either: (a) residues 12-52, followed by residues 133 - 145, followed by residues 175 - 400 of the said L protein; or (b) residue 12, followed by residues 14 - 52, followed by residues 133 - 145, followed by residues 175 - 400 of the said L protein. HBsAg may also refer to polypeptides described in EP 0 198 474 or EP 0 304 578. Normally the HBsAg will be in particle form. It may comprise S protein alone or may be as composite particles, for example (L*,S) wherein L* is as defined above and S denotes the S-protein of hepatitis B surface antigen. Preferably the HBsAg will be adsorbed on aluminium phosphate as described in W093/24148. Preferably the hepatitis B antigen is HBsAg S-antigen as used in the commercial product Engerix-B (SmithKline Beecham Biologicals). The vaccine formulations of the present invention will contain an immunoprotective quantity of the antigens and may be prepared by conventional techniques. Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757. The vaccine compositions of the invention are preferably administered in a 0, 6 month schedule, that is to say a first dose at 0 months and a second dose at 6 months. The vaccine compositions of the present invention are especially appropriate for adults and are also appropriate for administration to adolescents and children. The following examples illustrate but do not limit the invention. EXAMPLES Example 1. Specific formulations Specific formulations within the scope of the invention include the following: a) A vaccine composition for administration to adults which comprises: 40(ig HBsAg 1440EUHAV 0.85mg Aluminium salt (0.8mg aluminium phosphate plus 0.05mg aluminium hydroxide) 20µg formol in a 0.5ml dose b) A vaccine composition for administration to adults which comprises: 40µg HBsAg 1440EUHAV 0.85mg Aluminium salt (0.8mg aluminium phosphate plus 0.05mg aluminium hydroxide) 20µg formol in a 1.0ml dose c) A vaccine composition for administration to adolescents and/or children which comprises: 20µg HBsAg 720 EU HAV 0.45mg Aluminium salt (0.4mg aluminium phosphate plus 0.05mg aluminium hydroxide) 80µg formol in a lml dose d) A vaccine composition for administration to adolescents and/or children which comprises: 20µg HBsAg 720 EU HAV 0.45mg Aluminium salt (0.4mg aluminium phosphate plus 0.05mg aluminium hydroxide) 80µg formol in a 0.5ml dose Example 2: Study 'HAB 054' 2.1 One group of 47 healthy adult individuals was vaccinated with a vaccine composition (1ml volume) containing: 1440 EU HAV antigen 40µg HBsAg (S-antigen) 0.8mg A1P04 0.05mg Aluminium hydroxide 20µg/ml formol. This is abbreviated in the tables below to ' 1440/NF40' 2.2 One group of 47 healthy adult individuals was vaccinated : a) in one arm with a vaccine composition containing 40µg of HBsAg formulated with 0.8mg AIPO4, 20µg/ml formol (hereinafter this composition is abbreviated to 'NF 40') in a 1ml dose; and b) in the opposite arm with HAVRIX 1440 containing 1440 EU HAV antigen on 0.5mg Aluminium hydroxide and containing 20µg/ml formol in a 1 ml dose. The immunization schedule for HAB 054 was 0,6 (i.e. doses administered at month 0 and month 6). The Hepatitis B serological results at months 2,6 and 7 (seroconversion (SC), seroprotection (SP) and geometric mean titre (GMT) ) were unexpectedly high as compared to historical results obtained in another study (HBV NF 021) in which volunteers of the same age group were included. Study HBV NF 021 (see Table 1) included three groups : Group 1: Vaccinated with a formulation containing 40µg of HBsAg on 0.5^g AIPO4 in 0.5ml volume without formol, schedule 0, 6 months (this is abbreviated in the table to NF 40µg, 0, 6M) Group 2: Vaccinated with a formulation containing 20µg of HBsAg on 0.5|ag AIPO4 in 0.5ml volume without formol, schedule 0, 6 months (this is abbreviated in the table to NF 20µg, 0, 6M) Group 3: Vaccinated with an Engerix B formulation (20µg) using a classical 3 dose immunization schedule of 0, 1,6 months (third row of data in Table 1) Results 1) In study HBV NF 021, Hepatitis B serological results at month 7 in groups 1 and 2 were not satisfactory as compared to group 3 (lower SC, SP and GMT +/- 1/3 (500 to 600) of the levels obtained after three doses of Engerix -B (1500)). 2) In study HAB 054, the titres obtained after 2 doses of vaccine were of the same order of magnitude as those that would be obtained following three doses of Engerix B. TABLE 1: HBV-NF 021 STUDY TABLE 2: HAB 054 study HAB 2-dose program in adults (0, 6month schedule) N=94 TABLE 3: HAB 054 study HAB 2-dose program in adults (0,6month schedule) N=94 TABLE 4: HAB 054 study HAB 2-dose program in adults (0, 6month schedule) N=94 TABLE 5: HAB 054 study HAB 2-dose program in adults (0,6month schedule) N=94 TABLE 6: HAB 054 study HAB 2-dose program in adults (0,6month schedule) N=94 Example 3: Studies 'HAB 063, HAB071 and HAB 075' HAB 2 dose program in adolescents aged 11-18 3.1: Study HAB 063 Vaccine composition comprises: 720 HAV EU / 20µg HBs Ag 0.25µg Al Salt 40µg formol In 0.5ml dose Results are shown in Table 7. In 0.5ml dose and at a schedule of 0, 6 months Vaccine composition group 2 (Twinrix ™ Junior): 360HAVEU/10µg HBs Ag 0,225 mgr Al Salt 40µg formol In 0.5ml dose and at a schedule of 0, 1,6 months 3.3 Study HAB 075 Two groups were used in this study. Vaccine composition group 1 (Twinrix™): 720 HAV EU / 20µg HBs Ag 0,45µgAl Salt 80µg formol In 1ml dose and at a schedule of 0, 6 months N = 67 Vaccine composition group 2: 1440 HAV EU / 40µg HBs Ag 0.85µgAlSalt 80µg formol In 1ml dose and at a schedule of 0, 6 montns N = 55 Example 4: Study 'HAB 084' HAB 2 dose program in adolescents aged 12-15 WE CLAIM: 1. An aqueous vaccine composition comprising: hepatitis B surface antigen; an inactivated hepatitis A virus (HAV); and formol, in which there is 0.015 - 0.1 mg aluminium phosphate per µg hepatitis B surface antigen and 0.05 - 0.10 mg aluminium hydroxide per ml, for administration to adolescents or children in a 2 dose schedule. 2. The composition according to claim 1 in which the formol concentration is 10-200 µg per ml. 3. The composition according to claim 1 or 2 in which the concentration of HAV is 720-2880 EU per ml. 4. The composition according to claim 1 which comprises or consists of: 20µg HBsAg 720 EU HAV 0.45 mg Aluminium salt, consisting of 0.4 mg aluminium phosphate plus 0.05 mg aluminium hydroxide 80 µg formol in a 1 ml dose. 5. The composition according to claim 1 which comprises or consists of: 20µg HBsAg 720 EU HAV 0.45 mg Aluminium salt, consisting of 0.4 mg aluminium phosphate plus 0.05 mg aluminium hydroxide 80 µg formol in a 0.5 ml dose. 6. The composition according to any preceding claim wherein the hepatitis B surface antigen is the S-antigen. 7. The composition according to any preceding claim in which the hepatitis A antigen is derived from the HM-175 strain. 8. The composition according to any preceding in which the 2 doses are for administration at month 0 and 6.

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