AP567A

AP567A - Combined vaccines comprising hepatitis B surface antigen and other antigens.

Info

Publication number: AP567A
Application number: APAP/P/1993/000530A
Authority: AP
Inventors: Jean Petre
Original assignee: Smithkline Beecham Biologicals S A
Priority date: 1992-05-23
Filing date: 1993-05-20
Publication date: 1996-11-25
Also published as: LU91659I2; HK1011290A1; EP2156845A1; RU94046145A; DE69334297D1; AU2010200249B2; EP0642355B1; DK0835663T3; SG48365A1; KR100287083B1; AU785433B2; AU5062902A; CA2136429C; DE69319728D1; SK142194A3; AU2007201162A1; NZ253065A; JP3626996B2; KR950701822A; CA2136429A1

Abstract

Stable and effective multivalent vaccine compositions comprising hepatitis b surface antigen (hbsag)are described wherein the hbsag component is stable for one week at 37 degrees celcius and is highly immunogenic, for example when the vaccine is administered to infants. The compositions typically comprise hbsag adsorbed to aluminium phosphate and other antigens, especially those suitable for use in a paediatric vaccine, adsorbed to aluminium phosphate or aluminium hydroxide. Methods for preparing the vaccines and the use of aluminium phosphate to stabilise hbsag in a multivalent vaccine formulation are also described.

Description

Vaccine

The present invention relates to novel vaccine formulations, methods for preparing them and to their use in therapy. In particular the present invention relates to novel combination vaccine formulations including a Hepatitis B vaccine component for treating Hepatitis B infections.

Infection with Hepatitis Β (HB) virus is a widespread problem but vaccines which have been used for mass immunisation are now available, for example the product 'Engerix-B' (SmithKline Beecham pic). Engerix B has as the Hepatitis B antigenic component Hepatitis B surface antigen (HBsAg) which is obtained by genetic engineering techniques.

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However it is often necessary or desirable to administer Hepatitis B vaccine at the same time as other vaccines and this can involve multiple injections. Problems associated with multiple injections include a more complicated administration procedure and a large total injection volume.

There is therefore a need for a stable combined vaccine comprising a Hepatitis B antigen in combination with other antigens. The other antigens are in particular those capable in a vaccine formulation of preventing Hepatitis A (HA), diphtheria (D), tetanus (T), whole cell pertussis (Pw), acellular pertussis (Pa), Haemophilus influenzae b (Hib) and polio (P).

Aluminium hydroxide (AH) is widely used as an adjuvant in the formulation of o vaccines. For example, Engerix B uses Hepatitis B surface antigen (HBsAg) adsorbed to aluminium hydroxide. We have also used AH successfully in the formulation of Hepatitis A vaccine and in the combined vaccines DT, DTPw and DTPa. However, when AH -adsorbed HBsAg is used in combination with other vaccines in a combined formulation there is a substantial decrease of the immune response to HBsAg, resulting in lower or insufficient seroprotection after vaccination. In addition the stability of the HBsAg component of the combined vaccine is poor.

Aluminium phosphate (AP) adsorbed HBsAg has been used in a commercially available monovalent vaccine (HEPPACINE) made by Korean Cheil Sugar Co Ltd.

We have found that there is no significant difference in immunogenic properties between an AH- adsorbed HBsAg monovalent vaccine, Engerix B, and an APadsorbed HBsAg monovalent vaccine.

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- 2European patent application publication number 0 339 667 discloses a bivalent vaccine comprising HBsAg and a Hepatitis A antigen in which either aluminium hydroxide or aluminium phosphate is used as adjuvant. There appears, however, to be no appreciation of the need to avoid aluminium hydroxide as an adjuvant for a multivalent vaccine comprising HBsAg. Furthermore there appears to be no disclosure in this document or elsewhere of a bivalent or multivalent hepatitis B vaccine in which at least one antigen other than HBsAg is adsorbed on aluminium hydroxide and the HBsAg is adsorbed on aluminium phosphate.

Indeed there appears to be no prior enabling disclosure of a stable and effective multivalent vaccine comprising HBsAg at all.

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In one aspect the present invention provides a combined vaccine composition comprising Hepatitis B surface antigen (HBsAg) and a number (n) of other antigens in combination with an adjuvant comprising one or more aluminium salts in which the value of n is 1 or greater and in which the adjuvant used to adsorb the HBsAg is not aluminium hydroxide, with the proviso that when n is 1 the other antigen is not an antigen against Hepatitis A.

Preferably n is 2, 3,4, 5 or 6.

The advantage of the invention is that no substantial decrease in the immunogenicity of the HBsAg occurs in the combined vaccine formulation. Avoiding the use of AH to adsorb the HBsAg component in the vaccine formulation also gives rise to a product of markedly superior stability. A further advantage of the invention is that the aforesaid problems associated with multiple injections are overcome or at least mitigated and a stable, highly immunogenic combined formulation is provided. The compositions of the invention are particularly suitable for administration to children.

Preferably the HBsAg is adsorbed on AP. In particular we have found in human clinical studies that when AP-adsorbed HBsAg is combined with one or more AHadsorbed or AP-adsorbed antigens in a combined vaccine no substantial decrease in immunogenicity occurs. The stability of the AP-adsorbed HBsAg in the formulation is also greater than AH-adsorbed HBsAg.

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-3Accordingly in a further aspect the invention there is provided a vaccine composition according to the invention in which at least one of the antigens other than HBsAg is adsorbed to aluminium phosphate.

In a further preferred aspect at least one of the antigens other than HBsAg is adsorbed to AH.

In a further aspect, the invention provides a combined vaccine comprising Hepatitis B surface antigen (HBsAg) adsorbed to AP and an antigen adsorbed to AP or to AH selected from an antigen against one or more of the following viruses: diphtheria (D); tetanus (T); pertussis (P); Inactivated Polio (IPV); Haemophilus influenzae b (Hib); and Hepatitis A (HA).

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In a paediatric vaccine other compatible antigens may also be included, eg antigens known to be effective against meningitis B, meningitis A and C, and otitis media.

j As used herein the term 'bivalent' is used to refer to a vaccine comprising a combination of two antigens in total. The term 'multivalent' is applied to a vaccine j composition comprising more than two antigens, for example three, four or five or six

J 20 antigens.

The meaning of the terms 'aluminium phosphate' and 'aluminium hydroxide' as used herein includes all forms of aluminium hydroxide or aluminium phosphate which are suitable for adjuvanting vaccines.

For example, aluminium phosphate can be a precipitate of insoluble aluminium phosphate (amorphous, semi-crystalline or crystalline), which can be optionally but not exclusively prepared by mixing soluble aluminium salts and phosphoric acid salts. Aluminium hydroxide can be a precipitate of insoluble (amorphous, semi30 crystalline or crystalline) aluminium hydroxide, which can be optionally but not exclusively prepared by neutralizing a solution of aluminium salts. Particularly suitable are the various forms of aluminium hydroxide and aluminium phosphate gels available from commercial sources for example, Alhydrogel (aluminium hydroxide, 3% suspension in water) and Adju-fos (aluminium phosphate, 2% suspension in saline) supplied by Superfos (Vedbaek, 2950 Denmark).

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It will be appreciated that for the first time we are able to provide a stable and effective multivalent vaccine composition comprising HBsAg.

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- 4 Accordingly, in a further aspect of the invention there is provided a stable and effective combined vaccine composition directed to the prevention of more than two diseases, comprising HBsAg and at least two other antigens.

Excellent results are obtained when the HBsAg is adsorbed on AP and at least one of the antigens other than HBsAg is adsorbed to AH.

Preferred combination vaccines according to the invention are

Diphtheria-Tetanus- Pertussis-Hepatitis B (DTP-HB)

Diphtheria-Tetanus-Hepatitis B (DT-HB)

DTP - IPV (inactivated polio vaccine) - Hepatitis B Hib-Hepatitis B

DTP-Hib-Hepatitis B

IPV - DTP-Hib-Hepatitis B.

It will be appreciated that for a vaccine containing a Hib component the Hib antigen may be used extemporaneously by formulating the vaccine just prior to administration.

More specifically particular vaccines within the scope of the invention are:

CDiphtheria - Tetanus - Pertussis (DTP adsorbed on AH or AP) - Hepatitis B (HBsAg adsorbed on AP) ©

Diphtheria - Tetanus (DT adsorbed on AP or AH) - Hepatitis B (HBsAg adsorbed on AP).

By 'stable' as used herein to describe a vaccine according to the invention is meant a vaccine which can be kept a 37°C for one week without any substantial loss of immunogenicity of the HBsAg component.

By 'effective' as used herein is meant a vaccine composition, characterised in that the immunogenicity of the HBsAg in the combined vaccine is such that a geometric mean titre of at least 200 mlU/ml, preferably 300 mlU/ml or greater, is found in human infants after the third dose of the vaccine when given at one month intervals in an appropriate vaccination schedule.

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In a further aspect the invention provides a multivalent vaccine composition comprising HBsAg and a stabilising adjuvant selected such that the vaccine can be kept at 37° C for one week without any substantial loss in immunogenicity of the

HBsAg component. Preferably the multivalent vaccine composition is further characterised by giving rise to a geometric mean titre of at least 200 mlU/ml (one month post third dose), preferably 300 mlU/ml or greater, in human infants when a course of the vaccine is given at one month intervals in an appropriate vaccination schedule.

s-*.. As used herein the term 'appropriate vaccination schedule' means a schedule known to those of skill in the art for administering a course of doses of a vaccine, especially for ( paediatric doses. A schedule of 3,4 and 5 months may for example be used. This is particularly appropriate for example for DTP - HBsAg containing vaccines according to the invention.

In one aspect the HBsAg can be adsorbed to an aluminium salt other than aluminium hydroxide. Preferably it is adsorbed to AP. The other antigens in the multivalent vaccine formulation are advantageously adsorbed to AH as shown in the examples hereinbelow.

Advantageously the vaccine formulation according to the invention comprises a pertussis vaccine, c

The pertussis component is suitably a whole cell pertussis vaccine or an acellular pertussis vaccine containing partially or highly purified antigens.

The above combinations may optionally include a component which is protective against Hepatitis A, i.e. an HAV antigen.

Advantageously the Hepatitis B combination vaccine is a paediatric vaccine.

The preparation of the antigens and adsorption procedure with the adjuvants are well known in the art, see for example as given below.

The preparation of Hepatitis B surface antigen (HBsAg) is well documented. See for example, Harford et al.(1983) in Develop, Biol, Standard 54, page 125 , Gregg et al.

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- 6(1987) in Biotechnology $, page 479, 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 5 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. In particular the HBsAg may comprise a polypeptide comprising an amino acid sequence comprising residues 12-52 followed by residues 133-145 followed by residues 175-400 of the L-protein of HBsAg relative to the open reading frame on a Hepatitis B virus of ad serotype (this f polypeptide is referred to as L*; see EP 0 414 374). HBsAg within the scope of the invention may also include the preSl-preS2 -S polypeptide described in EP 0 198 474 (Endotronics) or analogues thereof such as those described in EP 0 304 578 (Me Cormick and Jones). HBsAg as herein described can also refer to mutants, for example the 'escape mutant' described in WO 91/14703 or European Patent Application Publication Number 0 511 855 Al, especially HBsAg wherein the amino acid substitution at position 145 is to arginine from glycine.

Normally the HBsAg will be in particle form. The particles may comprise for example S protein alone or may be composite particles, for example (L*,S) where L* is as defined above and S denotes the S-protein of HBsAg. The said particle is (C advantageously in the form in which it is expressed in yeast.

O Suitable antigens for use in vaccines according to the invention are already commercially available and details may be obtained from the World Health Organisation. For example the IPV component may be the Salk inactivated polio vaccine. The pertussis vaccine may comprise a whole cell product, an acellular product or a recombinantly produced product. In particular the pertussis component can be PT (pertussis toxins) or subfractions thereof, FHA (filamentous haemagglutinin antigen), agglutinogeous (fimbrial) and outer membrane proteins, including the 69kDa protein (pertactin, non fimbrial agglutinogen). References'. Robinson, A., Irons, L. I. & Ashworth, A. E., Vaccines, 1, 1985, 11-22; and

Brennan, H. J., Li, S. M., Cowell, J. L., Bishen, Μ. E., Steven, A. C. Novotny., P,

Manclarck, C. R., Infection and Immunity, 56. 1988, 3189-3195.

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-7The component affording protection against Hepatitis A is preferably the product known as 'Havrix' (SmithKline Beecham Biologicals) which is a killed attenuated vaccine derived from the HM-175 strain of HAV [see 'Inactivated Candidate Vaccines for Hepatitis A' by F.E. Andre, A. Hepburn and E.D'Hondt (1980), Prog.

Med, Virol, Vol 37. pages 72-95 and the product monograph 'Havrix' published by SmithKline Beecham Biologicals (1991).

Flehmig et al (loc cit., pages 56-71) have reviewed the clinical aspects, virology, immunology and epidemiology of Hepatitis A and discussed approaches to the development of vaccines against this common viral infection.

As used herein the expression 'HAV antigen' refers to any antigen capable of stimulating neutralising antibody to HAV in humans. The HAV antigen preferably comprises inactivated attenuated virus particles or may be, for example an HAV capsid or HAV viral protein, which may conveniently be obtained by recombinant DNA technology.

Vaccine preparation is generally described in New Trends and Developments in Vaccines(1978), edited by Voller et al., University Park Press, Baltimore, Maryland

U.S.A.

The amount of antigen in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical

O vaccinees. Such amount will vary depending on which specific immunogens are employed. Generally it is expected that each dose will comprise 1-1000 gg of total immunogen, preferably 2-100 gg, more preferably 1-40 gg, most preferably 1-5 gg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. A primary vaccination course may include 2 or 3 doses of vaccine, given one to two months apart, following the WHO recommendations for DTP immunization.

In a further aspect of the invention there is provided a vaccine composition according to the invention for use in medicine.

In a further aspect of the invention there is provided the use of HBsAg for the manufacture of a combination vaccine according to the invention for the prophylaxis of Hepatitis B viral infections.

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-8In a further aspect the invention provides the use of AP for the purpose of acting as a stabiliser for, and/or to maintain the efficacy of, HBsAg in a multivalent vaccine according to the invention.

Specifically the invention provides the use of aluminium phosphate for the purpose of preparing a stable combined vaccine comprising HBsAg and at least one other antigen whereby the stability and/or immunogenicity of the HBsAg component is greater than in the combined vaccine in which the HBsAg component is adsorbed on AH.

More specifically the invention provides the use whereby the vaccine can be kept at 37° C for 1 week without substantial loss of immunogenicity of the HBsAg. .

Also provided is the use whereby the geometric meant titre found one month after the third dose of a course of vaccinations given at one month intervals in an appropriate vaccination schedule to human infants is greater than 200, preferably greater than 300, mlU/ml.

In a further aspect of the present invention there is provided a method of manufacture of a combination vaccine effective in preventing hepatitis B infection, wherein the method comprises mixing AP- adsorbed HBsAg as defined herein with one or more other antigens, at least one of which is adsorbed on AP or AH.

In one preferred aspect the antigens other than HBsAg are all adsorbed on AH. A O very effective DTPa - Hepatitis B vaccine can, for example, be made in this way.

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The combined vaccines can be prepared as follows. The different DT, DTPw, DTPa or HA components are adsorbed onto AH or AP; HBsAg is adsorbed onto AP. After allowing time for complete and stable adsorption of the respective components, the different components are combined. The DT, DTPw and DTPa components can be combined separately before adding the adsorbed HBsAg components. Multivalent vaccines comprising other or additional antigens may be prepared in a similar manner. Accordingly there is provided a method of preparing a combination vaccine composition according to the invention wherein the method comprises mixing aluminium phosphate - adsorbed HBsAg with one or more aluminium hydroxide or aluminium phosphate adsorbed antigens.

The following examples illustrate the invention.

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-9EXAMPLES 1-5

Formulations

Particular formulations according to the present invention were prepared as described below.

Example 1 HBsAg adsorption on AIPO4 as concentrate for formulation of 10 combined vaccines.

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A suspension of aluminium phosphate containing 0.03 to 0.3 g aluminium (as ' f⁵' aluminium phosphate) in isotonic saline, is mixed with a HBsAg concentrate, containing 10 mg HBsAg protein, in a final volume of 10 to 100 ml. After adjusting 15 the pH to 5 - 6.5 the mixture is left 10 - 24 hrs at room temperature with stirring.

Antiseptic is then optionally added (i.e. merthiolate, 1 : 20,000 to 1 : 10,000 or 2) phenoxyethanol, 3 to 6 mg/ml) and the volume is brought to 50 ml with isotonic saline.

! 20 Example 2 Formulation of combined Diphteria-Tetanus-Hepatitis B vaccine.

A concentrate containing 25,000 Lf of diphtheria toxoid and 10,000 Lf of tetanus toxoid adsorbed to 0.35 g Al (as aluminium hydroxide or aluminium phosphate) is

C prepared in a final volume of 0.15 1 of isotonic saline and adjusted to between pH 6 and 7, as specified by WHO for DT and DTP vaccines. This concentrate is combined with 0.05 1 of the Hepatitis B concentrate of example 1.

This mixture is brought to a final volume of 0.5 1 with isotonic saline. Antiseptic media (c.c. merthiolate 1 : 20,000 to 0 : 10,000 or 2-phenoxyethanol, 3 to 6 mg/ml) can be optionally added. The final pH is between 6 and 7, as specified by WHO for DT and DTP vaccines.

One 0.5 ml dose of this bulk vaccine contains, as active ingredients:

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- 10D toxoid: 25Lf,

T toxoid: 10 Lf,

HBsAg: 10 pg protein

The procedure can be optionally amended to use higher or lower quantities of the active ingredients.

Example 3 Formulation of combined Diphtheria - Tetanus - pertussis (whole cell vaccine) - Hepatitis B vaccine

A concentrate ex Behringwerke containing 7,500 Lf of diphtheria toxoid, 3,250 Lf of Tetanus toxoid and 15,000 capacity units of B. pertussis antigen adsorbed to 0.45 mg Al (as aluminium hydroxide and aluminium phosphate) is prepared in a final volume of 0.4 1 of isotonic saline and adjusted to pH 6 - 7, as specified by WHO for DTP vaccines. This concentrate is combined with 0.05 I of Hepatitis B concentrate of example 1.

One 0.5 ml dose of this bulk vaccine contains, as active ingredients:

D toxoid: 7.5Lf,

T toxoid: 3.25 Lf

Pw antigen: 15OU

HBsAg: 10 pg protein.

Example 4 Formulation of Diphtheria-Tetanus-Pertussis (acellular component) vaccine.

A concentrate containing 25,000 Lf of diphtheria toxoid and 10,000 Lf of tetanus toxoid adsorbed to 0.35 g Al (as aluminium hydroxide or phosphate gel) is prepared bad

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- 11 in a final volume of 0.15 1 of isotonic saline and adjusted to between pH 6 and 7, as specified by WHO for DTP vaccines. 25 mg of inactivated pertussis toxin (DTPa), 25 mg of filamentous hemagglutinin (FHA) and optionally 8 mg of 69kDa outer membrane protein (pertactin), each combined with 0.05 g Al (as aluminium hydroxide or aluminium phosphate) are added. The B. pertussis antigens PT, FHA and pertactin can be prepared as described by methods known in the art, for example European patent application 427 462, PCT application WO 91/12020 or by other procedures giving physiologically acceptable and potent B. pertussis antigens.

This mixture is brought to a final volume of 0.5 1 with isotonic saline. Antiseptic media (c.c. merthiolate 1 : 20,000 to 0:10,000 or 2-phenoxyethanol, 3 to 6 mg/ml) can be optionally added. The final pH is between 6 and 7, as specified by WHO for DT and DTP vaccines.

One 0.5 ml dose of this bulk vaccine contains, as active ingredients:

D toxoid:

T toxoid: DTd toxoid: FHA toxoid: 69kDa OMP:

Lf,

Lf, gg, gg, gg (optional)

Example 5 Formulation of combined Diphtheria - Tetanus - Pertussis (acellular component) - Hepatitis B vaccine

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The procedure of example 4 is applied, with the exception that an additional 50 ml of 25 HBsAg adsorbed concentrate as prepared in example. 1 is added to the final mixture.

The resulting mixture is brought to a final volume of 0.5 1 with isotonic saline. Antiseptic media (c.c. merthiolate 1 : 20,000 to 0: 10,000 or 2-phenoxyethanol, 3 to 6 mg/ml) can be optionally added. The final pH is between 6 and 7, as specified by

WHO for DT and DTP vaccines.

One 0.5 ml dose of this bulk vaccine contains, as active ingredients:

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	- 12-
D toxoid:	25 Lf,
T toxoid:	10 Lf
PTd toxoid:	25 gg,
FHA toxoid:	25 gg.
69kDaOMP:	8 gg (optional)

EXAMPLES 6-10

Animal and Human Studies f

Example. 6 Formulation of combined Hepatitis A - Hepatitis B vaccines

An inactivated Hepatitis A virus concentrate (460,000 Elisa units), adsorbed to 0.02 to 0.2 g, preferably 0.04 - 0.1 g aluminium (as aluminium hydroxide) in a final , volume of about 125ml was combined to 50 ml of concentrate containing 10 mg

HBsAg adsorbed to aluminium phosphate as described in example 1.

The resulting mixture was supplemented with isotonic saline and an amino acid concentrate (Travasol, Baxter-Travenol Inc) to obtain a final volume of 0.5 1 containing 1.5 g amino acids. The resulting pH was between 6 and 7.

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Our 1 ml dose of this bulk vaccine contains, as active ingredients:

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Hepatitis A virus antigen: 800 Elisa units

HBsAg: 20 gg protein

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Results:

AP.00567

- 13Clinical studies comparing aluminium hydroxide (AH) and aluminium phosphate (AP) adsorbed HBsAg (Monovalent vaccine)

Initially seronegative healthy adult volunteers were immunised with 3 doses of 20 gg HBsAg protein given at one month interval. Antibody levels were determined in sera obtained one month post 2 and 3 doses using the Ausab (Abbott) test. Responses were defined as subjects with titres significantly above background. Titres were expressed in mlU/ml.

Results are expressed as Geometric Mean Titres (GMT) in mlU/ml.

HBsAg Lot	Adjuvant	N.Subj.	Post 2, month 2	Post	3, month 3
GMT	% responders	GMT	% responders
100	AH	43	32	86	141	100
101	AH	45	26	93	198	98
102	AH	46	30	84	147	93
105/P	AP	7	43	83	380	100

HBsAg Lot	Adjuvant	N.Subj.	Post 2, month 2	Post	3, month 3
GMT	% responders	GMT	% responders
102	AH	51	14	82	126	98
103	AH	50	15	83	110	98
102	AH	54	17	83	133	96
104/P	AP	54	18	96	270	98
105/P	AP	51	14	90	156	96

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- 14Example 7

Mouse immunogenicity tests and results of accelerated stability tests for combination vaccines comprising HBsAg with aluminium hydroxide (AH) or aluminium phosphate (AP) as adjuvant

Groups of 10 OF1 mice were immunised subcutaneously with 2 doses of 2.5 pg HBsAg (single component or combined) at days 0 and 14. Blood was drawn off at day 21 and titrated for anti-HBsAg using the Ausab (Abbott) test. Antibody titres 5} were calculated in mlU/ml. The number of responding animals was defined as the number of those with antibody levels significantly above background values. The geometric mean titres was also calculated (GMT).

The results of DT-HB, DTPw-HB, DTPa-HB show that AP adsorbed HBsAg performed better than AH adsorbed HBsAg both in terms of number of responding animals and GMTs. The response to AP adsorbed HBsAg in the combination was comparable to that obtained by monovalent HBsAg administration.

Vaccine	4°C	7 days, 37°C	7 days, 45°C
N.resp.	GMT	N.resp.	GMT	N.resp	GMT
Engerix B(HB+AH)	7/10	30	9/10	17	6/10	2.7
Engerix B(HB+AP)	9/10	54	8/10	13	5/10	6
HB(AH)	9/10	45	10/10	55	9/10	32
HB (AP)	9/10	54	10/10	50	7/10	6.9
DTPw(AH)HB(AH)	4/10	1.4	nd	nd	nd	nd
DTPw(AH)HB(AP)	9/10	52	8/10	16	8/10	26
DT(AH)HB(AH)	6/10	1.7	nd	nd	nd	nd
DT(AH)HB(AP)	8/10	44	9/10	21	10/10	36
DTPa(AH)HB(AH)	5/10	1.7	nd	nd	nd	nd
DTPa(AH)HB(AP)	10/10	18	8/10	8	9/10	24

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AP.00567 • 15Example 8

Immunogenicity of HBsAg combined to DTPw in monkeys

Results of aluminium hydroxide (AH) and aluminium phosphate (AP) adsorbed antigen

Cercopithcnus aethiops monkeys received two injections of 10 pg HBsAg (alone or combined) at days 0 and 30. Sera were withdrawn at days 30 and 57 and titrated (Ausab, Abbott) for anti-HbsAg. Animals with antibody levels significantly above background (pre-vaccination sera) were considered responders. GMT were calculated in mlU/ml.

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Results show AP adsorbed HBsAg performed better than AH adsorbed HBsAg.The response was comparable to that obtained by monovalent HBsAg administration.

Vaccine	Post 1, day 30	Post 2, day 57
N. resp.	GMT	N. resp.	GMT
Engerix B (MB)(AH)	4/5	10	5/5	666
DTPw(AH)HB(AH)	4/5	20	5/5	31
DTPw(AH)HB(AP)	5/5	12	5/5	414

Example 9

Clinical studies with combined DTPw vaccines using HBsAg adsorbed to aluminium hydroxide (AH) or aluminium phosphate (AP)

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Subjects were immunised with 3 doses of 0.5 ml containing DTPw and 10pg HBsAg protein given at the age of 3,4 and 5 months. Bleeding was at 6 months and sera were titrated with the Ausab test. Percentage responders (seroconversion) relates to subjects with antibody levels significantly above background. Percentage protection relates to subjects with titres equal to or greater than 10 mru/ml. GMT in mlU/ml.

Results for DTPw-ΗΒ show AP adsorbed HBsAg produced a satisfactory response as opposed to AH adsorbed HBsAg. Seroconversion rates and GMT were comparable to data typically seen with monovalent HBsAg vaccine (Engerix B).

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Vaccine	N. subj.	Bleeding Time	% resp.	% prot.	GMT
DTPw(AH).HB(AH)	32	post 2	nd	nd	nd
		post 3	94	84	38.5
DTPw(AH)HB(AP)	29	post 2*	97	97	63
	17	post 3*	100	100	469

Example 10

Immunogenicity and stability of HBsAg adsorbed to aluminium hydroxide (AH) or aluminium phosphate (AP) in a hepatitis A-Hepatitis B combined vaccine

Groups of 10 OF1 mice were immunised subcutaneously with 2 doses of 2.5 gg 10 HBsAg (single component or combined) at days 0 and 14. Blood was drawn at day 21 and titrated for anti-HBsAg as in Example 7.

Results for immunogenicity and stability of HA-HB combined product showed AP adsorbed HBsAg produced higher antibody levels and a more stable form.

Vaccine	Exposure	N. resp.	GMT
HA(AH).HB(AH)	4OC	9/10	41
	1 month, 37°C	6/10	5.6
	1 month, 45°C	5/10	6.4
HA(AH).HB(AP)	4°C	10/10	80
	1 month, 37°C	9/10	45
	1 month, 45°C	8/10	18
Engerix B HB(AH)	4°C	8/10	58

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- 17Example 11: Further Clinical Results in humans

1. Immunogenicity of DTPw - Hepatitis B vaccines in infants

Experiment A

Studies in Slovakia: Schedule: 3-4-5 months. lOgg HBsAg; DTPw ex Behringwerke (DT on AH; Pw on a mixture of AH and AP)

Anti-Hbs titres

HBsAg adjuvant	Time	N	GMT	SP(%)
Hydroxide	Post II (5 months)	44	45	79.5
Hydroxide	Post III (6 months)	13	34	69.2
Phosphate	Post II (5 months)	33	80	97.0
Phosphate	Post III (6 months)	32	396	100

In this and other examples Post II means after the second dose, post III after the third dose. GMT is always measured one month after the injection time shown in the schedule.

Anti-Diphtheria, Tetanus, B pertussis titres

Post III results	N	GMT	%>0.1 IU/ml	GMT Post/Pre
Anti-Diphtheria Anti-Tetanus Anti-B pertussis	38 38 38	2.302 3.281 61	100 100	37.4 38.4 7.7

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- 18 Experiment Β

Study in Greece: Schedule 2-4-6 months (same vaccine as for Experiment A) 5

Anti-HBs titres (interim results)

HBsAg adjuvant	Time	N	GMT	SP(%)
Hydroxide	Month 7	22	284	90.5
Hydroxide	Month 7	17	193	94.4
Phosphate	Month 7	23	1794	92.0

Experiment C

Study in Slovakia: Schedule 3-4-5 (HBsAg =5pg on aluminium phosphate; DTP ex Behringwerke as for Experiment A)

Anti-HBs titres

Time	N	GMT	SP(%)
Post II	21	94	90.5
Post III	18	311	100

Experiment D c

Study in Slovakia: Schedule 3-4-5 months of age (HBsAg =10gg on aluminium 20 phosphate; DTPw ex Behringwerke as for Experiment A)

Anti-HBs titres

Time	N	GMT	SP(%)
Pre	24	0	0
Post II (month 5)	13	259	92.3
Post III (month 6)	10	592	100.0

SAD

ORIGINAL $ j .......

ΑΡ.00567

- 19Anti-diphtheria antibodies

Timing	N	GMT	SP	GMT Post/Pre
Pre	32	0.054	6.3	1.0
Post II	16	1.094	93.8	20.4
Post III	11	2.314	100.0	43.1

Anti-tetanus antibodies

Timing	N	GMT	SP	GMT Post/Pre
Pre	32	0.083	34.4	1.0
Post II	16	3.146	100.0	37.9
Post III	11	7.989	100.0	96.4

Anti-B pertussis antibodies

Timing	N	GMT	GMT Post/Pre
Pre	32	8	1.0
Post II	16	20	2.7
Post III	11	50	6.6

AP/P/ 93/00530

BAD ORIGINAL jh

B45027

-202. Immunogenicity of DTPa - Hepatitis B vaccines in infants

Experiment A

Study in Turkey. HBsAg 10pg on AP; DTP (acellular) on AH. Preliminary results ιο

ΓGroup 1 (DTPa - Engerix B combination)

Timing	N	S+	%	GMT
Pre Post I Post II Post III	19 19 19 19	0 4 18 19	0 21.1 94.7 100.0	0 24 146 345

Group 2 (DTPa plus Engerix B; separate simultaneous injections)

Timing	N	S+	%	GMT
Pre Post I Post II Post III	8 8 8 7	0 2 5 6	0 25.0 62.5 83.7	0 37 33 385

Key: N = number of subjects tested; S+ = number of subjects sero positive at a given blood sampling time; % = seroconversion rate and GMT = geometric mean antibody titre of se reconvene rs

Claims

1. A vaccine composition comprising Hepatitis B surface antigen (HBsAg) and a number (n) of other antigens in combination with an adjuvant comprising one or more aluminium salts in which the value of n is 1 or greater and in which the adjuvant used to adsorb the HBsAg is aluminium phosphate (AP). with the proviso that when n is I the other antigen is not an antigen against Hepatitis A.

2. A vaccine composition as claimed in Claim 1 in which the said other antigens are selected to provide immunity against one or more of the following pathogens: diphtheria, tetanus, pertussis, polio, Haemophilus influenzae b, Hepatitis A, meningitis A, meningitis B, meningitis C, and otitis media.

3. A vaccine composition as claimed in Claim 1 or Claim 2 in which at least one of the said other antigens is adsorbed to AP.

4. A vaccine composition as claimed in Claim 1 or Claim 2 in which at at least one of the said other antigens is adsorbed to aluminium hydroxide (AH).

5. A vaccine composition as claimed in any preceding claim in which n is 2, 3, 4, 5 or

6.

6. A vaccine composition according to claim 5 wherein the said other antigens provide immunity against diphtheria (D), tetanus (T) and pertussis (P).

7. A vaccine composition as claimed in claim 6 wherein the pertussis component is whole cell pertussis (Pw) or acellular pertussis antigen (Pa).

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I

8. A vaccine composition as claimed in Claim 6 or Claim 7 additionally comprising an antigen against Haemophilus influenzae b (Hib).

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9. A vaccine composition as claimed in any one of Claims 6 to 8 additionally comprising inactivated polio (IPV). f

10. A vaccine composition as claimed in any one of claims 6 to 9 additionally comprising an antigen against Hepatitis A.

11. A vaccine composition according to any previous claim in which the stability of the vaccine is such that the vaccine can be kept at 37°C for one week without substantial loss of immunogenicity of the HBsAg component.

12. A vaccine composition according to any previous claim characterised in that the immunogenicity of the HBsAg in the vaccine is such that a geometric mean titre of 200 mlU/ml (one month post third dose) or greater is found in human infants when a course of the vaccine is given at one month intervals in an appropriate vaccination schedule.

0 2 5 0 0 /26 Zti/dV

BAD ORIGINAL

13. A composition as claimed in any one of Claims 1 to 12 for use in medicine.

14. Use of HBsAg in the manufacture of a vaccine composition according to any one of claims 1 to 12 for the prophylaxis of hepatitis B viral infections.

15. A method of preparing a vaccine composition as claimed in any one of claims 1 to 12 wherein the method comprises mixing AP-adsorbed HBsAg with one or more AH- or AP-adsorbed other antigens.

16. Use of aluminium phosphate (AP) as an adjuvant for adsorbing HBsAg, characterised in that the use is for the purpose of formulating a stable and effective combined vaccine comprising HBsAg and a number (n) of other antigens adsorbed on aluminium phosphate or aluminium hydroxide, wherein the value of n is 1 or greater, and whereby the stability and/or immunogenicity of the HBsAg component is greater than in a corresponding combined vaccine in which the HBsAg component is adsorbed on AH, with the proviso that when n is 1 the said other antigen is not an antigen against Hepatitis A.

17. Use according to claim 16 in which the stability of the vaccine is such that the vaccine can be kept at 37°C for 1 week without substantial loss of immunogenicity of the HBsAg.

18. Use according to claim 16 or claim 17 in which the immunogenicity in the combined vaccine is such that the geometric mean titre of 200 mlU/ml (one month post third dose) or greater is found in human infants when a course of the vaccine is given at one month intervals in an appropriate vaccination schedule.

19. Use according to any one of claims 16 to 18 in which there are at least 2 other antigens in the combined vaccine.

20. Use according to claim 19 In which the vaccine is a DTP - Hepatitis B vaccine.

21. Use according to claim 19 in which the vaccine is a DTP-Hepatitis B-Hib vaccine. _ t

22. Use according to claim 19 in which the vaccine is a DTP-Hepatitis B - Hib - IPV vaccine.

23. Use according to claim 19 in which the vaccine is a DTP-Hepatitis B - Hib - IPV - Hepatitis A vaccine.