Link:  Pharm/Biotech Resources

Title:  Hepatitis vaccines containing 3-O-deacylated monophoshoryl lipid A

United States Patent:  6,893,644

Issued:  May 17, 2005

Inventors:  Garcon-Johnson; Nathalie Marie-Josephe Claude (Wavre, BE); Hauser; Pierre (Chaumont-Gistoux, BE); Thiriart; Clothilde (Brussels, BE); Voet; Pierre (Izel, BE)

Assignee:  SmithKline Beecham Biologicals s.a. (Rixensart, BE)

Appl. No.:  465763

Filed:  June 19, 2003

A vaccine formulation for the treatment or prophylaxis of hepatitis, especially hepatitis, especially hepatitis B, infections is provided comprising the hepatitis antigen and a suitable carrier such as alum in combination with 3-O-deacylated monophosphoryl lipid. A combination vaccines including the vaccine formulation are also described.

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 formulations for treating Hepatitis infections and to combination vaccine formulations including a Hepatitis vaccine component.

Viral hepatitis, caused by the A, B, C, D, and E hepatitis viruses, is a very common viral illness. Via the B and C viruses, in particular, it is also responsible for many cases of liver cancer. Thus the development of effective vaccines is critical and, despite notable successes, is still an on-going task. A review on modern hepatitis vaccines, including a number of key references, may be found in the Lancet, May 12th 1990 at page 1142 ff (Prof A. L. W. F. Eddleston). See also ‘Viral Hepatitis and Liver Disease’ (Vyas, B. N., Dienstag, J. L., and Hoofnagle, J. L., eds, Grune and Stratton, Inc. (1984) and ‘Viral Hepatitis and Liver Disease’ (Proceedings of the 1990 International Symposium, eds F. B. Hollinger, S. M. Lemon and H. Margolis, published by Williams and Wilkins).

As used herein the expression ‘hepatitis antigen’ is used to refer to any antigenic material derived from a hepatitis virus which may be used to induce immunity to the virus in humans. The hepatitis antigen may be, for example, a polypeptide obtained by recombinant DNA techniques or an attenuated strain of hepatitis virus which has optionally been inactivated by known methods. The invention extends to all hepatitis antigens, whether A, B, C, D, or E, examples of which are discussed below.

Infection with hepatitis A virus (HAV) is a widespread problem but vaccines which can be used for mass immunisation are available, for example the product ‘Havrix’ (SmithKline Beecham Biologicals) which is a killed attenuated vaccine obtained from the HM-175 strain of HAV [see ‘Inactivated Candidate Vaccines for Hepatitis A’ by F. E. Andre, A Hepburn and E. D'Hondt, Prog Med. Virol. Vol 37, pages 72-95 (1990) 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 may comprise live attenuated virus particles or 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.

Infection with hepatitis B virus (HBV) is a widespread problem but vaccines which can be used for mass immunisation are now available, for example the product ‘Engerix-B’ (SmithKline Beecham plc) which is obtained by genetic engineering techniques.

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. 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 polypeptide is referred to as L*; see EP 0 414 374). HBsAg within the scope of the invention may also include the preS1-preS2-S polypeptide described in EP 0 198 474 (Endotronics) or analogues thereof such as those described in EP 0 304 578 (Mc Cormick and Jones). HBsAg as herein described can also refer to mutants, for example the ‘escape mutant’ described in WO 91/114703 or European Patent Application Publication Number 0 511 855 A1, 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 advantageously in the form in which it is expressed in yeast.

Hepatitis C virus (HCV) is specifically discussed in GB 2 212 511B and references therein. It has been reported that vaccines may be prepared from one or more immunogenic polypeptides derived from HCV c DNA.

Hepatitis D virus is discussed in ‘Viral Hepatitis and Liver Disease’ (1990 Symposium (loc. cit.).

Hepatitis E virus (HEV) is specifically discussed in WO 89/12462 and references therein. An example of a vaccine composition includes, in a pharmacologically acceptable adjuvant, a recombinant protein or protein mixture derived from HEV.

Whilst experimental and commercially available Hepatitis vaccines, for example Havrix and Engerix-B, afford excellent results it is an accepted fact that an optimal vaccine needs to stimulate not only neutralising antibody but also needs to stimulate as effectively as possible cellular immunity mediated through T-cells. There also exists a need for combination vaccines containing a Hepatitis component to stimulate cellular immunity in this way. The present invention achieves these aims.

The present invention provides a vaccine comprising a hepatitis antigen in conjunction with 3-O-deacylated monophosphoryl lipid A (abbreviated herein to MPL) and a suitable carrier.

3-O-deacylated monophosphoryl lipid A (or 3 De-O-acylated monophosphoryl lipid A) has formerly been termed 3D-MPL or d3-MPL to indicate that position 3 of the reducing end glucosamine is de-O-acylated. For preparation, see GB 2 220 211 A Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. Herein the term 3D-MPL (or d3-MPL) is abbreviated to MPL since ‘MPL’ is a Registered Trademark of Ribi Immunochem.,Montana which is used by Ribi to denote unambiguously their 3-O-deacylated monophosphoryl lipid A product.

GB 2 220 211A mentions that the endotoxicity of the previously used enterobacterial lipopolysacharides (LPS) is reduced while the immunogenic properties are conserved. However GB 2 220 211 cited these findings merely in connection with bacterial (Gram negative) systems. At the priority date of the present invention the suitability of 3-Deacylated monophosphoryl lipid A as an adjuvant for a vaccine containing a hepatitis viral antigen had not been suggested.

Surprisingly, however, it has been found that vaccine compositions according to the invention comprising hepatitis viral antigens have particularly advantageous properties as described herein.

A particular advantage is that the vaccine formulations of the invention are very effective in inducing protective immunity, even with very low doses of antigen.

They provide excellent protection against primary infection and stimulate advantageously both specific humoral (neutralising antibodies) and also effector cell mediated (DTH) immune responses.

A further important advantage is that vaccine compositions according to the invention may also be used as therapeutic vaccines.

The MPL as defined above will normally be present in the range of 10-100 ug, preferably 25-50 ug per dose wherein the Hepatitis antigen will be typically present in a range 2-50 ug per dose.

The carrier may be an oil in water emulsion, a lipid vehicle, or alum (aluminium salt).

Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalene and an emulsifier such as Tween 80, in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline.

One embodiment of the invention is HAV antigen (for example as in Havrix) in admixture with MPL and aluminium hydroxide as described hereinbelow.

A further embodiment of the invention is HBsAg S antigen (for example as in Engerix-B) in admixture with MPL and aluminium hydroxide as described hereinbelow.

A further specific embodiment of the invention is HBsAg antigen as (L*,S) particles, defined hereinabove, in admixture with MPL and aluminium hydroxide.

In the above embodiments an oil in water emulsion may be used instead of alum.

Other embodiments arm given in the examples hereinbelow.

The invention in a further aspect provides a vaccine formulation as described herein for use in medical therapy, particularly for use in the treatment or prophylaxis of hepatitis viral infections. In a preferred aspect the vaccine according to the invention is a therapeutic vaccine useful for the treatment of ongoing hepatitis infections, more especially hepatitis B and/or hepatitis C infections in humans suffering therefrom.

In view of the surprisingly efficaceous results obtained, in a further preferred aspect the invention provides a vaccine composition for the treatment or prophylaxis of Hepatitis A and/or Hepatitis B infections.

Advantagously the hepatitis vaccine composition of the invention contains other antigens so that it is effective in the treatment or prophylaxis of one or more other bacterial, viral or fungal infections.

Accordingly the hepatitis vaccine formulation according to the invention preferably contains at least one other component selected from non-hepatitis antigens which are known in the art to afford protection against one or more of the following: diphtheria, tetanus, pertussis, Haemophilus influenzae b (Hib), and polio.

Preferably the vaccine according to the invention includes HBsAg as hereinabove defined.

Particular combination vaccines within the scope of the invention include a DTP (diphtheria-tetanus-pertussis)-hepatitis B combination vaccine formulation, an Hib-Hepatitis B vaccine formulation, a DYP-Hib-Hepatitis B vaccine formulation and an IPV (inactivated polio vaccine)-DTP-Hib-Hepatitis B vaccine formulation.

The above combinations may advantageously include a component which is protective against Hepatitis A, especially the killed attenuated strain derived from the HM-175 strain as is present in Havrix.

Suitable components for use in such vaccines 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 whole cell or acellular product.

Advantageously the hepatitis or combination vaccine according to the invention is a paediatric vaccine.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md. U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Pat. No. 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No. 4,474,757.

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 vaccinees. Such amount will vary depending on which specific immunogens are employed. Generally it is expected that each dose will comprise 1-1000 ug of total immunogen, preferably 2-100 ug, most preferably 4-40 ug. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects may receive a boost in about 4 weeks.

In a further aspect of the present invention there is provided a method of manufacture of a vaccine effective in preventing or treating hepatitis infection, wherein the method comprises mixing the hepatitis antigen as defined herein with a carrier and MPL.

Using this method one or more additional components are preferably admixed with HBsAg to provide a combination vaccine, advantageously for paediatric use.

The following examples illustrate the invention and its advantages.

EXAMPLE 1

Hepatitis B Vaccine Formulation

MPL was obtained from Ribi Immunochem Research Inc. Aluminium hydroxide was obtained from Superfos (Alhydrogel).

MPL was resuspended in water for injection at a concentration varying from 0.2 to 1 mg/ml by sonication in a water bath until the particles reach a size of between 80 and 500 nm as measured by photo correlation light scattering.

1 to 20 ug of HBsAg (S-antigen as in Engerix B) in phosphate buffer solution at 1 mg/ml) is adsorbed on 30 to 100 ug of aluminium hydroxide (solution at 10.38 Al³⁺ mg/ml) for one hour at room temperature under agitation. To the solution is then added 30 to 50 ug of DMPL (solution 1 mg/ml). Volume and osmolarity are adjusted to 600 ul with water for injection and phosphate buffer 5× concentrated. Solution is incubated at room temperature for 1 hour and kept at 4° C. until use. Maturation of the formulation occurs during storage. This represents 10 injecting doses for testing in mice.

A similar formulation may be prepared by using as the HBsAg component the composite (L*,S) antigen as defined hereinabove.

EXAMPLE 2

Hepatitis A Vaccine Formulation

MPL was obtained from Ribi Immunochem Research Inc. Aluminium hydroxide was obtained from Superfos (Alhydrogel).

HAV (360 to 22 EU per dose) is preadsorbed on 10% of the aluminium hydroxide final concentration (0.5 mg/ml). MPL (12.5 to 100 ug per dose) is added to the solution.

The remaining aluminium hydroxide is added to the solution and left for one hour at room temperature. Volumes are adjusted with phosphate buffer (phosphate 10 mM, NaCl 15 mM) and the final formulation is then stored at 4° C. until use.

EXAMPLE 3

Combination Vaccine Formulation—Hepatitis B+Hepatitis A

HBsAg is adsorbed on 90% of the final amount of aluminium hydroxide (0.5 mg/ml) and incubated overnight at room temperature. The pH is adjusted to 6.2 and the preparation is left 14 days at room temperature for maturation.

Hepatitis A antigen at 360 to 22 EU per dose, in the form of an inactivated derivative of the HM-175 strain (as in Havrix) is preadsorbed on 10% of the aluminium hydroxide final concentration (0.5 mg/ml). The remaining aluminium hydroxide is then added to the solution and left for one hour at room temperature under agitation.

The HAV adsorbed on aluminium hydroxide is then added to the HBsAg formulation.

MPL is added to the HAV/HBsAg solution at a final concentration of 12.5 to 100 ug per 1 ml dose, the volume is adjusted to the final dose volume, and the formulation is stored at 4° C. until used.

EXAMPLE 4

Combination Vaccines Containing Additional Antigens

Combination vaccines containing DTP, IPV, Hib or acellular or whole cell pertussis antigens are prepared by adding one or more of the desired antigens to the formulations described in Example 1, Example 2 or Example 3 above.

Claim 1 of 13 Claims

1. A method of treating a human subject suffering from or susceptible to hepatitis infections, the subject being an elderly or immunocompromised subject, the method comprising administering a vaccine composition, said composition comprising a viral hepatitis antigen selected from the group consisting of: a killed attenuated Hepatitis A virus, a Hepatitis B antigen, and a combination of a killed attenuated Hepatitis A virus—Hepatitis B antigen, in conjunction with 3-O-deacylated monophoshoryl lipid A and a suitable carrier, wherein said carrier is selected from the group consisting of: an aluminium salt and an oil water emulsion.

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