A process for the production of inactivated Hepatitis A virus substantially free of host cell contamination is described, the process comprising: a) culturing Hepatitis A virus and harvesting a Hepatitis A preparation; b) treating said Hepatitis A preparation with a protease; and thereafter c) separating intact virus from protease digested material; d) inactivating said virus. Also described are vaccines comprising the inactivated Hepatitis A virus, preferably in combination with strong adjuvants.

The present invention relates to new vaccine compositions, processes for their manufacture and their use in medicine. In particular, the present invention relates to improved Hepatitis A vaccines adjuvanted with a potent immunostimulator, preferably such as monophosphoryl lipid A or a derivative thereof. The invention also relates to combination vaccines in which the Hepatitis A vaccine is a component.

Hepatitis A vaccines are known. For example the vaccine Havrix (Trade Mark), from SmithKline Beecham Biologicals can be used to prevent hepatitis A infections and is also formulated with aluminium hydroxide as adjuvant. This vaccine is produced according to the procedure of Andre et al. It 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. The vaccine Hcpatyrix (Trade Mark) which is a combination of the above hepatitis A antigen and a Salmonella typhimurium purified Vi polysaccharide may be used to protect against Hepatitis and typhoid simultaneously.

International patent application WO93/19780 (SmithKline Beecham Biologicals s.a.) discloses, inter alia, a Hepatitis A vaccine adjuvanted with 3D-MPL.

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.

It has now been found that traditional processes for producing and purifying inactivated virus for hepatitis A vaccines can leave a small residue of contaminants from the host cells in which the hepatitis A virus was grown. Such host cell contaminants, especially when they are from human origin, diploid in nature and at a low level, provide no concern when the vaccine is adjuvanted with aluminium salts. But when the vaccine is adjuvanted with strong immunostimulants there is a theoretical possibility that a vaccinee may raise an adverse immune response to the host contaminants.

Accordingly there is a need for a method of manufacture which removes substantially all traces of such host cell proteins.

Accordingly in one aspect of the invention there is provided a process for the production of inactivated Hepatitis A virus substantially free of host cell contamination, the process comprising:

• a) culturing Hepatitis A virus and harvesting a hepatitis A preparation;
• b) treating said hepatitis A preparation with a protease; and thereafter
• c) separating intact virus from protease-digested material;
• d) inactivating said virus.

Surprisingly, the protease digestion treatment does not adversely affect the Hepatitis A virus, but facilitates the breakdown and separation of host cell contaminants from the Hepatits A preparation.

Preferably the Hepatitis A virus is derived from HM-175 strain.

By substantially free of host cell contamination is meant that less than 10%, preferably less than 8%, more preferably less than 5% host cell protein can be detected by scanning of silver-stained SDS PAGE. More importantly and as determined by slot blot hybridisation one dose of HAV in the vaccine preferably contains less than 10 ng of host cell proteins.

Preferably the protease used is trypsin. Other proteases that may be utilised include pronase, papain, and pepsin.

The protease treatment is preferably carried out at above room temperature, e.g. at about 37° C for about 2 hrs.

The separation of the intact virus from the protease and the digested components can be achieved by a variety of suitable methods, for example by permeation chromatography.

Alternatively the protease and digested components may be separated by any separation method that separates on the basis of size, for example ultra filtration.

The product can then be further purified by other steps to remove other contaminants. For example, the product can be further purified by subjecting the product to ion-exchange chromatography to remove any nucleic acid residue.

It is believed that the protease digestion step of the method according to the invention can improve purification of the hepatitis A preparation due to two effects. First, the protease digests any contaminating host proteins such that they are easier to separate in the chromatographic separation step that follows the protease treatment. Second, the digestion of contaminating host proteins allows better separation of other contaminating materials which would otherwise be associated with undigested host proteins, in particular nucleic acid, in the ion exchange step. It will be appreciated that these observed effects do not necessarily limit the invention in any way.

In another aspect of the present invention there is provided an inactivated Hepatitis A virus substantially free of contaminating host proteins, as defined above.

The inactivated hepatitis A virus may then be formulated into a vaccine.

Thus the invention provides in a further aspect a Hepatitis A vaccine comprising an inactivated hepatitis A virus substantially free of host cell contaminants.

Such a vaccine may advantageously include a suitable adjuvant. Suitable adjuvants include an aluminium salt such as aluminium hydroxide gel or aluminium phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.

Advantageously, the highly purified hepatitis A virus may be formulated with strong adjuvant systems. Thus in the formulation of the invention, it is preferred that the adjuvant composition induces an immune response comprising Th1 aspects.

In general terms, a Th1-type response is characterised by the production of IFN-y as 10 opposed to a Th2-type response which is characterised by the production of cytokines such as IL-4, IL-5 and IL-10. The isotypic profile of the humoral response can also be used as a marker for Th1 or Th2-type responses. In mice Th1-type responses are often associated with the generation of antibodies of the IgG2a subtype while IgG1 are markers of a Th2-type response. The situation is not as clear in humans but data suggest that IgG1 and IgG4 could respectively be markers of Th1- and Th2-type responses.

Suitable adjuvant systems include for example a combination of monophosphoryl lipid A, preferably 3-O-de-acylated monophosphoryl lipid A (3D-MPL), and preferably formulated together with an aluminium salt.

An enhanced system involves the combination of monophosphoryl lipid A and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO 96/33739.

A particularly potent adjuvant formulation involving QS21, 3D-MPL and d,1-alpha-tocopherol in an oil in water emulsion is described in WO 95/17210.

Other known adjuvants which may be included are CpG containing oligonucleotides for example as disclosed in WO 96/02555.

Accordingly in a preferred embodiment of the present invention there is provided a vaccine comprising a virus of the present invention, adjuvanted with monophosphoryl lipid A or a derivative thereof.

Preferably the vaccine additionally comprises a saponin, more preferably QS21.

Preferably the formulation additionally comprises an oil in water emulsion and d, 1-alpha-tocopherol.

The present invention also provides a method for producing a vaccine formulation comprising mixing a purified virus of the present invention together with a pharmaceutically acceptable excipient or carrier, such as 3D-MPL.

The purified virus of the invention may advantageously be combined with other antigens so that it is effective in the prophylaxis or treatment of other diseases in addition to hepatitis A infections. A preferred combination involves a combination containing a hepatitis B antigen.

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 if 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 (McCormick and Jones). HBsAg as herein described can also refer to mutants, for example the 'escape mutant' described in WO 91/14703 or EP 0 511 855 A1, especially HBsAg wherein there is an amino acid substitution at position 145 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.

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 accordingly to the invention is a therapeutic vaccine useful for the treatment of ongoing hepatitis infections, more especially hepatitis A and/or hepatitis B infections in humans suffering therefrom.

In view of the surprisingly efficacious 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.

Advantageously 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 which may be selected from non-hepatitis antigens which are known in the art to afford protection against one or more of the following diseases:

• diphtheria, tetanus, pertussis, Haemophilus influenzae b (Hib), and polio.

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

Suitable components for use in such combination vaccines are already commercially available and details may be obtained from the World Health Organization. For example the polio component may be the Salk inactivated polio vaccine (IPV). The pertussis vaccine component may comprise a whole cell or acellular product.

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

Preferred combination vaccines according to the invention for adolescent use include one or more components selected from antigens which are known in the art to provide protection against one or more of the following diseases:

• human papillomavirus (HPV), herpes simplex virus (HSV), Epstein Barr virus (EBV), Varicella Zoster virus (VZV), human cytomegalovirus (HCMV), Toxoplasma gondii.

The amount of each antigen in the vaccine dose is selected as an amount that 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 0.01 to 1.0 μg protein/dose for Hepatitis A, most preferably from between 0.06 to 0.220 μg protein/dose. For antigens other than Hepatitis A, for example HBsAg, HSV etc, the amount of protein per dose may be higher, e.g. up to about 20 μg per dose. An optimal amount of each of one or more immunogens for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and/or other responses in subjects. Following an initial vaccination, subjects may receive a boost within 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 MPL or a derivative thereof.

Using this method one or more additional components are preferably admixed with the inactivated Hepatitis A vaccine to provide a combination vaccine.
 

Claim 1 of 5 Claims

1. A process for the production of inactivated Hepatitis A virus substantially free of host cell contamination, the process comprising:

a) a culturing Hepatitis A virus and harvesting a hepatitis A preparation;

b) treating said hepatitis A preparation with a protease, thereafter;

c) separating intact virus from protease-digested protein; and

d) inactivating said virus.

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