Title of Invention	A NOVEL VACCINE COMPOSITION
Abstract	ABSTRACT "A NOVEL VACCINE COMPOSITION" IN/PCT/2002/00335/CHE A vaccine composition comprising a hepatitis B surface antigen and a human papilloma virus {HPV) antigen, in conjunction with an adjuvant which is a preferential stimulator of TH1 call response, wherein the vaccine composition does not comprise a herpes simplex viral antigen, wherein the composition Is suitable for the treatment or prophylaxis of human papillomavirus Infections and hepatitis B infections, and wherein the HPV antigen comprises the major capsld protein L1 of HPV from HPV 18 and/or HPV 18, or a fusion of Haemophilus Influenza B protein 0 with £7 from HPV 16.

Title of Invention

A NOVEL VACCINE COMPOSITION

Abstract

ABSTRACT "A NOVEL VACCINE COMPOSITION" IN/PCT/2002/00335/CHE A vaccine composition comprising a hepatitis B surface antigen and a human papilloma virus {HPV) antigen, in conjunction with an adjuvant which is a preferential stimulator of TH1 call response, wherein the vaccine composition does not comprise a herpes simplex viral antigen, wherein the composition Is suitable for the treatment or prophylaxis of human papillomavirus Infections and hepatitis B infections, and wherein the HPV antigen comprises the major capsld protein L1 of HPV from HPV 18 and/or HPV 18, or a fusion of Haemophilus Influenza B protein 0 with £7 from HPV 16.

Full Text	This invention relates to novel vaccine formulations, methods for preparing them and their use in therapy. In particular the present invention relates to combination vaccines for administration to adolescents. Papillomaviruses are small DNA tumour viruses, which are highly species specific. So far, over 70 individual human papillomavirus (HPV) genotypes have been described. HPVs arc generally specific either for the skin (e.g. HPV-1 and -2) or mucosal surfaces (e.g. HPV-6 and -II) and usually cause benign tumours (warts) that persist for several months or years. Such benign tumours may be distressing for the individuals concerned but tend not to be life threatening, with a few exceptions. Some HPVs are also associated with cancers. The strongest positive association between an HPV and human cancer is that which exists between HPV-16 and HPV-18 and cervical carcinoma. Cervical cancer is the most common malignancy in developing cotmtries, with about 500.000 new cases occurring in the world each year. It is now technically feasible to actively combat primary HPV-I6 infections, and even established HPV-l6-containing cancers, using vaccines. For a review on the prospects for prophylactic and therapeutic vaccination against HPV-16 see Cason J., Clin. Immunother. 1994; 1(4) 293-306 and Hagenesee M.E., Infections in Medicine 1997 14(7) 555-556.559-564. Today, the different types of HPVs have been isolated and characterised with the help of cloning systems in bacteria and more recently by PCR amplification. The molecular organisation of the HPV genomes has been defined on a comparative basis with that of the well characterised bovine papillomavirus type 1 (BPVl). Other HPV serotypes of particular interest are 31, 33 and 45. Although minor variations do occur, all HPVs genomes described have at least seven early genes. El to E7 and two late genes LI and 12. In addition, an upstream regulatory region harbors the regulatory sequences which appear to control most transcriptional events of the HPV genome. El and E2 genes are involved in viral replication and transcriptional control, respectively and tend to be disrupted by viral integration. E6 and E7, and recent evidence implicate also E5 are involved in viral transformation. In the HPVs involved in cervical carcinoma such as HPV 16 and 18, the oncogenic process starts after mtegration of viral DNA. The integration results in the inactivation of genes codmg for the capsid proteins LI and L2 and in mstalling continuous over expression of the two early proteins E6 and E7 that will lead to gradual loss of the normal cellular di3erentiation and the development of the carcinoma. Carcinoma of the cervix is common in women and develops through a pre¬cancerous intermediate stage to the mvasive carcinoma which frequently leads to death. The intermediate stages of the disease is known as cervical intraepithelial neoplasia and is graded I to HI in terms of increasing severity. Clinically, HPV infection of the female anogenital traa manifests as cervical flat condylomas, the hallmark of which is the fcoilocytosis affecting predominantly the superficial and intermediate cells of the cervical squamous epithelium. KoUocytes which are the consequence of a cytopathic effea of the virus, appear as multinucleated cells with a perinuclear clear halo. The epithelium is thickened with abnormal keratinisation responsible for the warty appearance of the lesion. Such fiat condylomas when positive for the HPV 16 or 18 serotypes, are high-risk factors for the evolution toward cervical intraepithelial neoplasia (CIN) and carcinoma in situ (CIS) which are themselves regarded as precursor lesions of invasive cervix carcinoma. WO 96/19496 discloses variants of human papilloma vims E6 and E7 proteins, particularly fusion proteins of E6/E7 with a deletion in both the E6 and E7 proteins. These deleuon fusion proteins are said to be immunogenic. HPV LI based vaccines are disclosed in WO94/0O152, ■WO94/20137, ■WO93/02184 and WO94/0S792. Such a vaccine can comprise the LI antigen as a monomer, a capsomer or a virus like particle. Such particles may additionally comprise L2 proteins. L2 based vaccines are described for example in WO93/00436. Other HPV vaccines arc based on the Early proteins, such as E7 or fmion proietm such as L2-F7. Vaccines for the prophylaxis of hepatitis B infections, comprising one or more hepatitis B antigens, arc well known. For example the vaccine Edgerix-B (Trade Mark) from SmithKline Beccham Biologicals is used to prevent Hepatitis B. This vaccine comprises hepatitis B surfece antigen (specifically the 226 amino acid S-antigen described in Harford et. al. in Postgraduate Medical Journal, 19S7, 63 (Suppl. 2). p65-70) and is formulated using aluminium hydroxide as adjuvant. There is a need for effective combination vaccines to prevent diseases to which adolescents are particularly prone. The present invention provides a vaccine composition comprising; [a) a hepatitis B viral (HBV) antigen; and ;b) a human papillomavirus (HPV) antigen n combination with an adjuvant which is a preferential stunulator of THl cell esponse. The vaccine coroposition of the invention is of great benefit for administration to adolescents who may be particularly at risk of HBV, and/or HPV infection. Optionally the vaccine composition of the invention additionally comprises one or more of a number of other antigens as described below. It has been found that the vaccine compositions according to the invention surprisingly show no interference, that is to say that the immune response to each antigen in the composition of the invention is essentially the same as that which is obtained by each antigen given individually in conjunction with an adjuvant which is a preferential stimulator of THl cell response. The vaccine Havrix (Trade Mark), also from. SmithKline Beecham Biologicals is an example of a vaccine that can be used to prevent hepatitis A infections. It i$ formulated with aluminium hydroxide as adjuvant. This vaccine con3rises an attenuated strain of the HM-175 Hepatitis A virus inactivated with formol (formaldehyde); see Andre et. al. (Prog. med. Virol., vol. 37, pl-24). As used herein, the term hepatitis A viral (HAV) antigen is used to refer to either a protein derived from hepatitis A virus or an attenuated strain of HAV, optionally inactivated, e.g. with formaldehyde. If the HAV antigen is a protein derived from hepatitis A virus it may optionally be a recombinant protein. The vaccine Twinrix (Trade Mark) is a combination of a recombinant hepatitis B anitgen with the aforementioned inactivated attenuated hepatitis A virus. The vaccine 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. In a further aspect, the invention provides a vaccine composition comprising: (a) a hepatite B viral (HBV) antigen; (b) a human papillomavirus (HPV) antigen; and (c) a hepatitis A viral (HAV) antigen in combination with an adjuvant which is a preferential stimulator of TH1 cell response. Such a vaccine is of great benefit for administration to adolescents who may be particularly at risk of HBV, and/or HPV infection, and/or HAV infection. An immune response may be broadly divided into two extreme catagories, being a humoral or cell mediated immune response (traditionally characterised by antibody and cellular effeaor mechanisms of protection respectively). These categories of response have been termed THl-type responses (cell-mediated response), and TH2-type immune responses (humoral response). Extreme THl-type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and namral killer cell responses. In mice THl-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies. TH2-type immune responses are characterised by the generation of a range of immunoglobulin isotypes including in mice IgGl. It can be considered that the driving force behind the development of these two types of immune responses are cytokines. High levels of THl-type cytokines tend to favour the induction of cell mediated immune responses to the given antigen, whilst high levels of TH2-type cytokines tend to favour the induction of humoral immune responses to the antigen. The distinction of THl and TH2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominandy THl or predominantly TH2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4 +ve T cell clones by Mosmann and Coffman (Mosmann, T.R. and Coffinan, R.L. (1989) THl and TH2 cells: different paxtems oflymphokine secretion lead to different Junctional properties. Annual Review of Immunology, 7, pl45-173). Traditionally, THl-type responses are associated with the production of the INF-y cytoldnes by T-Iymphocytes. Other cytokines often directly associated with the induction of THl-type immune responses are not produced by T-ce!ls, such as IL-12. In contrast, TH2- type responses arc associated with the secretion of IL-4, IL-5, IL-6, IL-10 and tumour necrosis factor-P(TNF-p), It is known that certain vaccine adjuvants are particularly suited to the stimulation of either THl or TH2 - type cytokine responses. Traditionally the best indicators of the TH1:TH2 balance of the immune response after a vaccination or infection includes direct measurement of the production of THl or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or the measurement (at least in mice) of the IgGl:IgG2a ratio of antigen specific antibody responses. Thus, a THl-type adjuvant is one which stimulates isolated T-cell populations to produce high levels of THl-type cytokines when re-stimulated with antigen in vitro, and induces antigen specific immunoglobulin responses associated with THl-type isotype. Adjuvants which are capable of preferential stimulation of the TH 1 cell response are described in International Patent Application No. WO 94/00153 and WO 95/17209. 3 De-0-acylated moaophosphoryl lipid A Preferably, the particles of 3D-MPL are small enough to be sterile filtered through a 0.22micron membrane (as described in European Patent number 0 689 454). 3D-MPL will be present in the range of 103ig - lOO3ig preferably 25-50fj.g per dose wherein the antigen will typically be present in a range 2-50fig per dose. Another preferred adjuvant comprises QS21, an Hpic purified non-toxic fraction derived from the barfc of QuUlaja Saponaria Molina. Optionally this may be admixed with 3 De-0-acylated monophosphoryl lipid A (3D-MPL), optionally together with an carrier. The method of production of QS21 is disclosed m US patent No. 5,057,540. Non-reactogenic adjuvant formulations containing QS21 have been described previously (WO 96/33739). Such formulations comprising QS21 and cholesterol have been shown to be successful THI stimulating adjuvants when formulated together with an aatigea. Thus vaccine compositions which form part of the present invention may include a combination of QS21 and cholesterol. Further adjuvants which are preferential stimulators of THI cell response include unmunomodulatory oligonucleotides, for example unmethylated CpG sequences as disclosed in WO 96/02555. Combinations of different THI stimulating adjuvants, such as those mentioned hereinabove, are also contemplated as providing an adjuvant which is a preferential stimulator of THl cell response. For example. QS21 can be formulated together with 3D-MPL. The ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10 : 1; preferably 1:5 to 5 : 1 and often substantially 1 : 1. The preferred range for optima! synergy is 2.5 : I to I : 1 3D-MPL: QS21. Preferably a carrier is also present in the vaccine composition according to the invention. The carrier may be an oil in water emulsion, or an aluminium salt, such as aluminium phosphate or aluminium hydroxide. A preferred oil-in-water emulsion comprises a metabolisible oil, such as squalene, alpha tocopherol and Tweea 80. Additionally the oil in water emulsion may contain span 85 and/or lecithin and/or tricaprylin. In a particularly preferred aspect the antigens in the vaccine composition according to the invention are combined with 3D-MPL and alum. Typically for human administration QS21 and 3D-MPL will be present in a vaccine in the range of lug - 200jjg, such as 10-lOOjig, preferably lOiig - 503g per dose. Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is equal to or less than 1 as this provides a more stable emulsion. Span 85 may also be present at a level of I %. In some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser. Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in WO 95/17210. The HPV antigen in the composition of the invention is preferably derived from HPV 16 and/or IS, or from HPV 6 and/or U, or HPV 31, 33 or 45. In one preferred embodiment the HPV antigen in the vaccine composition according to the invention comprises the major capsid protein LI of HPV and optionally the L2 protein, particularly from HPV 16 and/or HPV 18. In this embodiment, the preferred form of the LI protein is a truncated LI protein. Preferably the LI is in the form of a virus-like particle (VLP). The LI protein may be fused to another HPV protein, in particular E7 to form an L1-E7 fusion. Chimeric VLPs comprising Ll-E or H-L2-E are particularly preferred. In another preferred embodiment, the HPV antigen in the composition of the invention is derived from an E6 or E7 protein, in particular E6 or E7 linked to an immunological fusion parmer having T cell epitopes. In a preferred form of this embodiment of the invention, the immunological fiision parmer is derived from protein D of Heamophilus influenza B. Preferably the protein D derivative comprises approximately the first 1/3 of the protein, in particular approximately the first N-termioal 100-110 amino acids. Preferred fusion proteins in this embodiment of the invention comprise Protein D -E6 from HPV 16, Protein D - E7 from HPV 16 Protein D - E7 from HPV 18 and Protein D - E6 from HPV 18. The protein D part preferably comprises the first 1/3 of protein D. In still another embodiment of the invention, the HPV antigen is in the form of an L2-E7 fusion, particularly from HPV 6 and/or HPV U. The proteins of the present invention preferably are expressed in E. coU. In a preferred embodiment the proteins are expressed with a Histidine tail comprising between 5 to 9 and preferably six Histidine residues. These are advantageous in aiding purification. The description of the manufacture of such proteins is fully described in co-pending UK patent application number GB 9717953.5. The HPV antigen in the vaccine composition may be adsorbed onto Al(0H)3. Preferably the LI VLP is adsorbed onto AKOH),. The hepatitis B viral (HBV) antigen in the composition of the invention is typically hepatitis B surfece 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 herem the expression 'Hepatitis B surface antigen', abbreviated herein to 'HBsAg' or 'HBS' includes any HBsAg antigen or fragment thereof displaying the antigenicity of HBV surfece antigen. It will be understood that in addition to the 226 amino acid sequence of the HBsAg S antigen (sec Tiollais et. al. Nature, 317, 489 (1985) and references therein) HBsAg as herein described niay, 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 fiirther aspect the HBsAg may comprise a protein described as L"* 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) protem (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 0 304 378. 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 defmed above and S denotes the S-protein of hepatitis B surface antigen. The HBsAg may be adsorbed on aluminium phosphate as described in W093/24148. Preferably the hepatitis B (HBV) antigen used in the formulation of the invention is HBsAg S-antigen as used in the commercial product Engerix-B (Trade Mark; SmithKline Beecham Biologicals). A vaccine comprising hepatitis B surface antigen in conjunction with 3D-MPL was described in European Patent Application 0 633 784. Examples of antigens from additional pathogens which may be included in the compositions according to the invention are now described. Epstein Barr Virus (EBV). a member of the herpesvirus group, causes infectious mononucleosis as a primary disease in humans. I3edominantly it affects children or young adults. More than 90% of the average adult population is infected by EBV that persists for lifetime in peripheral B-lymphocytes. The virus is lifelong produced in die parotid gland and spread primarily by exchange of saliva from individuals who shed the virus. Children infected with EBV are largely asymptomatic or have very mild symptoms, while adolescents and adults who become infected develop typical infectious mononucleosis, characterised by fever, pharyngitis, and adenopathy. People who have been infected maintain anti-EBV antibodies for the remainder of their lives, and are thus immune to further infection. In addition to its infectious qualities, EBV has been shown to transform lymphocytes into rapidly dividing cells and has therefore been implicated in several different iymphcmas, including African Burkitt's lymphoma (BL). EBV may also be involved in causing nasopharyngeal carcinoma (NPC). Worldwide it is estimated that 80,000 cases of nasopharyngeal carcinoma occur and it is more prevalent in ethnic Chinese populations. Infectious mononucleosis is a consequence of primary infection by EBV. It is not a life-threatening disease if additional risk fectors are absent. Four proteins of the EBV viral envelope coostituting the so-called membrane antigen complex have been described. They are usually referred to as gp 220/350 or gp 250/350 or simply as gp 250 or 350 (see EP-A-151079). There is convincing evidence that gp 350 and gp 250 induce the production of neutralising antibodies and that antibodies against gp 350 and gp 250 have neutralising capacity. These proteins are thus candidates for a possible EBV vaccine. For further inforraation about the application of gp 250/350 for prophylaxis and treatment of EBV-related diseases see EP 0 173 254. The major EBV surface glycoprotein gp350/220 infects human target cells through interaction with the cellular membrane protein. CD21. Gp350/220 is the primary target for EBV-neutralising antibodies in humans and some forms of gp350/220 have been shown to protect against EBV-related disease. Preferably a vaccine composition according to the invention comprises gp 350 of EBV although other protective antigens may be used. HSV-2 is the primary etiological agent of herpes genitalis. HSV-2 and HSV-1 (the causative agent of herpes labialis) are characterised by their ability to induce both acute diseases and to establish a latent infection, primarily in nem"onal ganglia cells. Genital herpes is estimated to occur in about 5 million people in the U.S.A. alone with 5CX),000 clinical cases recorded every year (primary and recurrent infection). Primary infection typically occurs after puberty and is characterised by the localised appearance of painful skin lesions, which persist for a period of between 2 to 3 weeks. Within the following six months after primary infection 50 % of patients will experience a recurrence of the disease. About 25 % of patients may experience between 10-15 recurrent episodes of the disease each year. In inununocompromised patients the incidence of high frequency recurrence is statistically higher than In the normal patient population. Both HSV-1 and HSV-2 virus have a number of glycoprotein corapoaents located on the surface of the virus. These are known as gB, gC, gD and gE etc. When an HSV antigen is included in the composition of the invention this is preferably derived from HSV-2, typically glycoprotein D. Glycoprotein D is located on the viral membrane, and is also found in the cytoplasm of infected ceils (Eisenberg R.J. et al; J of Virol 1980, 353, 428-435). It comprises 393 amino acids including a signal peptide and has a molecular weight of approximately 60 kD. Of all the HSV envelope glycoproteins this is probably the best characterised (Cohen et al; J. of Virology, 60, 157-166). In vivo it is known to play a central role in viral attachment to cell membranes. Moreover, glycoprotein D has been shown to be able to elicit neutralising antibodies in vivo (Eing et al J. Med. Virology 127: 59-65). However, latent HSV-2 virus can still be reactivated and induce recurrence of the disease despite the presence of high neutralising antibodies titre in the patients sera, In one embodiment of the invention there is present a truncated HSV-2 glycoprotein D of 308 amino acids which comprises amino acids 1 through 306 naturally occurring glycoprotein with the addition Asparagine and Glutamine at the C terminal end of the truncated protein devoid of its membrane anchor region. This form of the protein includes the signal peptide which is cleaved to yield a mature 283 amino acid protein. The production of such a protein in Chinese Hamster ovary cells has been described in Genentech's Etiropean patent EP-B-139 417. The recombinant mamre HSV-2 glycoprotein D truncate is preferably used in the vaccine formulations of the present invention and is designated rgD2t. A corr3ination of this HSV-2 antigen in combination with the adjuvant 3D-MPL has been described in WO 92/16231. In a preferred aspect the vaccine composition of the invention additionally comprises a Varicella Zoster viral antigen (VZV antigea). Suitable antigens of VZV for inclusion in the vaccine formulation include gpI-V described by Longnecfcer et al.. Proc Natl Acad Sci USA 84. 4303-4307 (1987). In a preferred embodiment gpl (see Ellis ei al., US patent 4.769,239) is used. See also European Patent No. 0 405 867 Bl. In another preferred aspect the vaccine composition of the invention additionally comprises a himian cytomegalovirus (HCMV) antigen. HCMV is a human DNA virus belonging to the femily of herpes viruses. HCMV is endemic in most parts of the world. Among two populatiotK, HCMV is responsible for serious medical conditions. HCMV is a major cause of congenital defects in new boms. The second population at risk are immunocompromised patients such as those suffering from HIV infection and those patients undergoing transplantations. The clinical disease causes a variety of symptoms including fever, hepatitis, pnemnonitis and mfectious mononucleosis. A preferred antigen for use in a vaccine against HCMV is gB685** as described in WO 95/31555. Immimogens for use in HCMV vaccines arc also provided by pp65, an HCMV Matrix Protein as described in WO 94/00150 (City of Hope). In one preferred aspect the vaccine composition of the invention additionally comprises both a VZV and an HCMV antigen, in particular those antigens described above. In another preferred aspect the vaccine composition of the invention additionally comprises a Toxoplasma gondii antigen. Toxoplasma gondii is an obligate intracellular protozoan parasite responsible for toxoplasmosis in warm-blooded animals, including man. AlthougU it is generally clinically asymptomatic in healthy individuals, toxoplasmosis may cause severe complications in pregnant women and unmunocomproraised patients. A. preferred antigen for use in a vaccine against Toxoplasma gondii is SAGl ( also known as P30) as described in WO96/02654 or Tg34 as described in W092/U366. In one preferred aspect the vaccine composition of the invention additionally comprises either a VZV antigen or an HCMV antigen combined with a Toxoplasma gondii antigen, in panicular those antigens described above. In a preferred aspect the vaccine composition of the invention is a multivalent vaccine, for example a tetra- or pentavalent vaccine. The formulations of the present invention are very effective in inducing protective immunity, even with very low doses of antigen (e.g. as low as 5\ig rgD2t). They provide excellent protection against primary infection and stimulate, advantageously both specific humoral (neutralising antibodies) and also effector cell mediated (DTH) immune responses. The present invention in a further aspect provides a vaccine formulation as herein described for use in medical therapy, particularly for use in the treatment or prophylaxis of human papillomavirus infections and hepatitis B virus infections. The vaccine of the present invention will contain an immunoproteciive quantity of the antigens and may be prepared by conventional techniques. Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol.61 Vaccine Design - the subunii and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, edited by VoUer et al.. University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by FuUerton, U.S. Patent 4,235,577. 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 amount of procem in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount wiii vary depending upon which specific immunogen is employed. Generally, it is expected that each dose will comprise I-lOOOjig of protein, preferably 2-lOOiJg, most preferably 4-403g. 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 addition to vaccination of persons susceptible to HPV or HBV infections, the pharmaceutical compositions of the present invention may be used to treat, immunotherapeutically, patients suffering from the said viral infections. In a further aspect of the present invention there is provided a method of manufacture as herein described, wherein the method comprises mixing a human papilloma virus antigen and a hepatitis B virus antigen with a TH-1 inducing adjuvant, for example 3D-MPL and, preferably, a carrier, for example alum. If desired, other antigens may be added, in any convenient order, to provide multivalent vaccine compositions as described herein. The following example illustrates but does not limit the invention. Example I: Comparative immopogenicity of HPV Ags / HBs combos formulated with Alum/3 D-MPL. INTRODUCTION An immunogenicity study was performed in Balb/C mice using four different antigens: 1. HPV16 LI Virus Like Particule (VLP-16) 2. HPV18L1 Virus Like PanicuIe(VLP18) 3. PD 1/3 16E7 2M from HPV-16 (E7) 4. HBsAg formulated with Alum/3D-MPL (AS04) using pre adsorbed monobulks of antigen or 3D-MPL on AKOH), or AIPO«. 3D-MPL/Al(OH)3 formulations are referred to as AS04D whereas SD-MPL/AIPO, based fonnuiations are refened to as AS04C. The following vaccines were assessed: 1. VLP16 + VLP18 AS04D; 2. E7 based formuialions, 3. HBs AS04C and the potential to combine these vaccines was evaluated. The aims of this experiment were as follows: 1) To compare the immunogenicity of different AS04 combinations of either VLP16 +VLP18 or E7 and HBs Ag. 2) As the monovalent vaccines are either formulated in AS04C or AS04Dr to compare the immunogenicity of different HBs AS04 formulations made of AIPOj or a mix of AlP04/A!(0H)j with different ratios of Alum forms; and to evaluate the effect of the adsorption of 3D-MPL on a fi-action of AI(0H)3 and ALPO4 versus 3D-MPL / A1(0H)3 in combination containing VLPs or E7 antigens. The experimental protocol is fully described in the Material and Methods section. In summary, groups of 10 mice were immunised intramuscularly twice at 3 week intervals with various Ag based formulations (I/IOHD). Antibody response to HBs, E7 and VLPs Ag and the isotypic profile induced by vaccination were monitored by ELISA at day 14 post 11. At die same timepoint, the CMI (lymphoproliferative response or the cytokine production (IFNy/IL5)) was analysed after in vitro restimulation of splenic cells with either HBs, VLPs or E7 antigen. MATERIALS AND METHODS Formulation Formulation compositions VLP16. VLP18, PDl/3-HPV16E7-His, and HBs on AS04C or AS04D. Adsorption. a) VLP adsorption. VLP 16 and VLP 18 purified bulk is added to Al(OH)i at 23g VLP/lO3g Al(OH)j. The mixture is stored between Z-S'C until final formulation. b) HBs adsorption. 2 fi% Hbs are mixed with 40;ig AIPO,. The mixture is stored between 2-8'C until final formulation. 2 /ig Hbs are mixed with 10/tg AIPO4. The mixmre is stored between 2-8'C until final formulation. c) PDl/3-HPV16E7-His adsorption. 2 ng E7 are mixed widi lO/ig Al(OH)). The mixture is stored between 2-8°C until final formulation. d) 3D-MPL adsorption. 5fig 3D-MPL are mixed with lO3g Ai(OH)j. The mixture is stored between 2-8'C until fmal formulation. 53g 3D-MPL are mixed with lO3ig AlPOj. The mixture is stored between 2-8°C until final formulation. 2.5/xg 3D-MPL are mixed with 53g A1{0H)3. The mixture is stored between 2-8'C until final formulation. 2.5fig 3D-MPL are mixed with 53g AIPO4. The mixture is stored between 28°C until final formulation. Formulation HjO and NaCl are mixed (lOx concentrated) and after 10 minutes of agitation at room temperature, the different components are added: adsorbed antigen, adsorbed 3D-MPL and A1(0H)3 (See table below). They are shaken at room temperamre for 10 minutes and stored at 4°C imtil injection. The in vitro characterisation of the formulation can then be performed. Mice Serology Aoti-HBs serology The quantitation of anti-HBs antibodies was performed by ELISA using HBs (Hep 286) as the coating antigen. Antigen and antibody solutions were 33ssd at 5031 per well. The antigen was diluted at a final concentration of Ijig/nil in PBS and was adsorbed overnight at 4°C to the wells of 96 wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plates were then incubated for Ihr at 37°C with PBS containing 1 % bovine serum albumin and 0.1% Tween 20 (saturation buffer). Two-fold dilutions of sera (staning at 1/100 dilution) in the saturation buffer were added to the HBs-coated plates and incubated for Ihr 30 min at ST3C. The plates were washed four times with PBS 0.1% Tween 20 and bioiin-conjugated anti-mouse Ig (Amersham, UK) diluted 1/1500 or IgGl, IgG2a, rgG2b GMTECH, USA) diluted respectively at 1/4000, 1/8000. 1/4000 in saturation buffer were added to each well and incubated for Ihr 30 min at ST3C. After a washing step, streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted 1/1000 in saturation buffer was added for an additional 30min at 21'C. Plates were washed as above and incubated for 20niin with a solution of o-phenylenediamine (Sigma) 0.04% HA0-P3% in 0.1% tween 20 0.05M citrate buffer pH4.5. The reaction was stopped with H.SO3 2N and read at 490/630 nm. ELISA liters were calculated from a reference by SoftmaxPro (using a four parameters equation) and expressed in EU/ml. ADti-E7 serology Quantitation of anti-E7 antibody was performed by ELISA using PDl/3 16E7 2M as coating antigen. Antigen and antibody solutions were used at lOO3il per weil. The antigen was diluted at a final concentration of 0.5/ig/ml in PBS and was adsorbed overnight at 4°C to the wells of 96 wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plates were then incubated for Ihr at 37°C with PBS containing 1 % bovine serum albumin and 0.1 % Tween 20 (saturation buffer). Two¬fold dilutions of sera (starting at 1/100 or 1/400 dilution) in the saturation buffer were added to the E7-coated plates and incubated for Ihr 30 min at 37'C. The plates were washed four times with PBS 0.1% Tween 20 and biotin-conjugated anti-mouse Ig, IgGl, IgG2a, IgG2b (Amersham. UK) diluted 1/1500 in saturation buffer were added to each well and incubated for Ihr 30 min at 37'C. After a washing step, streptavidin-biotmylated peroxydase complex (Amersham, UK) diluted 1/5(XX) m saturation buffer was added for an additional 30 min at 37'C. Plates were washed as above and incubated for 20min with a solution of Tetramethyl benzidine (TMB) (Biorad, USA) 2-fold diluted in Citrate buffer (O.IM pH=5.8). The reaction was stopped with H1SO4 0.5 N and read at 450/630 nm. ELISA titers were calculated from a reference by SoftmaxPro (using a four parameters equation) and expressed in EU/ml. Aiiti-VLP16 and anti-VLPlS serology The quantitation of anti-VLP16 and anti-VLPIS antibodies was performed by ELISA using VLP16 503/1 (20/12/99) ands VLP18 504/2 (25/10/99F) as coating antigens. The antigen and antibody solutions were used at 50;il per well. The antigen was diluted at a final concentration of 0.53g/m] in PBS and was adsorbed overnight at 4'C to the wells of 96 wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plates were then incubated for Ihr at ST'C with PBS containing 1% bovine serum albumin. Two-fold dilutions of sera (starting at 1/400 dilution) in the samration buffer were added to the VLPs-coated plates and incubated for Ihr 30min at 37'C. The plates were washed four times with PBS 0.1% Tween 20 and biotin-conjugated anti-mouse Ig (Amersham, UK) diluted 1/1500 in saturation buffer were added to each well and incubated for Ihr 30min at Sy3C. After a washing step, streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted 1/1000 in saturation buffer was added for an additional SOinin at 37'C. Plates were washed as above and incubated for 20min with a solution of o-phenylenediamine (Sigma) 0.04% HiOj 0.03% in 0.1% tween 20 0.05M citrate buffer pH4.5. The reaction was stopped with H3SO3 2N and read at 490/630 imi. ELISA titers were calculated from a reference by SoftmaxPro (using a four parameters equation) and expressed in EU/ml. T cell proliferation Two weeks after the second immunisation, mice were killed, spleens were removed aseptically and pooled (1 pool of 5 organs per group). Cell suspensions were prepared in RPMI1640 medium (GEBCO) containing 2mM L-glutamine, antibiotics, 5xI0"M 2-mercaptoethanoI, and 1% syngeneic normal mouse serum. Splenic cells were cultured at a final concentration of 2xI0cells/ml in 200/il in round-bottomed 96 wells-plates with different concentrations (10-0.03/tg/nil) of each of flie Ag (VLPs, E7 or HBs antigen). Each test was carried out in quadriplicate. After 96hr of culmre at 31'C under 5% CO,, the cells were pulsed for 18hr with 3H-Thymidine (Amersham, UK. 5Ci/mmoi) at 0.5MCi/well and then harvested on Unifilter plates (Packard) wiOi a cell harvester. Incorporated radioactivity was measured in a scintillation counter (Topconnt.Packard). Results are expressed in cpm (mean cpm in quadriplicate wells) or as stimulation indices (mean cpm in culmres of cells with antigen / mean cpm in cultures of cells without antigen). Cytokine prcduction Two weeks after the second immunisation, mice were killed, spleens were removed aseptically and pooled. Cell suspensions were prepared in RPMI 1640 medium (GIBCO) containing 2mM L-glutamine, antibiotics, 5xlO"M 2-mercaptoethanoi. and 5% foetal calf serum. Cells were cufmred at a final concentration of SxIO* cells/ml, in Iml per flat-bottomed 24 wells-plates with different concentrations (10-l3g/ml) of each of the Ag (VLPs, E7 or HBs antigen). Supematants were harvested 96 hrs later and frozen until tested for the presence of IFNy and IL5 by Elisa. IFNy (Genzyme) Quantitation of IFNy was performed by Elisa using reagents from Genzyme. Samples and antibody solutions were used at 50 3i per well. 96-weIls raicrotiter plates (Maxisorb Immuno-plate, Nunc, Denmark) were coated overnight at 4'C with 5031 of hamster anti-mouse IFNy diluted at 1.5;ig/ml in carbonate buffer pH 9.5. Plates were then incubated for Ihr at ST3C with 100/iI of PBS containing 1% bovine serum albumin and 0.1 % Tween 20 (samration buffer). Two-fold dilutions of supernatant from in vitro stimulation (starting at 1/2) in samration buffer were added to the anti- IFNy -coated plates and incubated for Ihr 30min at 37°C. The plates were washed 4 times with PBS Tween 0.1% (wash buffer) and biotin-conjugaied goat anti-mouse IFNy diluted in samration buffer at a final concentration of 0.53g/ml was added to each well and incubated for Ihr at 31°C. After a washing step, AMDEX conjugate (Amersham) diluted 1/10000 in samration buffer was added for 30 min at 37°C, Plates were washed as above and incubated with 50/iI of TMB (Biorad) for lOmin. The reaction was stopped with H3SO3 0.4N and read at 450/630nm. Concentrations were calculated using a standard curve (mouse IFNy standard) by SofimaxPro (four parameters equation) and expressed in pg/ml. IL5 (Pharmingen) Quantitation of IL5 was performed by Elisa using reagents from Pharmingen. Samples and antibody solutions were used at 50/xl per well. 96-welIs microtiier plates (Maxisorb Immuno-plate, Nunc, Denmark) were coated overnight at 4'C wiih 5031 of rat anti-mouse IL5 diluted at 1/xg/ml in carbonate buffer pH 9.5. Plates were then incubated for Ihr at 37°C with lOO/zl PBS containing 1% bovine serum albumin and 0.1% tweea 20 (saturation buffer).Two-fold dilutions of supernatant from in vitro stimulation (staning at 1/2) in saturation buffer were added to the anti-IL-5-coated plates and incubated for Ihr 30min at 37°C. The plates were washed 4 times with PBS Tween 0.1% (wash buffer) and biotin-conjugated rat anti-mouse IL5 diluted in saturation buffer at a fmal concentration of l3ig/ml was added to each well and incubated for Ihr at 37°C. After a washing step. AMDEX conjugate (Amersham) diluted 1/10000 in saturation buffer was added for 30min at ST3C. Plates were washed as above and incubated with 50/il of TMB (Biorad) for 15 min. The reaction was stopped with H2SO4 0.4N and read at 450/630 mn. Concentrations were calculated using a standard curve (recombinant mouse IL-5) by SofimaxPro (four parameters equation) and expressed in pg/ml. GROUPS Groups of 10 Balb/C mice were immunised intramuscularly with the following formulations: RESULTS 1. Serology a) Anti-HBs respoase: Humoral responses (Ig and isotypes) were measured by Elisa using HBsAg (Hep286) as coating antigen. Day 14 post II sera were analysed. Figure 1 shows the anii-HBs antibody responses measured on individual sera on day 14 post H. No difference was observed in the anti-HBs antibody response between the protocols applied to adsorb the 3D-MPL: on Al(OH)j alone or AIPO4 alone (groups C, D, E) with different ratios of A1(0H)3 and AlPO3 in the vaccine (GMT of 27905 EU/mi versus 30832 or 26670 EU/ml). A slightly lower anti-HBs antibody response is observed in the combination groups G and H containing the VLPs and the HBs antigen compared to HBs alone (group C) (GMT respectively of 10635 or 15589 EU/ml versus 27905 EU/ml). Anti-HBs GMT obtained in the E7/HBs combination reached 19235 EU/ml. Before statistical analysis, a T-Grubbs test was applied on each population for data exclusion. One mouse in group C was eliminated for analysis. A one-way-anaiysis of variance was performed on anti-HBs titers after log transformation of post n data. Significant differences were observed between formulations (p-vaiue= 0.0108) and the Smdent Newman Keuis test was then applied for multiple comparisons. No statistically significant difference was observed between the group H (VLP/HBs) or group F (HBs/E7) combination versus the group C (HBs AS04C). A statistically significant difference was shown between the group G (VI.P/HBs) and the group C (HBs AS04C) (p vaiue=0.0291) however the 95 % confidence intervals of the 2 groups overlap and the difference which reaches a 2.5 ratio might not be biologically relevant. The isotypic repanition analysed on pooled sera was as follows and showed QO major differences berween the 6 groups. b) Anti-E7 response: Humoral responses (Ig and isotypes) were measured by Elisa using PDl/3 I6E7 2M as the coating antigen. Day 14 post n sera of group B and F were analysed. Figure 2 shows the anti-E7 antibody responses measured on individual sera at day 14 post 11: A slight decrease was observed in the anti-E7 response with a two fold decrease in GMT for HBs/E7 combinations compared to E7 alone (9626 versus 22447 EU/ml). This was etablished as statistically insignificant using the Student Newman Keuls test. No difference was observed in the isotypic profile induced by the two formulations: mainly IgGl response (97-98% of IgGl) as reported in the table below. The isotypic repartition analysed on pooled sera was as follows: c) Anti-VLP16 response: Humoral responses (Ig ) were measured by Elisa using VLP16 503-1 (20/12/99) as the coating antigen. Day 14 post II sera were analysed. Figure 3 shows anti-VLP16 Ig antibody responses measured on individual sera on day 14 post II. Similar anti-VLP16 titers were obtained after iramunisaiion with the combination of HBs and VLPs (group G and H) as with the monovalent VLPs formulation (group A) (GMT of 19570 or 23448 EU/ml versus 303 U EU/ml) Equivalent liters were observed between the two combinations prepared using either ways to adsorb the 3D-MPL: A.I(0H)3 alone (group G) compared to mixed adsorption on AKOH), and AlPO3 (group H) (GMT of 19570 EU/ml versus 23448 EU/ml). These differences were shown as statistically not significant using one-way analysis of variance test. d) Anti-VLP18 response: Humoral responses (Ig ) were measured by Elisa using VLP18 504-2 (25/10/99) as the coating antigen. Day 14 post II sera were analysed. Figure 4 shows the anti-VLP18 Ig antibody response measured on individual sera on day 14 post II Similar anti-VLPlS liters were obtained after immunisation with the combination of HBs and VLPs {group G and H) or with the monovalent VLPc fonnulations (group A) (GMT of 37285 or 51202 EU/ml versus 56504 EU/mi) Equivalent titers (group G and H) were observed between the combination prepared using either ways to adsorb the 3D-MPL: AlCOH)3 alone (group G) compared to mixed adsorption on Al(OH), and AlPO3 (group H). These differences were shown as statistically not significant using one-way analysis of variance test. 2. Cell Mediated Immune Response Cell-mediated immune responses (iympboproliferalion, IFNy / IL5 production) were evaluated at day 14 post II after in vitro restimulation of splenic cells with either HBs, E7 or VLPs antigens. For each group of mice, pools of 5 organs were constimted. The experimental procedure is fully described above in Material and Methods. 3. Cytokine production a) In vitro restimulation with HBs Figure 5 shows the cytokine production monitored in splenic cells after 96h in vitro restimulation with HBs. Low IFN-y and IL5 production was observed for all groups but as shown in Table 2 higher production of IFN-Y 3re observed compared to IL-5 production with IFN-y/IL-S ratio indicating that a comparable THl response is induced wiih the monovalent and combined vaccines. The group C results should not be taken into account as data below the treshold may indicate absence of antigen for restimulation. Table 2 : IFN-y/IL-5 ratio after in vitro restimulation with HBs. b) In vitro restimulatioo with E7 Figure 6 shows the cytokine production monitored in splenic cells after 96h in vitro lestimulation with E7 antigen. A dose range effect was observed when comparing the lO\|i.g and l3g Ag dose for restimulation. A non-specific response was observed for HPV16/18 LI VLPs immunised groups using lO3g of Ag for restimulation. The IFN-y is produced in a much higher concentration compared to IL-5 (Table 3) indicating a clear TH-1 profile of the immune response in all groups evaluated (monovalent versus combination). Table 3 : IFN-//IL-5 ratio after in vitro restimulation with E7. c) In vitro restimuUtiou with VLP16 and 18 Figures 7 and 8 show the lymphoproliferation after in vitro restimulation with VLP16 or VLP18 on day 14 post H. Comparable profiles were observed for all the fonnulaiions containing VLPs (Imegraied Stimulated Indexes between 12-29) with cpm around 30000 for lO/zg Ag restimulation dose, indicating the absence of interference between the different formulations on this read-out. Figure 9 shows the cytokine production monitored in splenic cells after 96h in vara restimulation with VLP16. Figure 10 shows the cytokine production monitored in splenic cells after 96h in vitro restimulation with VLP18. No dose range effect has been observed using lO3g and Ifig Ag dose for restimulation with either VLP antigens on both cytoldne production. A clear THI profile was observed with all formulations. Table 5 :IFN-y/IL-5 ratio after in vitro restimulanon with VLP16 and VLP18. CONCLUSIONS The effect of the combination of VLPs/HBs or E7/HBs Ag formulated in AS04 on the immunogenicity was evaluated in Balb/C mice; Regarding the serological analysis, no interference of the Ag combination was observed on anti-HBs, anti-E7 and anti-VLPs serology. The combination of VLPs and HBs or E7 and HBs antigens did not interfere with the isotypic profile of the antibody response displayed by the monovalent vaccine. The method of adsorption of 3D-MPL (A1(0H)3, AlPO,, or mixmres of Al(OH)j and A1P04) did not interfere with the serological results. In the lymphoproliferadon assays, results were available after restimulation with the VLPs. In these groups, no negative effect of the combination of Ag was observed on the proliferative response. For the cytokines evaluation, low cytokine production (IL-5 and IFN-y) was obtained after restimulation with HBs Ag but responses were comparable in the monovalent and combined vaccines. After restimulation with E7 or with VLPs, comparable cytokine levels were produced respectively in the E7/HBs or in the VLP/HBs combination as compared to monovalent groups. The TH-1 profile observed with each monovalent vaccine was conserved in the combination vaccine groups. We claim: 1. A vaccine composition comprising a hepatitis B surface antigen and a human papilloma virus (HPV) antigen, in conjunction with an adjuvant which is a preferential stimulator of TIHI cell response, wherein the vaccine composition does not comprise a herpes simplex viral antigen, wherein the composition is suitable for the treatment or prophylaxis of human papillomavirus infections and hepatitis B infections, and wherein the HPV antigen comprises the major capsid protein LI of HPV from HPV 16 and/or HPV 18, or a fusion of Haemophilus influenza B protein D with E7 from HPV 16. 2. The vaccine composition according to claim 1 which additionally comprises a canier. 3. The vaccine composition according to claim 1 or claim 2 in which the preferential stimulator of THI-cell response is selected from the group of adjuvants comprising: 3D-MPL, 3D-MPL wherein the size of the particles of 3D-MPL is preferably about or less than lOOnm, QS21, a mixture of QS21 and cholesterol, and a CpG oligonucleotide. 4. The vaccine composition according to claim 3 in which the preferential stimulator of TH1-cell response is 3D-MPL. 5. The vaccine composition according to any one of claims 1 to 4 in which an EBV antigen is additionally present. 6. The vaccine composition as defined in claim 5 in which the EBV antigen is gp 350. 7. The vaccine composition according to any one of claims 1 to 4 in which a hepatitis A antigen is additionally present. 8. The vaccine composition according to claim 7 in which the HAV antigen is derived from the HM-175 strain. 9. The vaccine composition according to any one of claims 1 to 8 in which the carrier is selected from the group comprising aluminium hydroxide, aluminium phosphate and tocopherol and an oil in water emulsion. 10. The vaccine composiyon according to claim 1-9 which additionally comprises a VZV antigen. 11. The vaccine composition according to claim 10 in which the VZV antigen is gpl. 12. The vaccine composition according to any one of claims 1 to 11 which additionally comprises a HCMV antigen. 13. T^^s vaccine composition according to claim 12 in which the HCMV antigen is gB685 or pp65. 14. The vaccine composition according to any one of claims 1 to 13 which additionally comprises a Toxoplasma gondii antigen. 15. The vaccine composition according to claim 14in which the Toxop/asma gone///antigen isSAG1orTG34. 16. The vaccine composition acconjing to any one of claims 1 to 4 comprising L2, E6, E7, protein D-E6, protein D-E7 or L2-E7 of HPV and optionally in addition one or more of HSV-2 gDt; EBVgp 350; VZVgpl; HAV HM-175 inactivated strain; gB685" or pp65 of HCMV and SAG1 orTG34 antigens of Toxoplasma gondii.

Full Text

This invention relates to novel vaccine formulations, methods for preparing them and their use in therapy. In particular the present invention relates to combination vaccines for administration to adolescents.
Papillomaviruses are small DNA tumour viruses, which are highly species specific. So far, over 70 individual human papillomavirus (HPV) genotypes have been described. HPVs arc generally specific either for the skin (e.g. HPV-1 and -2) or mucosal surfaces (e.g. HPV-6 and -II) and usually cause benign tumours (warts) that persist for several months or years. Such benign tumours may be distressing for the individuals concerned but tend not to be life threatening, with a few exceptions.
Some HPVs are also associated with cancers. The strongest positive association between an HPV and human cancer is that which exists between HPV-16 and HPV-18 and cervical carcinoma. Cervical cancer is the most common malignancy in developing cotmtries, with about 500.000 new cases occurring in the world each year. It is now technically feasible to actively combat primary HPV-I6 infections, and even established HPV-l6-containing cancers, using vaccines. For a review on the prospects for prophylactic and therapeutic vaccination against HPV-16 see Cason J., Clin. Immunother. 1994; 1(4) 293-306 and Hagenesee M.E., Infections in Medicine 1997 14(7) 555-556.559-564.
Today, the different types of HPVs have been isolated and characterised with the help of cloning systems in bacteria and more recently by PCR amplification. The molecular organisation of the HPV genomes has been defined on a comparative basis with that of the well characterised bovine papillomavirus type 1 (BPVl).
Other HPV serotypes of particular interest are 31, 33 and 45.

Although minor variations do occur, all HPVs genomes described have at least seven early genes. El to E7 and two late genes LI and 12. In addition, an upstream regulatory region harbors the regulatory sequences which appear to control most transcriptional events of the HPV genome.
El and E2 genes are involved in viral replication and transcriptional control, respectively and tend to be disrupted by viral integration. E6 and E7, and recent evidence implicate also E5 are involved in viral transformation.
In the HPVs involved in cervical carcinoma such as HPV 16 and 18, the oncogenic process starts after mtegration of viral DNA. The integration results in the inactivation of genes codmg for the capsid proteins LI and L2 and in mstalling continuous over expression of the two early proteins E6 and E7 that will lead to gradual loss of the normal cellular di3erentiation and the development of the carcinoma.
Carcinoma of the cervix is common in women and develops through a pre¬cancerous intermediate stage to the mvasive carcinoma which frequently leads to death. The intermediate stages of the disease is known as cervical intraepithelial neoplasia and is graded I to HI in terms of increasing severity.
Clinically, HPV infection of the female anogenital traa manifests as cervical flat condylomas, the hallmark of which is the fcoilocytosis affecting predominantly the superficial and intermediate cells of the cervical squamous epithelium.
KoUocytes which are the consequence of a cytopathic effea of the virus, appear as multinucleated cells with a perinuclear clear halo. The epithelium is thickened with abnormal keratinisation responsible for the warty appearance of the lesion.
Such fiat condylomas when positive for the HPV 16 or 18 serotypes, are high-risk factors for the evolution toward cervical intraepithelial neoplasia (CIN) and

carcinoma in situ (CIS) which are themselves regarded as precursor lesions of invasive cervix carcinoma.
WO 96/19496 discloses variants of human papilloma vims E6 and E7 proteins, particularly fusion proteins of E6/E7 with a deletion in both the E6 and E7 proteins. These deleuon fusion proteins are said to be immunogenic.
HPV LI based vaccines are disclosed in WO94/0O152, ■WO94/20137, ■WO93/02184 and WO94/0S792. Such a vaccine can comprise the LI antigen as a monomer, a capsomer or a virus like particle. Such particles may additionally comprise L2 proteins. L2 based vaccines are described for example in WO93/00436. Other HPV vaccines arc based on the Early proteins, such as E7 or fmion proietm such as L2-F7.
Vaccines for the prophylaxis of hepatitis B infections, comprising one or more hepatitis B antigens, arc well known. For example the vaccine Edgerix-B (Trade Mark) from SmithKline Beccham Biologicals is used to prevent Hepatitis B. This vaccine comprises hepatitis B surfece antigen (specifically the 226 amino acid S-antigen described in Harford et. al. in Postgraduate Medical Journal, 19S7, 63 (Suppl. 2). p65-70) and is formulated using aluminium hydroxide as adjuvant.
There is a need for effective combination vaccines to prevent diseases to which adolescents are particularly prone.
The present invention provides a vaccine composition comprising;
[a) a hepatitis B viral (HBV) antigen; and
;b) a human papillomavirus (HPV) antigen
n combination with an adjuvant which is a preferential stunulator of THl cell
esponse.

The vaccine coroposition of the invention is of great benefit for administration to adolescents who may be particularly at risk of HBV, and/or HPV infection.
Optionally the vaccine composition of the invention additionally comprises one or more of a number of other antigens as described below.
It has been found that the vaccine compositions according to the invention surprisingly show no interference, that is to say that the immune response to each antigen in the composition of the invention is essentially the same as that which is obtained by each antigen given individually in conjunction with an adjuvant which is a preferential stimulator of THl cell response.
The vaccine Havrix (Trade Mark), also from. SmithKline Beecham Biologicals is an example of a vaccine that can be used to prevent hepatitis A infections. It i$ formulated with aluminium hydroxide as adjuvant. This vaccine con3rises an attenuated strain of the HM-175 Hepatitis A virus inactivated with formol (formaldehyde); see Andre et. al. (Prog. med. Virol., vol. 37, pl-24).
As used herein, the term hepatitis A viral (HAV) antigen is used to refer to either a protein derived from hepatitis A virus or an attenuated strain of HAV, optionally inactivated, e.g. with formaldehyde. If the HAV antigen is a protein derived from hepatitis A virus it may optionally be a recombinant protein.
The vaccine Twinrix (Trade Mark) is a combination of a recombinant hepatitis B anitgen with the aforementioned inactivated attenuated hepatitis A virus. The vaccine 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.
In a further aspect, the invention provides a vaccine composition comprising:
(a) a hepatite B viral (HBV) antigen;
(b) a human papillomavirus (HPV) antigen; and
(c) a hepatitis A viral (HAV) antigen
in combination with an adjuvant which is a preferential stimulator of TH1 cell response.
Such a vaccine is of great benefit for administration to adolescents who may be particularly at risk of HBV, and/or HPV infection, and/or HAV infection.
An immune response may be broadly divided into two extreme catagories, being a humoral or cell mediated immune response (traditionally characterised by antibody and cellular effeaor mechanisms of protection respectively). These categories of response have been termed THl-type responses (cell-mediated response), and TH2-type immune responses (humoral response).
Extreme THl-type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and namral killer cell responses. In mice THl-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies. TH2-type immune responses are characterised by the generation of a range of immunoglobulin isotypes including in mice IgGl.
It can be considered that the driving force behind the development of these two types of immune responses are cytokines. High levels of THl-type cytokines tend to favour the induction of cell mediated immune responses to the given antigen,

whilst high levels of TH2-type cytokines tend to favour the induction of humoral immune responses to the antigen.
The distinction of THl and TH2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominandy THl or predominantly TH2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4 +ve T cell clones by Mosmann and Coffman (Mosmann, T.R. and Coffinan, R.L. (1989) THl and TH2 cells: different paxtems oflymphokine secretion lead to different Junctional properties. Annual Review of Immunology, 7, pl45-173). Traditionally, THl-type responses are associated with the production of the INF-y cytoldnes by T-Iymphocytes. Other cytokines often directly associated with the induction of THl-type immune responses are not produced by T-ce!ls, such as IL-12. In contrast, TH2- type responses arc associated with the secretion of IL-4, IL-5, IL-6, IL-10 and tumour necrosis factor-P(TNF-p),
It is known that certain vaccine adjuvants are particularly suited to the stimulation of either THl or TH2 - type cytokine responses. Traditionally the best indicators of the TH1:TH2 balance of the immune response after a vaccination or infection includes direct measurement of the production of THl or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or the measurement (at least in mice) of the IgGl:IgG2a ratio of antigen specific antibody responses.
Thus, a THl-type adjuvant is one which stimulates isolated T-cell populations to produce high levels of THl-type cytokines when re-stimulated with antigen in vitro, and induces antigen specific immunoglobulin responses associated with THl-type isotype.
Adjuvants which are capable of preferential stimulation of the TH 1 cell response are described in International Patent Application No. WO 94/00153 and WO 95/17209.

3 De-0-acylated moaophosphoryl lipid A Preferably, the particles of 3D-MPL are small enough to be sterile filtered through a 0.22micron membrane (as described in European Patent number 0 689 454). 3D-MPL will be present in the range of 103ig - lOO3ig preferably 25-50fj.g per dose wherein the antigen will typically be present in a range 2-50fig per dose.
Another preferred adjuvant comprises QS21, an Hpic purified non-toxic fraction derived from the barfc of QuUlaja Saponaria Molina. Optionally this may be admixed with 3 De-0-acylated monophosphoryl lipid A (3D-MPL), optionally together with an carrier.
The method of production of QS21 is disclosed m US patent No. 5,057,540.
Non-reactogenic adjuvant formulations containing QS21 have been described previously (WO 96/33739). Such formulations comprising QS21 and cholesterol have been shown to be successful THI stimulating adjuvants when formulated together with an aatigea. Thus vaccine compositions which form part of the present invention may include a combination of QS21 and cholesterol.
Further adjuvants which are preferential stimulators of THI cell response include unmunomodulatory oligonucleotides, for example unmethylated CpG sequences as disclosed in WO 96/02555.
Combinations of different THI stimulating adjuvants, such as those mentioned hereinabove, are also contemplated as providing an adjuvant which is a preferential

stimulator of THl cell response. For example. QS21 can be formulated together with 3D-MPL. The ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10 : 1; preferably 1:5 to 5 : 1 and often substantially 1 : 1. The preferred range for optima! synergy is 2.5 : I to I : 1 3D-MPL: QS21.
Preferably a carrier is also present in the vaccine composition according to the invention. The carrier may be an oil in water emulsion, or an aluminium salt, such as aluminium phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as squalene, alpha tocopherol and Tweea 80. Additionally the oil in water emulsion may contain span 85 and/or lecithin and/or tricaprylin.
In a particularly preferred aspect the antigens in the vaccine composition according to the invention are combined with 3D-MPL and alum.
Typically for human administration QS21 and 3D-MPL will be present in a vaccine in the range of lug - 200jjg, such as 10-lOOjig, preferably lOiig - 503g per dose. Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is equal to or less than 1 as this provides a more stable emulsion. Span 85 may also be present at a level of I %. In some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser.
Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline.
A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in WO 95/17210.

The HPV antigen in the composition of the invention is preferably derived from HPV 16 and/or IS, or from HPV 6 and/or U, or HPV 31, 33 or 45.
In one preferred embodiment the HPV antigen in the vaccine composition according to the invention comprises the major capsid protein LI of HPV and optionally the L2 protein, particularly from HPV 16 and/or HPV 18. In this embodiment, the preferred form of the LI protein is a truncated LI protein. Preferably the LI is in the form of a virus-like particle (VLP). The LI protein may be fused to another HPV protein, in particular E7 to form an L1-E7 fusion. Chimeric VLPs comprising Ll-E or H-L2-E are particularly preferred.
In another preferred embodiment, the HPV antigen in the composition of the invention is derived from an E6 or E7 protein, in particular E6 or E7 linked to an immunological fusion parmer having T cell epitopes.
In a preferred form of this embodiment of the invention, the immunological fiision parmer is derived from protein D of Heamophilus influenza B. Preferably the protein D derivative comprises approximately the first 1/3 of the protein, in particular approximately the first N-termioal 100-110 amino acids.
Preferred fusion proteins in this embodiment of the invention comprise Protein D -E6 from HPV 16, Protein D - E7 from HPV 16 Protein D - E7 from HPV 18 and Protein D - E6 from HPV 18. The protein D part preferably comprises the first 1/3 of protein D.
In still another embodiment of the invention, the HPV antigen is in the form of an L2-E7 fusion, particularly from HPV 6 and/or HPV U.
The proteins of the present invention preferably are expressed in E. coU. In a preferred embodiment the proteins are expressed with a Histidine tail comprising between 5 to 9 and preferably six Histidine residues. These are advantageous in

aiding purification. The description of the manufacture of such proteins is fully described in co-pending UK patent application number GB 9717953.5.
The HPV antigen in the vaccine composition may be adsorbed onto Al(0H)3. Preferably the LI VLP is adsorbed onto AKOH),.
The hepatitis B viral (HBV) antigen in the composition of the invention is typically hepatitis B surfece 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 herem the expression 'Hepatitis B surface antigen', abbreviated herein to 'HBsAg' or 'HBS' includes any HBsAg antigen or fragment thereof displaying the antigenicity of HBV surfece antigen. It will be understood that in addition to the 226 amino acid sequence of the HBsAg S antigen (sec Tiollais et. al. Nature, 317, 489 (1985) and references therein) HBsAg as herein described niay, 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 fiirther aspect the HBsAg may comprise a protein described as L"* 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) protem (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 0 304 378.
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 defmed above and S denotes the S-protein of hepatitis B surface antigen.
The HBsAg may be adsorbed on aluminium phosphate as described in W093/24148.
Preferably the hepatitis B (HBV) antigen used in the formulation of the invention is HBsAg S-antigen as used in the commercial product Engerix-B (Trade Mark; SmithKline Beecham Biologicals).
A vaccine comprising hepatitis B surface antigen in conjunction with 3D-MPL was described in European Patent Application 0 633 784.
Examples of antigens from additional pathogens which may be included in the compositions according to the invention are now described.
Epstein Barr Virus (EBV). a member of the herpesvirus group, causes infectious mononucleosis as a primary disease in humans. I3edominantly it affects children or young adults. More than 90% of the average adult population is infected by EBV that persists for lifetime in peripheral B-lymphocytes. The virus is lifelong produced in die parotid gland and spread primarily by exchange of saliva from individuals who shed the virus. Children infected with EBV are largely asymptomatic or have very mild symptoms, while adolescents and adults who become infected develop typical infectious mononucleosis, characterised by fever, pharyngitis, and adenopathy. People who have been infected maintain anti-EBV antibodies for the remainder of their lives, and are thus immune to further infection.
In addition to its infectious qualities, EBV has been shown to transform lymphocytes into rapidly dividing cells and has therefore been implicated in several

different iymphcmas, including African Burkitt's lymphoma (BL). EBV may also be involved in causing nasopharyngeal carcinoma (NPC). Worldwide it is estimated that 80,000 cases of nasopharyngeal carcinoma occur and it is more prevalent in ethnic Chinese populations. Infectious mononucleosis is a consequence of primary infection by EBV. It is not a life-threatening disease if additional risk fectors are absent.
Four proteins of the EBV viral envelope coostituting the so-called membrane antigen complex have been described. They are usually referred to as gp 220/350 or gp 250/350 or simply as gp 250 or 350 (see EP-A-151079). There is convincing evidence that gp 350 and gp 250 induce the production of neutralising antibodies and that antibodies against gp 350 and gp 250 have neutralising capacity. These proteins are thus candidates for a possible EBV vaccine. For further inforraation about the application of gp 250/350 for prophylaxis and treatment of EBV-related diseases see EP 0 173 254.
The major EBV surface glycoprotein gp350/220 infects human target cells through interaction with the cellular membrane protein. CD21. Gp350/220 is the primary target for EBV-neutralising antibodies in humans and some forms of gp350/220 have been shown to protect against EBV-related disease. Preferably a vaccine composition according to the invention comprises gp 350 of EBV although other protective antigens may be used.
HSV-2 is the primary etiological agent of herpes genitalis. HSV-2 and HSV-1 (the causative agent of herpes labialis) are characterised by their ability to induce both acute diseases and to establish a latent infection, primarily in nem"onal ganglia cells.
Genital herpes is estimated to occur in about 5 million people in the U.S.A. alone with 5CX),000 clinical cases recorded every year (primary and recurrent infection). Primary infection typically occurs after puberty and is characterised by the localised appearance of painful skin lesions, which persist for a period of between 2 to 3 weeks. Within the following six months after primary infection 50 % of patients

will experience a recurrence of the disease. About 25 % of patients may experience between 10-15 recurrent episodes of the disease each year. In inununocompromised patients the incidence of high frequency recurrence is statistically higher than In the normal patient population.
Both HSV-1 and HSV-2 virus have a number of glycoprotein corapoaents located on the surface of the virus. These are known as gB, gC, gD and gE etc.
When an HSV antigen is included in the composition of the invention this is preferably derived from HSV-2, typically glycoprotein D. Glycoprotein D is located on the viral membrane, and is also found in the cytoplasm of infected ceils (Eisenberg R.J. et al; J of Virol 1980, 353, 428-435). It comprises 393 amino acids including a signal peptide and has a molecular weight of approximately 60 kD. Of all the HSV envelope glycoproteins this is probably the best characterised (Cohen et al; J. of Virology, 60, 157-166). In vivo it is known to play a central role in viral attachment to cell membranes. Moreover, glycoprotein D has been shown to be able to elicit neutralising antibodies in vivo (Eing et al J. Med. Virology 127: 59-65). However, latent HSV-2 virus can still be reactivated and induce recurrence of the disease despite the presence of high neutralising antibodies titre in the patients sera,
In one embodiment of the invention there is present a truncated HSV-2 glycoprotein D of 308 amino acids which comprises amino acids 1 through 306 naturally occurring glycoprotein with the addition Asparagine and Glutamine at the C terminal end of the truncated protein devoid of its membrane anchor region. This form of the protein includes the signal peptide which is cleaved to yield a mature 283 amino acid protein. The production of such a protein in Chinese Hamster ovary cells has been described in Genentech's Etiropean patent EP-B-139 417.
The recombinant mamre HSV-2 glycoprotein D truncate is preferably used in the vaccine formulations of the present invention and is designated rgD2t.

A corr3ination of this HSV-2 antigen in combination with the adjuvant 3D-MPL has been described in WO 92/16231.
In a preferred aspect the vaccine composition of the invention additionally comprises a Varicella Zoster viral antigen (VZV antigea). Suitable antigens of VZV for inclusion in the vaccine formulation include gpI-V described by Longnecfcer et al.. Proc Natl Acad Sci USA 84. 4303-4307 (1987).
In a preferred embodiment gpl (see Ellis ei al., US patent 4.769,239) is used. See also European Patent No. 0 405 867 Bl.
In another preferred aspect the vaccine composition of the invention additionally comprises a himian cytomegalovirus (HCMV) antigen. HCMV is a human DNA virus belonging to the femily of herpes viruses. HCMV is endemic in most parts of the world. Among two populatiotK, HCMV is responsible for serious medical conditions. HCMV is a major cause of congenital defects in new boms. The second population at risk are immunocompromised patients such as those suffering from HIV infection and those patients undergoing transplantations. The clinical disease causes a variety of symptoms including fever, hepatitis, pnemnonitis and mfectious mononucleosis. A preferred antigen for use in a vaccine against HCMV is gB685** as described in WO 95/31555. Immimogens for use in HCMV vaccines arc also provided by pp65, an HCMV Matrix Protein as described in WO 94/00150 (City of Hope).
In one preferred aspect the vaccine composition of the invention additionally comprises both a VZV and an HCMV antigen, in particular those antigens described above.
In another preferred aspect the vaccine composition of the invention additionally comprises a Toxoplasma gondii antigen. Toxoplasma gondii is an obligate intracellular protozoan parasite responsible for toxoplasmosis in warm-blooded animals, including man. AlthougU it is generally clinically asymptomatic in healthy

individuals, toxoplasmosis may cause severe complications in pregnant women and unmunocomproraised patients. A. preferred antigen for use in a vaccine against Toxoplasma gondii is SAGl ( also known as P30) as described in WO96/02654 or Tg34 as described in W092/U366.
In one preferred aspect the vaccine composition of the invention additionally comprises either a VZV antigen or an HCMV antigen combined with a Toxoplasma gondii antigen, in panicular those antigens described above.
In a preferred aspect the vaccine composition of the invention is a multivalent vaccine, for example a tetra- or pentavalent vaccine.
The formulations of the present invention are very effective in inducing protective immunity, even with very low doses of antigen (e.g. as low as 5\ig rgD2t).
They provide excellent protection against primary infection and stimulate, advantageously both specific humoral (neutralising antibodies) and also effector cell mediated (DTH) immune responses.
The present invention in a further aspect provides a vaccine formulation as herein described for use in medical therapy, particularly for use in the treatment or prophylaxis of human papillomavirus infections and hepatitis B virus infections.
The vaccine of the present invention will contain an immunoproteciive quantity of the antigens and may be prepared by conventional techniques.
Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol.61 Vaccine Design - the subunii and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, edited by VoUer et al.. University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by FuUerton, U.S. Patent

4,235,577. 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 amount of procem in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount wiii vary depending upon which specific immunogen is employed. Generally, it is expected that each dose will comprise I-lOOOjig of protein, preferably 2-lOOiJg, most preferably 4-403g. 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 addition to vaccination of persons susceptible to HPV or HBV infections, the pharmaceutical compositions of the present invention may be used to treat, immunotherapeutically, patients suffering from the said viral infections.
In a further aspect of the present invention there is provided a method of manufacture as herein described, wherein the method comprises mixing a human papilloma virus antigen and a hepatitis B virus antigen with a TH-1 inducing adjuvant, for example 3D-MPL and, preferably, a carrier, for example alum.
If desired, other antigens may be added, in any convenient order, to provide multivalent vaccine compositions as described herein.
The following example illustrates but does not limit the invention.

Example I: Comparative immopogenicity of HPV Ags / HBs combos formulated with Alum/3 D-MPL.
INTRODUCTION
An immunogenicity study was performed in Balb/C mice using four different antigens:
1. HPV16 LI Virus Like Particule (VLP-16)
2. HPV18L1 Virus Like PanicuIe(VLP18)
3. PD 1/3 16E7 2M from HPV-16 (E7)
4. HBsAg
formulated with Alum/3D-MPL (AS04) using pre adsorbed monobulks of antigen or 3D-MPL on AKOH), or AIPO«.
3D-MPL/Al(OH)3 formulations are referred to as AS04D whereas SD-MPL/AIPO, based fonnuiations are refened to as AS04C.
The following vaccines were assessed:
1. VLP16 + VLP18 AS04D;
2. E7 based formuialions,
3. HBs AS04C
and the potential to combine these vaccines was evaluated.
The aims of this experiment were as follows:
1) To compare the immunogenicity of different AS04 combinations of either VLP16 +VLP18 or E7 and HBs Ag.
2) As the monovalent vaccines are either formulated in AS04C or AS04Dr

to compare the immunogenicity of different HBs AS04 formulations made of AIPOj or a mix of AlP04/A!(0H)j with different ratios of Alum forms; and
to evaluate the effect of the adsorption of 3D-MPL on a fi-action of AI(0H)3 and ALPO4 versus 3D-MPL / A1(0H)3 in combination containing VLPs or E7 antigens.
The experimental protocol is fully described in the Material and Methods section.
In summary, groups of 10 mice were immunised intramuscularly twice at 3 week intervals with various Ag based formulations (I/IOHD). Antibody response to HBs, E7 and VLPs Ag and the isotypic profile induced by vaccination were monitored by ELISA at day 14 post 11. At die same timepoint, the CMI (lymphoproliferative response or the cytokine production (IFNy/IL5)) was analysed after in vitro restimulation of splenic cells with either HBs, VLPs or E7 antigen.
MATERIALS AND METHODS
Formulation Formulation compositions
VLP16. VLP18, PDl/3-HPV16E7-His, and HBs on AS04C or AS04D.

Adsorption.
a) VLP adsorption.
VLP 16 and VLP 18 purified bulk is added to Al(OH)i at 23g VLP/lO3g Al(OH)j. The mixture is stored between Z-S'C until final formulation.
b) HBs adsorption.
2 fi% Hbs are mixed with 40;ig AIPO,. The mixture is stored between 2-8'C until final formulation.
2 /ig Hbs are mixed with 10/tg AIPO4. The mixmre is stored between 2-8'C until final formulation.
c) PDl/3-HPV16E7-His adsorption.
2 ng E7 are mixed widi lO/ig Al(OH)). The mixture is stored between 2-8°C until final formulation.
d) 3D-MPL adsorption.
5fig 3D-MPL are mixed with lO3g Ai(OH)j. The mixture is stored between 2-8'C until fmal formulation.

53g 3D-MPL are mixed with lO3ig AlPOj. The mixture is stored between 2-8°C
until final formulation.
2.5/xg 3D-MPL are mixed with 53g A1{0H)3. The mixture is stored between 2-8'C
until final formulation.
2.5fig 3D-MPL are mixed with 53g AIPO4. The mixture is stored between 2*8°C
until final formulation.
Formulation
HjO and NaCl are mixed (lOx concentrated) and after 10 minutes of agitation at room temperature, the different components are added: adsorbed antigen, adsorbed 3D-MPL and A1(0H)3 (See table below). They are shaken at room temperamre for 10 minutes and stored at 4°C imtil injection. The in vitro characterisation of the formulation can then be performed.

Mice Serology
Aoti-HBs serology
The quantitation of anti-HBs antibodies was performed by ELISA using HBs (Hep 286) as the coating antigen. Antigen and antibody solutions were 33ssd at 5031 per well. The antigen was diluted at a final concentration of Ijig/nil in PBS and was adsorbed overnight at 4°C to the wells of 96 wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plates were then incubated for Ihr at 37°C with PBS containing 1 % bovine serum albumin and 0.1% Tween 20 (saturation buffer). Two-fold dilutions of sera (staning at 1/100 dilution) in the saturation buffer were added to the HBs-coated plates and incubated for Ihr 30 min at ST3C. The plates were washed four times with PBS 0.1% Tween 20 and bioiin-conjugated anti-mouse Ig (Amersham, UK) diluted 1/1500 or IgGl, IgG2a, rgG2b GMTECH, USA) diluted respectively at 1/4000, 1/8000. 1/4000 in saturation buffer were added to each well and incubated for Ihr 30 min at ST3C. After a washing step, streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted 1/1000 in saturation buffer was added for an additional 30min at 21'C. Plates were washed as above and incubated for 20niin with a solution of o-phenylenediamine (Sigma)

0.04% HA0-P3% in 0.1% tween 20 0.05M citrate buffer pH4.5. The reaction was stopped with H.SO3 2N and read at 490/630 nm. ELISA liters were calculated from a reference by SoftmaxPro (using a four parameters equation) and expressed in EU/ml.
ADti-E7 serology
Quantitation of anti-E7 antibody was performed by ELISA using PDl/3 16E7 2M as coating antigen. Antigen and antibody solutions were used at lOO3il per weil. The antigen was diluted at a final concentration of 0.5/ig/ml in PBS and was adsorbed overnight at 4°C to the wells of 96 wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plates were then incubated for Ihr at 37°C with PBS containing 1 % bovine serum albumin and 0.1 % Tween 20 (saturation buffer). Two¬fold dilutions of sera (starting at 1/100 or 1/400 dilution) in the saturation buffer were added to the E7-coated plates and incubated for Ihr 30 min at 37'C. The plates were washed four times with PBS 0.1% Tween 20 and biotin-conjugated anti-mouse Ig, IgGl, IgG2a, IgG2b (Amersham. UK) diluted 1/1500 in saturation buffer were added to each well and incubated for Ihr 30 min at 37'C. After a washing step, streptavidin-biotmylated peroxydase complex (Amersham, UK) diluted 1/5(XX) m saturation buffer was added for an additional 30 min at 37'C. Plates were washed as above and incubated for 20min with a solution of Tetramethyl benzidine (TMB) (Biorad, USA) 2-fold diluted in Citrate buffer (O.IM pH=5.8). The reaction was stopped with H1SO4 0.5 N and read at 450/630 nm. ELISA titers were calculated from a reference by SoftmaxPro (using a four parameters equation) and expressed in EU/ml.
Aiiti-VLP16 and anti-VLPlS serology
The quantitation of anti-VLP16 and anti-VLPIS antibodies was performed by ELISA using VLP16 503/1 (20/12/99) ands VLP18 504/2 (25/10/99F) as coating antigens. The antigen and antibody solutions were used at 50;il per well. The antigen was diluted at a final concentration of 0.53g/m] in PBS and was adsorbed overnight at 4'*C to the wells of 96 wells microtiter plates (Maxisorb Immuno-plate,

Nunc, Denmark). The plates were then incubated for Ihr at ST'C with PBS containing 1% bovine serum albumin. Two-fold dilutions of sera (starting at 1/400 dilution) in the samration buffer were added to the VLPs-coated plates and incubated for Ihr 30min at 37'C. The plates were washed four times with PBS 0.1% Tween 20 and biotin-conjugated anti-mouse Ig (Amersham, UK) diluted 1/1500 in saturation buffer were added to each well and incubated for Ihr 30min at Sy3C. After a washing step, streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted 1/1000 in saturation buffer was added for an additional SOinin at 37'*C. Plates were washed as above and incubated for 20min with a solution of o-phenylenediamine (Sigma) 0.04% HiOj 0.03% in 0.1% tween 20 0.05M citrate buffer pH4.5. The reaction was stopped with H3SO3 2N and read at 490/630 imi. ELISA titers were calculated from a reference by SoftmaxPro (using a four parameters equation) and expressed in EU/ml.
T cell proliferation
Two weeks after the second immunisation, mice were killed, spleens were removed aseptically and pooled (1 pool of 5 organs per group). Cell suspensions were prepared in RPMI1640 medium (GEBCO) containing 2mM L-glutamine, antibiotics, 5xI0"*M 2-mercaptoethanoI, and 1% syngeneic normal mouse serum. Splenic cells were cultured at a final concentration of 2xI0*cells/ml in 200/il in round-bottomed 96 wells-plates with different concentrations (10-0.03/tg/nil) of each of flie Ag (VLPs, E7 or HBs antigen). Each test was carried out in quadriplicate. After 96hr of culmre at 31'C under 5% CO,, the cells were pulsed for 18hr with 3H-Thymidine (Amersham, UK. 5Ci/mmoi) at 0.5MCi/well and then harvested on Unifilter plates (Packard) wiOi a cell harvester. Incorporated radioactivity was measured in a scintillation counter (Topconnt.Packard). Results are expressed in cpm (mean cpm in quadriplicate wells) or as stimulation indices (mean cpm in culmres of cells with antigen / mean cpm in cultures of cells without antigen).

Cytokine prcduction
Two weeks after the second immunisation, mice were killed, spleens were removed aseptically and pooled. Cell suspensions were prepared in RPMI 1640 medium (GIBCO) containing 2mM L-glutamine, antibiotics, 5xlO"*M 2-mercaptoethanoi. and 5% foetal calf serum. Cells were cufmred at a final concentration of SxIO* cells/ml, in Iml per flat-bottomed 24 wells-plates with different concentrations (10-l3g/ml) of each of the Ag (VLPs, E7 or HBs antigen). Supematants were harvested 96 hrs later and frozen until tested for the presence of IFNy and IL5 by Elisa.
IFNy (Genzyme)
Quantitation of IFNy was performed by Elisa using reagents from Genzyme. Samples and antibody solutions were used at 50 3i per well. 96-weIls raicrotiter plates (Maxisorb Immuno-plate, Nunc, Denmark) were coated overnight at 4'C with 5031 of hamster anti-mouse IFNy diluted at 1.5;ig/ml in carbonate buffer pH 9.5. Plates were then incubated for Ihr at ST3C with 100/iI of PBS containing 1% bovine serum albumin and 0.1 % Tween 20 (samration buffer). Two-fold dilutions of supernatant from in vitro stimulation (starting at 1/2) in samration buffer were added to the anti- IFNy -coated plates and incubated for Ihr 30min at 37°C. The plates were washed 4 times with PBS Tween 0.1% (wash buffer) and biotin-conjugaied goat anti-mouse IFNy diluted in samration buffer at a final concentration of 0.53g/ml was added to each well and incubated for Ihr at 31°C. After a washing step, AMDEX conjugate (Amersham) diluted 1/10000 in samration buffer was added for 30 min at 37°C, Plates were washed as above and incubated with 50/iI of TMB (Biorad) for lOmin. The reaction was stopped with H3SO3 0.4N and read at 450/630nm. Concentrations were calculated using a standard curve (mouse IFNy standard) by SofimaxPro (four parameters equation) and expressed in pg/ml.
IL5 (Pharmingen)
Quantitation of IL5 was performed by Elisa using reagents from Pharmingen. Samples and antibody solutions were used at 50/xl per well. 96-welIs microtiier plates (Maxisorb Immuno-plate, Nunc, Denmark) were coated overnight at 4'*C

wiih 5031 of rat anti-mouse IL5 diluted at 1/xg/ml in carbonate buffer pH 9.5. Plates were then incubated for Ihr at 37°C with lOO/zl PBS containing 1% bovine serum albumin and 0.1% tweea 20 (saturation buffer).Two-fold dilutions of supernatant from in vitro stimulation (staning at 1/2) in saturation buffer were added to the anti-IL-5-coated plates and incubated for Ihr 30min at 37°C. The plates were washed 4 times with PBS Tween 0.1% (wash buffer) and biotin-conjugated rat anti-mouse IL5 diluted in saturation buffer at a fmal concentration of l3ig/ml was added to each well and incubated for Ihr at 37°C. After a washing step. AMDEX conjugate (Amersham) diluted 1/10000 in saturation buffer was added for 30min at ST3C. Plates were washed as above and incubated with 50/il of TMB (Biorad) for 15 min. The reaction was stopped with H2SO4 0.4N and read at 450/630 mn. Concentrations were calculated using a standard curve (recombinant mouse IL-5) by SofimaxPro (four parameters equation) and expressed in pg/ml.
GROUPS
Groups of 10 Balb/C mice were immunised intramuscularly with the following formulations:

RESULTS
1. Serology
a) Anti-HBs respoase:
Humoral responses (Ig and isotypes) were measured by Elisa using HBsAg (Hep286) as coating antigen. Day 14 post II sera were analysed.
Figure 1 shows the anii-HBs antibody responses measured on individual sera on day 14 post H.
No difference was observed in the anti-HBs antibody response between the protocols applied to adsorb the 3D-MPL: on Al(OH)j alone or AIPO4 alone (groups C, D, E) with different ratios of A1(0H)3 and AlPO3 in the vaccine (GMT of 27905 EU/mi versus 30832 or 26670 EU/ml).
A slightly lower anti-HBs antibody response is observed in the combination groups G and H containing the VLPs and the HBs antigen compared to HBs alone (group C) (GMT respectively of 10635 or 15589 EU/ml versus 27905 EU/ml). Anti-HBs GMT obtained in the E7/HBs combination reached 19235 EU/ml.
Before statistical analysis, a T-Grubbs test was applied on each population for data exclusion. One mouse in group C was eliminated for analysis.
A one-way-anaiysis of variance was performed on anti-HBs titers after log transformation of post n data. Significant differences were observed between formulations (p-vaiue= 0.0108) and the Smdent Newman Keuis test was then applied for multiple comparisons. No statistically significant difference was observed between the group H (VLP/HBs) or group F (HBs/E7) combination versus the group C (HBs AS04C). A statistically significant difference was shown between

the group G (VI.P/HBs) and the group C (HBs AS04C) (p vaiue=0.0291) however the 95 % confidence intervals of the 2 groups overlap and the difference which reaches a 2.5 ratio might not be biologically relevant.
The isotypic repanition analysed on pooled sera was as follows and showed QO major differences berween the 6 groups.

b) Anti-E7 response:
Humoral responses (Ig and isotypes) were measured by Elisa using PDl/3 I6E7 2M as the coating antigen. Day 14 post n sera of group B and F were analysed.
Figure 2 shows the anti-E7 antibody responses measured on individual sera at day 14 post 11:
A slight decrease was observed in the anti-E7 response with a two fold decrease in GMT for HBs/E7 combinations compared to E7 alone (9626 versus 22447 EU/ml). This was etablished as statistically insignificant using the Student Newman Keuls test.
No difference was observed in the isotypic profile induced by the two formulations: mainly IgGl response (97-98% of IgGl) as reported in the table below.
The isotypic repartition analysed on pooled sera was as follows:

c) Anti-VLP16 response:
Humoral responses (Ig ) were measured by Elisa using VLP16 503-1 (20/12/99) as the coating antigen. Day 14 post II sera were analysed.
Figure 3 shows anti-VLP16 Ig antibody responses measured on individual sera on day 14 post II.
Similar anti-VLP16 titers were obtained after iramunisaiion with the combination of HBs and VLPs (group G and H) as with the monovalent VLPs formulation (group A) (GMT of 19570 or 23448 EU/ml versus 303 U EU/ml)
Equivalent liters were observed between the two combinations prepared using either ways to adsorb the 3D-MPL: A.I(0H)3 alone (group G) compared to mixed adsorption on AKOH), and AlPO3 (group H) (GMT of 19570 EU/ml versus 23448 EU/ml).
These differences were shown as statistically not significant using one-way analysis of variance test.
d) Anti-VLP18 response:
Humoral responses (Ig ) were measured by Elisa using VLP18 504-2 (25/10/99) as the coating antigen. Day 14 post II sera were analysed.
Figure 4 shows the anti-VLP18 Ig antibody response measured on individual sera on day 14 post II

Similar anti-VLPlS liters were obtained after immunisation with the combination of HBs and VLPs {group G and H) or with the monovalent VLPc fonnulations (group A) (GMT of 37285 or 51202 EU/ml versus 56504 EU/mi)
Equivalent titers (group G and H) were observed between the combination prepared using either ways to adsorb the 3D-MPL: AlCOH)3 alone (group G) compared to mixed adsorption on Al(OH), and AlPO3 (group H).
These differences were shown as statistically not significant using one-way analysis of variance test.
2. Cell Mediated Immune Response
Cell-mediated immune responses (iympboproliferalion, IFNy / IL5 production) were
evaluated at day 14 post II after in vitro restimulation of splenic cells with either
HBs, E7 or VLPs antigens. For each group of mice, pools of 5 organs were
constimted.
The experimental procedure is fully described above in Material and Methods.
3. Cytokine production
a) In vitro restimulation with HBs
Figure 5 shows the cytokine production monitored in splenic cells after 96h in vitro
restimulation with HBs.
Low IFN-y and IL5 production was observed for all groups but as shown in Table 2 higher production of IFN-Y 3re observed compared to IL-5 production with IFN-y/IL-S ratio indicating that a comparable THl response is induced wiih the monovalent and combined vaccines. The group C results should not be taken into account as data below the treshold may indicate absence of antigen for restimulation.

Table 2 : IFN-y/IL-5 ratio after in vitro restimulation with HBs.

b) In vitro restimulatioo with E7
Figure 6 shows the cytokine production monitored in splenic cells after 96h in vitro lestimulation with E7 antigen.
A dose range effect was observed when comparing the lO|i.g and l3g Ag dose for restimulation.
A non-specific response was observed for HPV16/18 LI VLPs immunised groups using lO3g of Ag for restimulation.
The IFN-y is produced in a much higher concentration compared to IL-5 (Table 3) indicating a clear TH-1 profile of the immune response in all groups evaluated (monovalent versus combination).
Table 3 : IFN-//IL-5 ratio after in vitro restimulation with E7.

c) In vitro restimuUtiou with VLP16 and 18
Figures 7 and 8 show the lymphoproliferation after in vitro restimulation with VLP16 or VLP18 on day 14 post H.

Comparable profiles were observed for all the fonnulaiions containing VLPs (Imegraied Stimulated Indexes between 12-29) with cpm around 30000 for lO/zg Ag restimulation dose, indicating the absence of interference between the different formulations on this read-out.
Figure 9 shows the cytokine production monitored in splenic cells after 96h in vara restimulation with VLP16.
Figure 10 shows the cytokine production monitored in splenic cells after 96h in vitro restimulation with VLP18.
No dose range effect has been observed using lO3g and Ifig Ag dose for restimulation with either VLP antigens on both cytoldne production. A clear THI profile was observed with all formulations.
Table 5 :IFN-y/IL-5 ratio after in vitro restimulanon with VLP16 and VLP18.

CONCLUSIONS
The effect of the combination of VLPs/HBs or E7/HBs Ag formulated in AS04 on the immunogenicity was evaluated in Balb/C mice;
Regarding the serological analysis, no interference of the Ag combination was observed on anti-HBs, anti-E7 and anti-VLPs serology.

The combination of VLPs and HBs or E7 and HBs antigens did not interfere with the isotypic profile of the antibody response displayed by the monovalent vaccine.
The method of adsorption of 3D-MPL (A1(0H)3, AlPO,, or mixmres of Al(OH)j and A1P04) did not interfere with the serological results.
In the lymphoproliferadon assays, results were available after restimulation with the VLPs. In these groups, no negative effect of the combination of Ag was observed on the proliferative response.
For the cytokines evaluation, low cytokine production (IL-5 and IFN-y) was obtained after restimulation with HBs Ag but responses were comparable in the monovalent and combined vaccines. After restimulation with E7 or with VLPs, comparable cytokine levels were produced respectively in the E7/HBs or in the VLP/HBs combination as compared to monovalent groups. The TH-1 profile observed with each monovalent vaccine was conserved in the combination vaccine groups.

We claim:
1. A vaccine composition comprising a hepatitis B surface antigen and a human
papilloma virus (HPV) antigen, in conjunction with an adjuvant which is a preferential
stimulator of TIHI cell response, wherein the vaccine composition does not comprise a
herpes simplex viral antigen, wherein the composition is suitable for the treatment or
prophylaxis of human papillomavirus infections and hepatitis B infections, and wherein the
HPV antigen comprises the major capsid protein LI of HPV from HPV 16 and/or HPV 18, or
a fusion of Haemophilus influenza B protein D with E7 from HPV 16.
2. The vaccine composition according to claim 1 which additionally comprises a canier.
3. The vaccine composition according to claim 1 or claim 2 in which the preferential stimulator of THI-cell response is selected from the group of adjuvants comprising: 3D-MPL, 3D-MPL wherein the size of the particles of 3D-MPL is preferably about or less than lOOnm, QS21, a mixture of QS21 and cholesterol, and a CpG oligonucleotide.

4. The vaccine composition according to claim 3 in which the preferential stimulator of TH1-cell response is 3D-MPL.
5. The vaccine composition according to any one of claims 1 to 4 in which an EBV antigen is additionally present.
6. The vaccine composition as defined in claim 5 in which the EBV antigen is gp 350.
7. The vaccine composition according to any one of claims 1 to 4 in which a hepatitis A antigen is additionally present.
8. The vaccine composition according to claim 7 in which the HAV antigen is derived from the HM-175 strain.
9. The vaccine composition according to any one of claims 1 to 8 in which the carrier is selected from the group comprising aluminium hydroxide, aluminium phosphate and tocopherol and an oil in water emulsion.
10. The vaccine composiyon according to claim 1-9 which additionally comprises a VZV
antigen.

11. The vaccine composition according to claim 10 in which the VZV antigen is gpl.
12. The vaccine composition according to any one of claims 1 to 11 which additionally comprises a HCMV antigen.
13. T^^s vaccine composition according to claim 12 in which the HCMV antigen is gB685* or pp65.

14. The vaccine composition according to any one of claims 1 to 13 which additionally comprises a Toxoplasma gondii antigen.
15. The vaccine composition according to claim 14in which the Toxop/asma gone///antigen isSAG1orTG34.
16. The vaccine composition acconjing to any one of claims 1 to 4 comprising L2, E6, E7,
protein D-E6, protein D-E7 or L2-E7 of HPV and optionally in addition one or more of HSV-2
gDt; EBVgp 350; VZVgpl; HAV HM-175 inactivated strain; gB685" or pp65 of HCMV and
SAG1 orTG34 antigens of Toxoplasma gondii.

Documents:

in-pct-2002-0335-che abstract.pdf

in-pct-2002-0335-che claims-duplicate.pdf

in-pct-2002-0335-che claims.pdf

in-pct-2002-0335-che correspondence-others.pdf

in-pct-2002-0335-che correspondence-po.pdf

in-pct-2002-0335-che description (complete)-duplicate.pdf

in-pct-2002-0335-che description (complete).pdf

in-pct-2002-0335-che drawings.pdf

in-pct-2002-0335-che form-1.pdf

in-pct-2002-0335-che form-18.pdf

in-pct-2002-0335-che form-26.pdf

in-pct-2002-0335-che form-3.pdf

in-pct-2002-0335-che form-5.pdf

in-pct-2002-0335-che pct search report.pdf

in-pct-2002-0335-che pct.pdf

in-pct-2002-0335-che petition.pdf

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Patent Number

222559

Indian Patent Application Number

IN/PCT/2002/335/CHE

PG Journal Number

47/2008

Publication Date

21-Nov-2008

Grant Date

14-Aug-2008

Date of Filing

05-Mar-2002

Name of Patentee

SMITHKLINE BEECHAM BIOLOGICALS S A

Applicant Address

RUE DE L'INSTITUT 89, B-1330 RIXENSART,

Inventors:

#	Inventor's Name	Inventor's Address
1	WETTENDORFF MARTINE, ANNE CECILE	RUE DE L'INSTITUT 89, B-1330 RIXENSART,

PCT International Classification Number

A61K39/00

PCT International Application Number

PCT/EP00/08728

PCT International Filing date

2000-09-06

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	GB 99211477.6	1999-09-07	U.K.