Vaccine against HPV

Abstract: HPV vaccines consisting of an HPV 16 L1 VLP, an HPV 18 L1 VLP and the combination of both with aluminium hydroxide and 3D MPL.

Description: 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 are generally specific either for the skin (e.g. HPV-1 and -2) or mucosal surfaces (e.g. HPV-6 and -11) 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 countries, with about 500,000 new cases occurring in the world each year. It is now technically feasible to actively combat primary HPV-16 infections, and even established HPV-16-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. Other HPVs of particular interest are serotypes 31,33 and 45. 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 (BPV1). Although minor variations do occur, all HPVs genomes described have at least seven early genes, E1 to E7 and two late genes L1 and L2. In addition, an upstream regulatory region harbors the regulatory sequences which appear to control most transcriptional events of the HPV genome. E1 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 integration of viral DNA. The integration results in the inactivation of genes coding for the capsid proteins L1 and L2 and in installing continuous over expression of the two early proteins E6 and E7 that will lead to gradual loss of the normal cellular differentiation and the development of the carcinoma. Carcinoma of the cervix is common in women and develops through a pre-cancerous intermediate stage to the invasive carcinoma which frequently leads to death. The intermediate stages of the disease is known as cervical intraepithelial neoplasia and is graded I to III in terms of increasing severity. Clinically, HPV infection of the female anogenital tract manifests as cervical flat condylomas, the hallmark of which is the koilocytosis affecting predominantly the superficial and intermediate cells of the cervical squamous epithelium. Koilocytes which are the consequence of a cytopathic effect 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 flat 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 virus E6 and E7 proteins, particularly fusion proteins of E6/E7 with a deletion in both the E6 and E7 proteins. These deletion fusion proteins are said to be immunogenic. HPV L1 based vaccines are disclosed in WO94/00152, WO94/20137, WO93/02184 and WO94/05792. Such a vaccine can comprise the L1 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 are based on the Early proteins, such as E7 or fusion proteins such as L2-E7. Roden R B S et al: ("Assessment of the serological relatedness of genital human papillomaviruses by hemagglutination inhibition" Journal of Virology, The American Society for Microbiology, us, vol. 70, no. 5, May 1996 (1996-05), pages 3298-3301) discloses experiments to consider cross protection among genital HPV types in VLP based vaccinations. Wheeler C M: (Preventive Vaccines for Cervical Cancer" Salud Publica de Mexico, Mexico,, mx, vol. 39, no. 4, July 1997(1997-07), pages 283-287) provides a review of preventative vaccines for cervical cancer. Schiller J T et al: (Papillomavirus-like particles and HPV vaccine development" Seminars in cancer biology, Saunders scientific publications, Philadelphia, Pa, us, vol. 7, no. 6, December 1996 (1996-12), pages 373-382), discloses the use of VLPs in animals to induce antibodies. Suzich J A et al: (Systemic immunization with papilomavirus 11 protein completely prevents the development of viral mucosal papillomas" Proceedings of the National Academy of Sciences of USA, national academy of science. Washington, us, vol. 92, 1 December 1995 (1995-12-01), pages 11553-11557), discloses the use of L1 particles from canine oral papillomavirus to vaccinate beagles Lowe R S et al: (Human papillomavirus type 11 (hpv-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with hpv-11 virus-like particles expressed in yeast" Journal of infectious diseases, Chicago, IL, us, vol. 176, no. 5, 1997, pages 1141-1145) discloses vaccination of monkeys with HPV 11 VLPs formulated on an aluminium adjuvant. W09517209 discloses vaccines comprising an oil in water emulsion. The present invention provides a vaccine composition according to claim 1. 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 effector mechanisms of protection respectively). These categories of response have been termed TH1-type responses (cell-mediated response), and TH2-type immune responses (humoral response). Extreme TH1-type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice TH1-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgG1 type antibodies. TH2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgG1, IgA, and IgM. It can be considered that the driving force behind the development of these two types of immune responses are cytokines. High levels of TH1-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 TH1 and TH2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominantly TH1 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 Coffman, R.L. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, p145-173 ). Traditionally, TH1-type responses are associated with the production of the INF-γ and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of TH1-type immune responses are not produced by T-cells, such as IL-12. In contrast, TH2- type responses are associated with the secretion of IL-4, IL-5, IL-6, IL-10 and tumour necrosis factor-β(TNF-β). It is known that certain vaccine adjuvants are particularly suited to the stimulation of either TH1 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 TH1 or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or (at least in mice) the measurement of the IgG1:IgG2a ratio of antigen specific antibody responses. Thus, a TH1-type adjuvant is one which stimulates isolated T-cell populations to produce high levels of TH1-type cytokines when re-stimulated with antigen in vitro, and induces antigen specific immunoglobulin responses associated with TH1-type isotype. Adjuvants which are capable of preferential stimulation of the TH1 cell response are described in International Patent Application No. WO 94/00153 and WO 95/17209. 3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is known from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 Bl (SmithKline Beecham Biologicals SA). 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 10µg - 100µg preferably 25-50µg per dose wherein the antigen will typically be present in a range 2-50µg per dose. A carrier is also present in the vaccine composition according to the invention. The carrier is an aluminium salt, such as aluminium phosphate or aluminium hydroxide. The antigens in the vaccine composition according to the invention are combined with 3D-MPL and alum. Typically for human administration 3D-MPL will be present in a vaccine in the range of 1µg - 200µg, such as 10-100µg, preferably 10µg - 50µg per dose. In one preferred embodiment the HPV antigen in the vaccine composition according to the invention comprises the major capsid protein L1 of HPV and optionally the L2 protein, particularly from HPV 16 and/or HPV 18. In this embodiment, the preferred form of the L1 protein is a truncated L1 protein. The L1, optionally in a L1-L2 fusion, is in the form of a virus-like particle (VLP). The L1 protein may be fused to another HPV protein, in particular E7 to form an Ll-E7 fusion. Chimeric VLPs comprising L1-E or L1-L2-E are particularly preferred. The proteins of the present invention preferably are expressed in E. coli. 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(OH) 3 . 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. 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. The vaccine of the present invention will contain an immunoprotective quantity of the antigens and may be prepared by conventional techniques. Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol.61 Vaccine Design - the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757. The amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed. Generally, it is expected that each dose will comprise 1-1000µg of protein, preferably 2-100µg, most preferably 4-40µg. 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 infections, the pharmaceutical compositions of the present invention may be used to treat, immunotherapeutically, patients suffering from the said viral infections. 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 1: Comparative immunogenicity of HPV Ags / HBs / gD in monovalent or combination vaccines formulated with AS04. INTRODUCTION An immunogenicity study was performed in Balb/C mice using four different antigens: 1. HPV16 L1 Virus Like Particule (VLP-16) 2. HPV18 L1 Virus Like Particule (VLP-18) 3. gD antigen of HSV-2 4. HBsAg formulated with Alum/3D-MPL (AS04) using pre adsorbed monobulks of antigen or 3D-MPL on Al(OH) 3 or AlPO 4 . 3D-MPL/Al(OH) 3 formulations are referred to AS04D whereas 3D-MPL/AlPO 4 based formulations are referred to AS04C. The following vaccines were assessed: 1. VLP16 + VLP18 AS04D; 2. gD AS04D; 3. HBs AS04C. and the potential to combine these vaccines was evaluated. The aim of this experiment was to compare the immunogenicity of two different AS04 combinations made of either: 1. VLP16 +VLP18 and gD. 2. VLP16 +VLP18 and gD and HBs Ag. 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. Antibody response to VLPs, gD and HBs Ag and the isotypic profile induced by vaccination were monitored by ELISA at day 14 post II. At the same timepoint, the cytokine production (IFNγ/IL5) was analysed after in vitro restimulation of splenic cells with either VLPs, gD or HBs antigens. MATERIALS AND METHODS Formulation Formulation compositions VLP16, VLP18, gD and HBs formulated with 3D-MPL on Aluminium salt. Components used Adsorption. a) VLP adsorption. VLP 16 and VLP 18 purified bulk are adjusted to 500 µg/ml with the purification buffer, then Al(OH) 3 is added to obtain a ratio of 2µg VLP/10µg Al(OH) 3 . Magnetic stirring is carried out at room temperature for 60 minutes and the mixture stored between 2-8°C until final formulation. b) gD adsorption. 2µg gD are mixed with 10µg Al(OH) 3 . Magnetic stirring is carried out for 60 minutes at room temperature and the mixture stored between 2-8°C until final formulation. c) HBs absorption. 2µg Hbs are mixed with 10µg AlPO 4 . Magnetic stirring is carried out for 60 minutes at room temperature and the mixture stored between 2-8°C until final formulation. d) 3D-MPL adsorption. 5µg 3D-MPL are mixed with 10µg Al(OH) 3 . Magnetic stirring is carried out for 60 minutes at room temperature and the mixture stored between 2-8°C until final formulation. 5µg 3D-MPL are mixed with 10µg AlPO 4 . Magnetic stirring is carried out for 60 minutes at room temperature and the mixture stored between 2-8°C until final formulation. Formulation. H 2 O and NaCl are mixed (10x concentrated) and after 10 minutes of agitation at room temperature, the differents components are added: adsorbed antigen, 3D-MPL adsorbed and Al(OH) 3 (See table below). They are shaken at room temperature for 10 minutes and stored at 4°C until injection. Mice Serology Anti-VLP-16 and anti-VILP-18 serology The quantitation of anti-VLP16 and anti-VLP 18 antibodies was performed by ELISA using VLP16 503/1 (20/12/99) and VLP18 504/2 (25/10/99F) as coating antigens. The antigen and antibody solutions were used at 50 µl per well. The antigen was diluted at a final concentration 0.5µg/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 1hr at 37°C with PBS containing 1% bovine serum albumin. Two-fold dilutions of sera (starting at 1/400 dilution) in the saturation buffer were added to the VLP-coated plates and incubated for 1 hr 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 1hr 30min at 37°C. After a washing step, streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted 1/1000 in saturation buffer was added for an additional 30min at 37°C. Plates were washed as above and incubated for 20min with a solution of o-phenylenediamine (Sigma) 0.04% H 2 O 2 0.03% in 0.1% tween 20 0.05M citrate buffer pH4.5. The reaction was stopped with H 2 SO 4 2N and read at 490/630 nm. ELISA titers were calculated from a reference by SoftmaxPro (using a four parameters equation) and expressed in EU/ml. Anti-gD response: Quantitation of anti-gD antibody was performed by ELISA using gD (gD 43B318) as the coating antigen. The antigen and antibody solutions were used at 50µl per well. The antigen was diluted at a final concentration of 1 µg/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 1hr at 37°c with PBS containing 1% bovine serum albumin and 0.1% Tween 20 (saturation buffer; 100µl/well). Two-fold dilutions of sera (starting at 1/100 dilution) in the saturation buffer were added to the gD-coated plates and incubated for 1hr 30min at 37°C. The plates were washed four times with PBS 0.1% Tween 20 and biotin-conjugated anti-mouse IgG1, IgG2a, IgG2b or Ig (Amersham, UK) diluted 1/1000 in saturation buffer was added to each well and incubated for 1hr 30 min at 37°C. After a washing step, streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted 1/1000 in saturation buffer was added for an additional 30min at 37°C. Plates were washed as above and incubated for 20min with a solution of o-phenylenediamine (Sigma) 0.04% H 2 O 2 0.03% in 0.1 % tween 20 0.05M citrate buffer pH 4.5. The reaction was stopped with H 2 SO 4 2N and read at 490/630 nm. ELISA titers were calculated from a reference by SoftmaxPro (using a four parameters equation) and expressed in EU/ml. Anti-HBs serology The quantitation of anti-HBs antibody was performed by ELISA using HBs (Hep 286) as the coating antigen. Antigen and antibody solutions were used at 50µl per well. The antigen was diluted at a final concentration of 1µg/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 1hr 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 dilution) in the saturation buffer were added to the HBs-coated plates and incubated for 1hr 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 or IgG1, IgG2a, IgG2b (IMTECH, USA) diluted respectively at 1/4000, 1/8000, 1/4000 in saturation buffer were added to each well and incubated for 1hr 30min at 37°C. After a washing step, streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted 1/1000 in saturation buffer was added for an additional 30min at 37°C. Plates were washed as above and incubated for 20min with a solution of o-phenylenediamine (Sigma) 0.04% H 2 O 2 0.03% in 0.1% tween 20 0.05M citrate buffer pH4.5. The reaction was stopped with H 2 SO 4 2N and read at 490/630 nm. ELISA titers were calculated from a reference by SoftmaxPro (using a four parameters equation) and expressed in EU/ml. Cytokine production 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 2 mM L-glutamine, antibiotics, 5x10 -5 M 2-mercaptoethanol, and 5% foetal calf serum. Cells were cultured at a final concentration of 5x10 6 cells/ml, in 1ml per flat-bottomed 24 well-plates with different concentrations (10-1 µg/ml) of each of the Ag (VLPs, gD or HBs antigen). Supernatants were harvested 96hrs later and frozen until tested for the presence of IFN γ and IL5 by ELISA. IFNγ (Genzyme) Quantitation of IFNγ was performed by ELISA using reagents from Genzyme. Samples and antibody solutions were used at 50µl per well. 96-well microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark) were coated overnight at 4°C with 50µl of hamster anti-mouse IFNγ diluted at 1.5µg/ml in carbonate buffer pH 9.5. Plates were then incubated for 1hr at 37°C with 100µl of PBS containing 1% bovine serum albumin and 0.1% Tween 20 (saturation buffer).Two-fold dilutions of supernatant from in vitro stimulation (starting at 1/2) in saturation buffer were added to the anti-IFNγ -coated plates and incubated for 1hr 30min at 37°C. The plates were washed 4 times with PBS Tween 0.1% (wash buffer) and biotin-conjugated goat anti-mouse IFNγ diluted in saturation buffer at a final concentration of 0.5µg/ml was added to each well and incubated for 1hr at 37°C. After a washing step, AMDEX conjugate (Amersham) diluted 1/10000 in saturation buffer was added for 30min at 37°C. Plates were washed as above and incubated with 50µl of TMB (Biorad) for 10min. The reaction was stopped with H 2 SO 4 0.4N and read at 450/630 nm. Concentrations were calculated using a standard curve (mouse IFNγ standard) by SoftmaxPro (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 50µl per well. 96-well microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark) were coated overnight at 4°C with µl of rat anti-mouse IL5 diluted at 1 µg/ml in carbonate buffer pH 9.5. Plates were then incubated for 1hr at 37°C with 100µl PBS containing 1% bovine serum albumin and 0.1% tween 20 (saturation buffer).Two-fold dilutions of supernatant from in vitro stimulation (starting at 1/2) in saturation buffer were added to the anti-IL-5-coated plates and incubated for 1hr 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 final concentration of 1 µg/ml was added to each well and incubated for 1hr at 37°C. After a washing step, AMDEX conjugate (Amersham) diluted 1/10000 in saturation buffer was added for 30min at 37°C. Plates were washed as above and incubated with 50µl of TMB (Biorad) for 15min. The reaction was stopped with H 2 SO 4 0.4N and read at 450/630 nm. Concentrations were calculated using a standard curve (recombinant mouse IL-5) by SoftmaxPro (four parameters equation) and expressed in pg/ml. GROUPS Groups of 10 Balb/C mice were immunised intramuscularly with the following formulations: Groups and formulations GROUP FORMULATION A VLP16 2µg / VLP18 2µg / gD 2 µg / 3D-MPL 5µg / Al(OH) 3 50µg B VLP16 2µg / VLP18 2µg / HBs 2 µg / gD 2 µg / 3D-MPL 5µg/ Al(OH) 3 40µg / AIPO 4 10µg C gD 2 µg / 3D-MPL 5µg / Al(OH) 3 50µg D VLP16 2µg / VLP18 2µg / 3D-MPL 5µg / AI(OH) 3 50µg E HBs 2µg / 3D-MPL 5µg / AlPO 4 10µg / Al(OH) 3 40µg Details of formulations are described above in Materials and Methods. RESULTS 1. Serology: a) 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 1 shows anti-VLP16 Ig antibody responses measured on individual sera on day 14 post II. The anti-VLP16 titers obtained after immunisation with the combination of VLPs, gD and HBs Ag (group B), were slightly lower than the one obtained with either the combination of VLPs and gD (group A) or the monovalent VLPs formulation (group D) (GMT respectively of 27578 versus 48105 EU/ml versus 44448 EU/ml). Before statistical analysis a T-Grubbs test was applied on each population for data exclusion. Two non-responder mice in groups A and D were eliminated for analysis. The differences observed between the groups were shown as statistically not significant using the Student Newman Keuls Test. b) Anti-VLP18 response: Humoral responses (Ig) were measured by ELISA using VLP18 504-2 (25/10/99) as coating antigen. Day 14 post II sera were analysed. Figure 2 shows the anti-VLP18 Ig antibody response measured on individual sera on day 14 post II The anti-VLP18 titers obtained after immunisation with the combination of VLPs, gD and HBs Ag (group B), were in the same magnitude as the titers obtained with either the combination of VLPs and gD (group A) or the monovalent VLPs formulation (group D) (GMT respectively of 56078 versus 88786 EU/ml versus 76991 EU/ml) Before statistical analysis a T-Grubbs test was applied on each population for data exclusion. Two non-responder mice in groups A and D were eliminated for analysis. The differences observed were shown as statistically not significant using one-way analysis of variance test. c) Anti-gD response: Humoral responses (Ig and isotypes) were measured by ELISA using gD as coating antigen. Day 14 post II sera were analysed. Figure 3 shows the anti-gD antibody responses measured on individual sera at day 14 post II: Regarding the anti-gD response, a slight decrease was observed in the GMT obtained with the VLPs/gD/HBs combination (group B) compared to gD alone (Group C) or VLPs/gD combination (Group A) (GMT respectively of 18631 versus 32675 versus 27058 EU/ml). Before statistical analysis a T-Grubbs test was applied on each population for data exclusion. Two non-responder mice in group A were eliminated for analysis. A one-way-analysis of variance was performed on anti-gD titers after log transformation of post II data. No statistically significant difference was observed between the three formulations. The isotypic repartition analysed on pooled sera was as follows: Isotypic repartition (%) IgG1 IgG2a IgG2b Group A 96 3 2 Group B 96 3 2 Group C 97 1 1 No difference was observed in isotypic profile induced by the three formulations: mainly IgGl response (96-97% of IgGl) were induced in the 3 groups as reported in the table below. d) Anti-HBs response: Humoral responses (Ig and isotypes) were measured by ELISA using HBsAg (Hep286) as coating antigen. Day 14 post II sera were analysed. Figure 4 shows the anti-HBs antibody responses measured on individual sera on day 14 post II. A slightly lower anti-HBs antibody response is observed in the combination group B containing the VLPs, gD and HBs antigens compared to HBs alone (group E) (GMT of 28996 EU/ml versus 20536 EU/ml). A one-way-analysis of variance was performed on anti-HBs titers after log transformation of post II data. No statistically significant difference was observed between the group B (VLP/HBs/gD) versus the group E (HBs AS04) using Student Newman Keuls test. The isotypic repartition analysed on pooled sera was as follows and showed no differences between the 2 groups with a proportion of IgG2a preserved in the combination vaccine. Isotypic repartition (%) IgG1 IgG2a IgG2b Group B 54 24 21 Group E 56 23 21 2. Cell Mediated Immune Response Cell-mediated immune responses (IFNγ / IL5 production) were evaluated at day 14 post II after in vitro restimulation of splenic cells with either VLPs, gD or HBs antigens. For each group of mice, pools of 5 organs were constituted. The experimental procedure is fully described in Material and Methods. 3. Cytokine production a) In vitro restimulation with VLP16 and VLP18 Figure 5 shows the cytokine production monitored in splenic cells after 96h in vitro restimulation with VLP16. Figure 6 shows the cytokine production monitored in splenic cells after 96h in vitro restimulation with VLP 18. No clear dose range effect has been observed using 10µg and 1 µg Ag dose for restimulation with either VLP antigens on both cytokine production. A clear TH1 profile was observed with all formulations. IFN γ / IL-5 ratio after in vitro restimulation with VLP 16 and VLP18. Ratio IFN / IL-5 Group A Group B Group D VLP1610µg/ml 5.2 8.9 11.8 VLP16 1µg/ml 15.1 14.3 16.5 Ratio IFN / IL-5 Group A Group B Group D VLP18 10µg/ml 19.6 11.1 16.1 VLP18 1µg/ml 23.2 14.3 18.2 b) In vitro restimulation with gD Figure 7 shows the cytokine production monitored in splenic cells after 96h in vitro restimulation with gD antigen. No clear dose range effect was observed when comparing the 10 and 1µg Ag dose for restimulation The IFN-γ is produced in much higher concentration as compared to IL-5 (Table 3) indicating a clear TH-1 profile of the immune response in all groups evaluated (monovalent versus combination). IFN-γ / IL-5 ratio after in vitro restimulation with gD. Ratio IFN / IL-5 Group A Group B Group C gD 10µg/ml 6.2 7.2 3.1 gD 1µg/ml 6.2 11.2 2.3 c) In vitro restimulation with HBs Figure 8 shows the cytokine production monitored in splenic cells after 96h in vitro restimulation with HBs. A significant level of IFN-γ but no IL5 production was observed for group B. As shown in Table 2 higher production of IFN-γ was observed in group E as compared to group B. However high background value of IFN-γ was observed in the group E (HBs monovalent) for the control with no antigen for restimulation. A very high IFN-γ/IL-5 ratio was observed with the monovalent vaccine indicating that a strong TH1 response is induced. Similarly, a high IFN-γ /IL-5 ratio was measured with the combined vaccine confirming the ability of this formulation to also induce a TH-1 response. IFN-γ / IL-5 ratio after in vitro restimulation with HBs. Ratio IFN / IL-5 Group B Group E HBs 10µg/ml 15.8 65.3 HBs 1µg/ml 7.6 67.6 CONCLUSIONS The effect of the combination of VLPs/gD or VLPs/gD/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-VLPs, anti-gD and anti-HBs serology. The combination of VLPs and gD or VLPs, gD and HBs antigens did not interfere with the isotypic profile of the antibody response displayed by the gD and HBs monovalent vaccines. In the cytokines evaluation, the TH-1 profile (IFN-γ/IL-5 ratio) observed with each monovalent vaccines was confirmed with the combination vaccine groups.

Claims: A vaccine comprising a human papillomavirus 16 L1 virus like particle, a human papillomavirus 18 L1 virus like particle, aluminium hydroxide and 3 De-O-acylated monophosphoryl lipid A. A vaccine according to claim 1 wherein the virus like particles are formulated using pre-adsorbed monobulks of antigen and 3 De-O-acylated monophosphoryl lipid A on aluminium hydroxide. A vaccine according to claim 1 or 2 wherein the human papillomavirus 16 L1 virus like particle and human papillomavirus 18 L1 virus like particle purified bulk are added to aluminium hydroxide to obtain a ratio of 2 µg VLP/10 µg Al(OH) 3 . A vaccine consisting essentially of a human papillomavirus 16 L1 virus like particle, a human papillomavirus 18 L1 virus like particle, aluminium hydroxide and 3 De-O-acylated monophosphoryl lipid A. A human papillomavirus 16 L1 virus like particle formulated with alum and 3 De-O-acylated monophosphoryl lipid A using pre-adsorbed monobulks of antigen and 3 De-O-acylated monophosphoryl lipid A on aluminium hydroxide or AlPO 4 . A human papillomavirus 18 L1 virus like particle formulated with alum and 3 De-O-acylated monophosphoryl lipid A using pre-adsorbed monobulks of antigen and 3 De-O-acylated monophosphoryl lipid A on aluminium hydroxide or AlPO 4 .

Inventors: SLAOUI MONCEF MOHAMMED, SLAOUI, MONCEF MOHAMMED, WETTENDORFF, MARTINE ANNE CECILE
Applicants: GLAXOSMITHKLINE BIOLOG SA, GLAXOSMITHKLINE BIOLOGICALS SA
Application year: 2000
Main classification: A61K39
IPC codes: A61K39, A61P31, A61P37, A61P35