WO2011074006A2

WO2011074006A2 - Vaccine composition

Info

Publication number: WO2011074006A2
Application number: PCT/IN2010/000814
Authority: WO
Inventors: Jadhav Suresh Sakharam; Gairola Sunil Jagdishprasad; Gautam Manish Maheshkumar
Original assignee: Serum Institute Of India Ltd.
Priority date: 2009-12-16
Filing date: 2010-12-16
Publication date: 2011-06-23
Also published as: BR112012014689A2; WO2011074006A4; CN102655879B; CN102655879A; WO2011074006A3; BR112012014689B1

Abstract

The instant invention provides cost effective and immunogenic IPV formulations.The said formulations contain an adjuvant that can enable 5 to 10 fold dose reduction for inactivated polio virus vaccines.

Description

VACCINE COMPOSITION

Field of invention The present invention relates to the field of vaccines for protection against polio type 1 , 2 and 3 viruses particular to vaccines containing a reduced dose of inactivated polio virus D antigen in monovalent or multivalent forms with an adjuvant to induce protective immunity against polio, p Background Art

Inactivated poliovirus vaccines (IPVs) that are currently licensed and in use are based on non attenuated (Salk)polio virus vaccine and virus strains: they are therefore also referred to as wild-type IPV (wtlPV). IPV is delivered by intramuscular (IM) or deep subcutaneous (SC) injection. IPV is currently available either as a non-adjuvanted standalone formulation, or in various combinations, including DT-IPV (with diphtheria and tetanus toxoids) and hexavalentDTPHepB- Hib-IPV vaccines (additionally with pertussis, hepatitis B, and Haemophilus influenzae b). The future global demand for IPV following eradication of polioviruses could increase from the current level of 80 million doses to 450 million doses per year. Consequently, approaches to "stretch" supplies of IPV are likely to be required. Additionally, various strategies to make IPV available at more affordable prices needs to be evaluated.^"

Reduced-dose vaccine formulations which provide protection against infection using a lower dose of IPV antigen are desirable in situations where the supply of conventional vaccine is insufficient to meet global needs or where the cost of manufacture of the conventional vaccine prevents the vaccine being sold at a price which is affordable for developing countries.

Reduced-dose vaccines to permit antigen sparing for pandemic influenza vaccines are known (see for instance WO 2008/128939 ). Oil in water emulsions per se are well known in the art, and have been suggested to be useful as adjuvants. (EP 399843; WO 95/17210).

The currently acceptable standard dose of polio vaccnes contains D antigens as 40 Units of inactivated poliovirus type 1 (Mahoney), 8 units of inactivated poliovirus type 2 (MEF-1) and 32 units of inactivated poliovirus type 3 (Saukett) (e.g. Infanrix-IPV™).

Existing preparations of stand-alone IPV do not contain adjuvant. In combination vaccines it has been reported that aluminum based adjuvant enable 3 to 4 fold reduction in IPV dose,please refer Eighth WHO/U ICEF consultation with OPV/IPV manufacturers and NRAs 30^th Oct 2009,Geneva, WHO/HQ. The three to 4 fold reduction is predicted to be insufficient for stretching global vaccine supplies and significant reduction in cost of manufacture. Thus there is an urgent need in prior art for strategies to allow significant reduction of doses (more than 5 fold) so as to increase vaccine supplies and reduce the cost of manufacture. Such reduction will make such vaccine affordable to developing countries.

The present inventors have surprisingly found that by combining an oil-in-water emulsion as adjuvant with the inactivated polio antigen, reduced doses of IPV can be used to induce an adequate or improved level of protection against polio. Such vaccines carry considerable advantages including the ability to provide more doses of IPV vaccines for the individuals in need thereof.

Summary Of The Invention

The present invention provides various reduced-dose IPV vaccines comprising antigens (which may only have IPV components or may have IPV components combined with other antigens) and an oil-in-water emulsion. Accordingly, in one aspect the present invention provides an IPV vaccine of the invention comprising inactivated poliovirus type 1 at a dose greater than 1 D- antigen units and less than 10 D-antigen units.

In one embodiment, the present invention provides an IPV vaccine of the invention comprising inactivated poliovirus type 3 at a dose of between 1 and 7 D-antigen units.

In another embodiment, the present invention provides an IPV vaccine of the invention comprising inactivated poliovirus type 2 at a dose of between 0.2 and 2 D-antigen units.

In a further aspect, the present invention provides an IPV vaccine of the invention which is a comprising inactivated poliovirus type and an oil-in-water emulsion.

Also according to the instant invention use of said adjuvant results in reduction of effective dose of inactivated polio virus D antigen by 5,6,7,8,9 or 10 fold

Said oil-in-water emulsion of present invention comprises a metabolisable oil, an emulsifying agent, and optionally a tocopherol, such as alpha tocopherol.

Definitions:

The term "vaccine" is optionally substitutable with the term "immunogenic composition" and vice versa.

"D-antigen units" (also referred to as "international units" or IU): The D antigenic form of the poliovirus induces protective neutralising antibodies. D antigen units referred to herein (for instance in the vaccines of the invention) are the measured total D antigen units of each bulk IPV antigen type prior to formulation of the final vaccine which are added in each human dose of formulated vaccine (typically 0.5mL final volume). Reliable methods of measuring D-antigen units are well known in the art and are published, for instance, by the European Pharmacopoeia. For instance, D-antigen units may be measured using the ELISA test as described in Example 1 ("D-antigen quantification by ELISA") below. European Pharmacopoeia provides a test sample (European Pharmacopoeia Biological Reference Preparation - available from Ph. Eur. Secretariat, e.g. Code P 216 0000) for standardization of such methods between manufacturers (Pharmeuropa Special Issue, Bio 96-2). Thus the D-antigen unit value is well understood in the art.

The term "dose" herein is typically one administration of the vaccine of the invention, which is typically one injection. A typical human dose is 0.5mL. Of course various doses may be administered in a vaccine administration schedule.

The term "IPV" or a vaccine comprising these components herein is intended to mean inactivated polio virus type 1 (e.g. Mahoney, as preferably used, or Brunhilde as marketed by Statens Serum Institut under the name of DiTeKiPoI), type 2 (e.g. MEF-I), or type 3 (e.g. Saukett), or a combination of either two or all three of these types. An example of a full (or standard) dose (40-8-32 D antigen units of IPV types 1, 2 and 3 respectively) IPV vaccine for the purposes of this invention could be Poliorix® (GSK Biologicals S. A.). Thus, where it is stated herein that fold reduction of a standard dose of IPV is present in a vaccine of the invention, it is meant D-antigen units equating to atleast 5 fold reduction and preferably 10 fold reduction of 40, 8, and/or 32 D-antigen units of IPV types 1 , 2 and/or 3 respectively (as measured in each bulk IPV antigen type) are formulated within each dose of said vaccine.

The term "component(s)" from a pathogen or "component(s) affording protection to such a pathogen" within the vaccines of the invention herein is intended to mean one or more antigen(s) from that pathogen. The terms "around" or "approximately" herein are taken to mean ±10% of the stated value, but should be in keeping with the context of use.

Detailed description

The present invention provides a vaccine comprising antigens from poliovirus (IPV) and an oil-in-water emulsion.

The antigens of the invention may comprise of IPV type 1 or IPV type 2 or IPV type 3, or IPV types 1 and 2, or IPV types 1 and 3, or IPV types 2 and 3, or IPV types 1 , 2 and 3.

Methods of preparing inactivated poliovirus (IPV) are well known in the art. In one embodiment, IPV should comprise types 1 , 2 and 3 as is common in the vaccine art, and may be the Salk polio vaccine which is inactivated with formaldehyde (see for example, Sutter et al, 2000, Pediatr. Clin. North Am. 47:287; Zimmerman & Spahn 1999, Am Fam Physician 59: 1 13; Salk et al., 1954, Official Monthly Publication of the American Public Health Association 44(5):563; Hennesen, 1981 , Develop. Biol. Standard 47: 139; Budowsky, 1991 , Adv. Virus Res. 39:255).

Poliovirus may be grown in cell culture. The cell culture may be a VERO cell line or PMKC, which is a continuous cell line derived from monkey kidney. VERO cells can conveniently be cultured on microcarriers. Culture of the VERO cells before and during viral infection may involve the use of bovine-derived material, such as calf serum, and this material should be obtained from sources which are free from bovine spongiform encephalitis (BSE). Culture may also involve materials such as lactalbumin hydro lysate. After growth, virions may be purified using techniques such as ultrafiltration, diafiltration, and chromatography. Prior to administration to patients, the viruses must be inactivated, and this can be achieved by treatment with formaldehyde. Viruses may be grown, purified and inactivated individually, and then combined to give a concentrate bulk mixture for IPV vaccine use.

Antigens in vaccines of the invention will be present in "immunologically effective amounts" i.e. the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention of disease. Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses).

Standard doses of polio vaccines today tend to contain 40 D antigen units of inactivated poliovirus type 1 , 8 D antigen units of inactivated poliovirus type 2 and 32 D antigen units of inactivated poliovirus type 3 (e.g. Infanrix-IPV™).

However, the present inventors have surprisingly found that by combining the antigens with an oil-in-water emulsion adjuvant, reduced doses of IPV can be used to obtain a good immune response.

In one embodiment, an IPV vaccine dose of the present invention may comprise between 1 and 10 D-antigen units of IPV type 1.

In another embodiment, a vaccine dose of the present invention may comprise approximately or exactly one tenth of a standard 40 D-antigen unit dose of IPV type 1.

In a further embodiment, the vaccines of the present invention may comprise less than 2 D-antigen units, 0.2-2 D-antigen units (equivalent to 2.5-25% of a standard 8 D-antigen unit dose) or around or exactly 0.8 D-antigen units of IPV type 2 (equivalent to 10% of a standard 8 D-antigen unit dose). In a further embodiment the vaccines of the present invention may comprise more than 1 and less than 8 D-antigen units, for instance around or exactly 3.2 D-antigen units of IPV type 3.

In another embodiment, the vaccine of the present invention may comprise approximately or exactly one tenth of a standard 32 D-antigen unit dose of IPV type 3 (equivalent to approximately 3.2 D-antigen units).

Also according to the instant invention use of said adjuvant can result in atleast 10 fold reduction in IPV antigen dose.

In yet another aspect there is provided a method of preparing an immunogenic composition, or a kit, for inducing an improved humoral response, against said polio antigen or antigenic composition in a human, that comprises of (a) a low amount, as herein defined, of polio virus antigen or antigenic preparation thereof and (b) an oil-in- water emulsion adjuvant.

Suitably said oil-in-water emulsion comprises a metabolisable oil, an emulsifying agent, and optionally a a tocopherol, such as alpha tocopherol.

In another specific embodiment, said oil-in-water emulsion adjuvant comprises at least one metabolisable oil in an amount of 0.5% to 20% of the total volume, and has oil droplets of which at least 70% by intensity have diameters of less than 1 μπι.

In one embodiment the polio antigen and the oil-in-water emulsion adjuvant are contained in the same container. It is referred to as One vial approach'. In an alternative embodiment, the polio antigen and the oil-in-water emulsion adjuvant are contained in separate containers or vials or units and admixed shortly before or upon administration into the subject. It is referred to as 'two vials approach'. By way of example, when the vaccine is a 2 components vaccine for a total dose volume of injected dose of 0.5 ml, the polio antigen may be presented as a standard polio vaccine containing approximately 40- 8-32 D antigen units of IPV types 1 , 2 and 3 respectively in one vial (500 μΐ) (antigen container, such as a vial) and another vial contains the adjuvant (4500 μΐ). The contents of the antigen container are mixed with the contents of the adjuvant container. Typically, the polio vaccine is a 0.5 ml injected dose and multidose vials contain a 1 : 10 vial mixture prior to first subject.

Oil-in-water emulsion adjuvant

The adjuvant composition of the invention contains an oil-in-water emulsion adjuvant, suitably said emulsion comprises a metabolisable oil in an amount of 0.5% to 20% of the total volume, and having oil droplets of which at least 70 % by intensity have diameters of less than 1 μιη.

In order for any oil in water composition to be suitable for human administration, the oil phase of the emulsion system has to comprise a metabolisable oil. The oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils.

Synthetic oils are also part of this invention and can include commercially available oils such as NEOBEE® and others. A particularly suitable metabolisable oil is squalene. Squalene (2, 6, 10, 15, 19, 23-Hexamethyl-2, 6,10, 14, 18,22-tetracosahexaene) is an unsaturated oil which is found in large quantities in shark-liver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly suitable oil for use in this invention. Squalene is a metabolisable oil by virtue of the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th Edition, entry no.8619). Oil in water emulsions per se are well known in the art, and have been suggested to be useful as adjuvant compositions (EP 399843; WO 95/17210).

Suitably the metabolisable oil is present in an amount of 0.5% to 20% (final concentration) of the total volume of the immunogenic composition, suitably an amount of 1.0% to 10% of the total volume, suitably in an amount of 2.0% to 6.0% of the total volume.

In a specific embodiment, the metabolisable oil is present in a final amount of about 0.5%, 1 %, 3.5% or 5% of the total volume of the immunogenic composition. In another specific embodiment, the metabolisable oil is present in a final amount of 0.5%, 1 %, 3.57% or 5% of the total volume of the immunogenic composition. A suitable amount of squalene is about 10.7 mg per vaccine dose, suitably from 10.4 to 1 1 .0 mg per vaccine dose.

Suitably the oil-in-water emulsion systems of the present invention have a small oil droplet size in the sub-micron range. Suitably the droplet sizes will be in the range 120 to 750 nm, suitably sizes from 120 to 600 nm in diameter.

Typically the oil-in water emulsion contains oil droplets of which at least 70% by intensity are less than 500 nm in diameter, in particular at least 80% by intensity are less than 300 nm in diameter, suitably at least 90% by intensity are in the range of 120 to 200 nm in diameter.

The oil droplet size, i.e. diameter, according to the present invention is given by intensity. There are several ways of determining the diameter of the oil droplet size by intensity. Intensity is measured by use of a sizing instrument, suitably by dynamic light scattering such as the Malvern Zetasizer 4000 or suitably the Malvern Zetasizer 3000HS. A detailed procedure is given in Example II.2. A first possibility is to determine the z average diameter ZAD by dynamic light scattering (PCS-Photon correlation spectroscopy); this method additionally give the polydispersity index (PDI), and both the ZAD and PDI are calculated with the cumulants algorithm. These values do not require the knowledge of the particle refractive index. A second mean is to calculate the diameter of the oil droplet by determining the whole particle size distribution by another algorithm, either the Contin, or NNLS, or the automatic "Malvern" one (the default algorithm provided for by the sizing instrument). Most of the time, as the particle refractive index of a complex composition is unknown, only the intensity distribution is taken into consideration, and if necessary the intensity mean originating from this distribution.

The oil in water emulsion according to the invention comprises a tocopherol, such as alpha tocopherol.

Suitably alpha-tocopherol or a derivative thereof such as alpha-tocopherol succinate is present. Suitably alpha-tocopherol is present in an amount of between 0.2% and 5.0% (v/v) of the total volume of the immunogenic composition, suitably at an amount of 2.5% (v/v) in a 0.5 ml vaccine dose volume, or 0.5% (v/v) in 0.5 ml vaccine dose volume or 1 .7- 1.9% (v/v), suitably 1 .8% in 0.7 ml vaccine dose volume. By way of clarification, concentrations given in v/v can be converted into concentration in w/v by applying the following conversion factor: a 5% (v/v) alpha-tocopherol concentration is equivalent to a 4.8% (w/v) alpha-tocopherol concentration. A suitable amount of alpha-tocopherol is about 1 1.9 mg per vaccine dose, suitably from 1 1.6 to 12.2 mg per vaccine dose.

The oil in water emulsion comprises an emulsifying agent. The emulsifying agent may be present at an amount of 0.01 to 5.0% by weight of the immunogenic composition (w/w), suitably present at an amount of 0.1 to 2.0% by weight (w/w). Suitable concentration are 0.5 to 1.5% by weight (w/w) of the total composition.

The emulsifying agent may suitably be polyoxyethylene sorbitan monooleate (Tween 80). In a specific embodiment, a 0.5 ml vaccine dose volume contains 1 % (w/w) Tween 80, and a 0.7 ml vaccine dose volume contains 0.7% (w/w) Tween 80. In another specific embodiment the concentration of Tween 80 is 0.2% (w/w). A suitable amount of polysorbate 80 is about 4.9 mg per vaccine dose, suitably from 4.6 to 5.2 mg per vaccine dose.

Suitably a vaccine dose comprises alpha-tocopherol in an amount of about 1 1.9 mg per vaccine dose, squalene in an amount of 10.7 mg per vaccine dose, and polysorbate 80 in an amount of about 4.9 mg per vaccine dose.

The oil in water emulsion adjuvant may be utilised with other adjuvants or immuno- stimulants and therefore an important embodiment of the invention is an oil in water formulation comprising squalene or another metabolisable oil, a tocopherol, such as alpha tocopherol, and tween 80. The oil in water emulsion may also contain span 85 and/or Lecithin.

Typically the oil in water will comprise from 2 to 10% squalene of the total volume of the immunogenic composition, from 2 to 10% alpha tocopherol and from 0.3 to 3% Tween 80, and may be produced according to the procedure described in WO 95/17210. Suitably the ratio of squalene: alpha tocopherol is equal or less than 1 as this provides a more stable emulsion. Span 85 (polyoxyethylene sorbitan trioleate) may also be present, for example at a level of 1 %. The vaccines of the instant invention can comprise of a monovalent composition comprising a polio virus D antigen or antigenic preparation from atleast one inactivated polio virus type selected from a group consisiting of polio virus type 1 , type 2 and type 3.

The vaccines of the instant invention can comprise of a multivalent composition comprising a polio virus D antigen or antigenic preparation from atleast 2 or atleast 3 inactivated polio virus types.

The said multivalent composition can be selected from a group consisting^'of a) D antigens as 8 Units of inactivated poliovirus type 1 , 1.6 units of inactivated poliovirus type 2 and 6.4 units of inactivated poliovirus type 3 .b) D antigens as 6.6 Units of inactivated poliovirus type 1 , 1.3 units of inactivated poliovirus type 2 and 5.3 units of inactivated poliovirus type 3 .c) D antigens as 5.7 Units of inactivated poliovirus type 1 , 1.1 units of inactivated poliovirus type 2 and 4.5 units of inactivated poliovirus type 3 .d) D antigens as 5 Units of inactivated poliovirus type 1 , 1 units of inactivated poliovirus type 2 and 4 units of inactivated poliovirus type 3 .e)D antigens as 4.4 Units of inactivated poliovirus type 1, 0.88 units of inactivated poliovirus type 2 and 3.5 units of inactivated poliovirus type 3 or f) D antigens as 4 Units of inactivated poliovirus type 1 , 0.8 units of inactivated poliovirus type 2 and 3.2 units of inactivated poliovirus type 3 .

Vaccines of the invention will typically, in addition to the antigenic and adjuvant components mentioned above, comprise one or more "pharmaceutically acceptable carriers or excipients", which include any excipient that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable excipients are typically large, slowly metabolised macromolecules such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et ah, 2001 , Vaccine, 19:21 18), trehalose (WO 00/56365), lactose and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, phosphate buffered physiologic saline is a typical carrier. A thorough discussion of pharmaceutically acceptable excipients is available in reference Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20^th edition, ISBN:0683306472. Compositions may be presented in vials, or they may be presented in ready filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses (e.g. 2 doses). In one embodiment the dose is for human. In a further embodiment - - the dose is for an adult, adolescent, toddler, infant or less than one year old human and may be administered by injection. Vaccines of the invention may be packaged in unit dose form or in multiple dose form (e.g. 2 doses). The said multidose composition can be selected from a group consisting of 2 dose,5 dose and 10 dose .

For multiple dose forms, vials are preferred to pre-filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of the composition for injection has a volume of 0.5mL.

Compositions of the invention may be isotonic with respect to humans. Vaccines of the invention may include an antimicrobial, particularly when packaged in a multiple-dose format. Thiomersal should be avoided as this leads to loss of potency of the IPV component. Other antimicrobials may be used, such as 2- phenoxyethanol or parabens (methyl, ethyl, propyl parabens). Any preservative is preferably present at low levels. Preservative may be added exogenously and/or may be a component of the bulk antigens which are mixed to form the composition (e.g. present as a preservative in pertussis antigens). In one embodiment, vaccines of the invention are thiomersal free or substantially thiomersal free. By "thiomersal free" or "substantially thiomersal free" it is meant that there is not enough thiomersal present in the final formulation to negatively impact the potency of the IPV component.

Vaccines of the invention may comprise detergent e.g. a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels e.g. <0.01 %. Vaccines of the invention may include sodium salts (e.g. sodium chloride) to give tonicity. The composition may comprise sodium chloride.

In one embodiment, the concentration of sodium chloride in the composition of the invention is in the range of 0.1 to 100 mg/mL (e.g. l-50mg/mL, 2-20mg/mL, 5-15mg/mL) and in a further embodiment the concentration of sodium chloride is 10±2mg/mL NaCl e.g. about 9mg/mL.

Vaccines of the invention will generally include a buffer. A phosphate,citrate or histidine buffer is typical. Vaccines of the invention may include free phosphate ions in solution (e.g. by the use of a phosphate buffer) in order to favour non-adsorption of antigens. The concentration of free phosphate ions in the composition of the invention is in one embodiment between 0.1 and 10.0 mM, or in another embodiment between 1 and 5mM, or in a further embodiment about 2.5mM. Polio Potency as measured by seroneutralisation test on rats: For the purposes of the invention, the assay for the IPV quantitative evaluation of the vaccine potency of the IPV containing vaccines of the invention should be carried out using a single dose of vaccine and should be done by determining the ratio of test vaccine geometric mean titre (GMT) to reference vaccine GMT and is reported as the relative response (RR) or relative potency (RP). Reference GMT may be the GMT obtained with any IPV vaccine comprising 40-8-32 D-antigen units of IPV types 1-2-3 respectively, and may be the GMT obtained with the known vaccine Poliorix®. Typically, the RP test is carried out by as follows:

The potency of poliovirus Types 1, 2 and 3 is determined on rats by seroneutralisation : Groups of 10 healthy rats (Sprague-Dawley (OF A) or any beforehand validated strain) are inoculated intramuscularly with dilutions (1/1.25; 1/3.125; 1/7.81) of the test samples or reference material in phosphate buffer saline. If necessary, the dilution range may be extended to 4 dilutions by inoculating undiluted vaccine and the three previous mentioned dilutions. Ten rats inoculated with the diluent are used as negative controls. Rats are observed once a week to detect any abnormal reaction. 20 to 22 days after the inoculation, each animal is deeply anesthetized, and bled and the serum is collected to be analysed by seroneutralisation test. For the seroneutralisation test, sera are inactivated by incubation at 56°C for 30 minutes in a water bath. Three dilution series of the sera, one for each polio type, are prepared in microplates using the appropriate dilution medium. Plates are stored at + 4°C. For the three polio virus types, a predetermined amount of virus (30-300 CCID50) is added to the sera dilutions. The three virus suspensions are diluted taking into account their respective titers. The final dilution is called 'working dilution'. Each working dilution is added to the corresponding microplates. Plates are then sealed and incubated at 37°C ± 1 °C for 16 hours. Hep-2 cells are then added and microplates are incubated at 37°C ± 1 °C for 7 days. The cytopathogenic effect (CPE) of the virus is read using an inverted microscope after Coomassie blue coloration. The presence of anti-poliomyelitis antibodies inhibits the growth of the virus and the appearance of the corresponding CPE. The anti-polio virus titers (type 1 , 2 and 3) correspond to the reciprocal of the last dilution without any CPE. In each group, animals with neutralising antibodies are recorded and the antibodies titer of each serum sample is determined for the different type of poliovirus. The neutralizing antibody titer is expressed as the Iog2 of the inverse of the highest dilution of the serum sample that totally inhibits the cytopathic effect of poliovirus on Hep-2 cells.

The geometric mean titer per dilution (GMT) and per virus type is also determined for each group of rats.

Packaging of vaccines of the invention

Vaccines of the invention can be packaged in various types of container e.g. in vials, in syringes, etc. A multidose vial will typically comprise a re-sealable plastic port through which a sterile needle can be inserted to remove a dose of vaccine, which reseals once the needle has been removed.

The vaccine may be supplied in various containers (e.g. 2 or 3). The contents of the containers may be mixed extemporaneously before administering to a host in a single injection or may be administered concomitantly at different sites. The dose of the vaccine or each vaccine if a kit is administered concomitantly (in two or more containers) will typically be 0.5mL.

Administration of vaccines of the invention

The invention provides a method for raising an immune response in a mammal, comprising the step of administering an effective amount of a vaccine of the invention. The vaccines can be administered prophylactically (i.e. to prevent infection). The immune response is preferably protective and preferably involves antibodies. The method may raise a booster response. Following an initial vaccination, subjects may receive one or several booster (subsequent) immunisations adequately spaced. Dosing treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule, which may be in the first year of life, may be followed by a booster dose schedule. Suitable timirig between priming doses (e.g. between 4-16 weeks), and between priming and boosting can be routinely determined. In one embodiment, the mammal is a human. Where the vaccine is for prophylactic use, the human is preferably a child (e.g. a toddler of infant) or a teenager; where the vaccine is for therapeutic use, the human is preferably an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc. The vaccine preparations of the present' invention may be used to protect or treat a mammal susceptible to infection, by means of administering said vaccine directly to a patient. Direct delivery may be accomplished by parenteral administration (intramuscularly, intraperitoneally, intradermally, subcutaneously, intravenously, or to the interstitial space of a tissue); or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration. In one embodiment, administration is by intramuscular injection to the thigh or the upper arm. Injection may be via a needle (e.g. a hypodermic needle), but needle free injection may alternatively be used. A typical intramuscular dose is 0.5mL. Bacterial infections affect various areas of the body and so the compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as spray, drops, gel or powder (see e.g. Almeida & Alpar, 1996, J Drug Targeting, 3:455; Bergquist et al, 1998, APMIS, 106:800). Successful intranasal administration of DTP vaccines has been reported (Ryan et al., 1999, Infect. Immun., 67:6270; Nagai et al., 2001 , Vaccine, 19:4824). In one embodiment the vaccines of the first and second (and third where applicable) containers are administered concomitantly at different sites, and in an alternative embodiment the inventors envision that the contents of the first and second containers may be mixed (optionally extemporaneously) before administration as a single vaccine. The invention may be used to elicit systemic and/or mucosal immunity.

All cited references and publications are incorporated by reference herein. EXAMPLES Examples are provided solely for the purposes of illustration and are not intended to limit the scope of the invention.

Example 1:

Method of making an oil-in-water emulsion and combining the emulsion with IPV vaccine. Oil in water emulsion was made according to the method described in the document [Ott et al. The adjuvant MF59: a 10-year perspective. In- vaccine adjuvants: preparation methods and research protocols. Methods in Molecular medicine, vol 42, chapter 12, p21 1 -228. Edited by D.T. O'Hagan (2000)]. Briefly, 9.75g squalene (Sigma, derived from shark liver) was combined with 1 .175 g of sorbitan trioleate. Separately 1 .175 g of polysorbate 80 was combined with 240 ml of l OmM citrate buffer pH6.5, and then added to the squalene- sorbitan trioleate solution.This mixture was further homogenised for 1 minute with a bench-top homogeniser (Silverson). This material was then immediately subject to five passages of high-pressure microfluidisation using a Microfluidics M1 10H microfluidiser (Microfluidics, MA, USA). The size of the oil-droplets was confirmed to be less than 150nm using a Malvern Zetasizer. The resulting emulsion was sterile-filtered and stored at 2-8°C.

1 vial (1 human dose) of IPV (NVI, Holland) was diluted five-fold with PBS, and to this was then added an equal volume of the emulsion, resulting in a total 10-fold dilution of the vaccine. Example 2:

Immunization of rats and evaluation of immunogenicity

Wistar rats (n=5) were immunized twice (day 0, 28) intramuscularly with 0.1 ml of A) IPV full strength; B) IPV diluted 10-fold with PBS and C) IPV diluted 10-fold with emulsion as above. Sera was collected on day 42 and evaluated for polio virus neutralization titers against each of the three strains in the IPV vaccine.

Geometric mean titres from virus neutralization are shown in the table below Table 1 Geometric Mean Titres (N=5)

(Value in parenthesis indicates geometric standard deviation) This demonstrates that 1/10 of a dose of IPV vaccine, when combined with oil-in-water emulsion, induces an immune response at least as potent as that induced by the full-dose vaccine without adjuvant.

Thus addition of adjuvant to IPV vaccine permits a dose reduction of the vaccine and will permit immunization with a lower dose of the antigen.

Claims

We claim:

1. An immunogenic composition for inducing improved humoral response against polio antigen comprising of a) a low amount of polio virus antigen or antigenic preparation and b) an oil-in-water emulsion adjuvant.

2. The immunogenic composition of claim 1 , wherein said oil-in-water emulsion comprises a metabolisable oil .

3. The immunogenic composition of claim 3, wherein said metabolisable oil is squalene.

4. The immunogenic composition of claim 1 or claim 3, wherein said metabolisable oil is present at an amount of 0.5-10, 0.5-9, 1-10, 2-10, 4-8, 1-2, 2-3, 4.5-5.5, 5-6 or 9- 10 or

10-1 1 mg, per human dose. (Other composition in tocopherol)

5. The immunogenic composition of claim 1 , wherein said emulsifying agent is a non- ionic surfactant.

6. The immunogenic composition of claims 5, wherein said emulsifying agent is a polyoxyethylene sorbitan monooleate or a sorbitan trioleate or a mixture of both.

7. The immunogenic composition of claim 6, wherein said polyoxyethylene sorbitan monooleate is selected from the group comprising: polysorbate 80 or Tween® 80, and sorbitan trioleate is Span85.

8. The immunogenic composition of any one of claims 1 to 7, wherein said emulsifying agent is present at an amount of 0.1 -5, 0.2-5, 0.3-5, 0.4-5, 0.4-1.2, 0.5-4, 1 -2, 2-3 or 4-5.5 mg per human dose.

9. The immunogenic composition of any one of claims 1 to 8, wherein said adjuvant further comprises a tocol.

10. The immunogenic composition of claim 9, wherein said tocol is alpha-tocopherol.

1 1 . The immunogenic composition of claim or claim 10, wherein said tocol is present at an amount of 0.5-12, 1 -1 1 , 2-10, 4-9, 5-6, 5-7, 2.5-3.5, 1 -2, 1 -3 or 10-1 1 or 1 1 -12 mg per human dose.

12. The immunogenic composition of claims 1 -1 1 , wherein the adjuvant is selected from group consisting of

i) 10-1 l mg metabolisable oil, 4.5-5.5 mg emulsifying agent , and when present 1 1 .5-12.5 mg tocol per human dose

ii) 10.5-1 1 mg metabolisable oil, 4.6-5.2 mg emulsifying agent, and when present 1 1.6- 12.2 mg tocol per human dose

13. The immunogenic composition of claims 1 to 12 wherein said composition is a monovalent composition comprising a polio virus D antigen or antigenic preparation from atleast one inactivated polio virus type selected from a group consisiting of polio virus type 1 , type 2 and type 3.

14. The immunogenic composition of claims 1 to 12 wherein said composition is a multivalent composition comprising a polio virus D antigen or antigenic preparation from atleast 2 or atleast 3 inactivated polio virus types.

15. An IPV dose reduced composition according to claim 14, wherein the dose for multivalent composition is selected from a group consisting of i) D antigens as 8 Units of inactivated poliovirus type 1 , 1.6 units of inactivated poliovirus type 2 and 6.4 units of inactivated poliovirus type 3 .

ii) D antigens as 6.6 Units of inactivated poliovirus type 1 , 1 .3 units of inactivated poliovirus type 2 and 5.3 units of inactivated poliovirus type 3 . iii) D antigens as 5.7 Units of inactivated poliovirus type 1 , 1 .1 units of inactivated poliovirus type 2 and 4.5 units of inactivated poliovirus type 3 . iv) D antigens as 5 Units of inactivated poliovirus type 1, 1 units of inactivated poliovirus type 2 and 4 units of inactivated poliovirus type 3 .

v) D antigens as 4.4 Units of inactivated poliovirus type 1 , 0.88 units of inactivated poliovirus type 2 and 3.5 units of inactivated poliovirus type 3 . vi) D antigens as 4 Units of inactivated poliovirus type 1 , 0.8 units of inactivated poliovirus type 2 and 3.2 units of inactivated poliovirus type 3 .

16. A vaccine kit comprising a polio virus D antigen component or antigenic preparation as defined in any of the preceeding claims ,that comprises of (a) a low amount, as herein defined, of polio virus antigen or antigenic preparation thereof and (b) an oil-in-water emulsion adjuvant.

17. The immunogenic composition according to any preceding claim wherein said human dose is selected from: 0.5 ml or less, between 0.5 and 1.5 ml, between 0.2 and 1.2 ml, between 0.2 and 0.7 ml and less than or exactly 0.25, or 0.5, or 0.7, or 1 ml. .

18. The immunogenic composition according to any preceeding claim, wherein the polio antigen and the oil-in-water emulsion adjuvant are contained in the same container.

19. The immunogenic composition according to any preceeding claim, wherein the polio antigen and the oil-in-water emulsion adjuvant are contained in separate containers or vials or units and admixed shortly before or upon administration into the subject.

20. An immunogenic composition of any preceding claim characterized in that the composition is single dose or multidose.

21 . An immunogenic composition according to claim 20, characterized in that the multidose composition is selected from a group consisting of 2 dose,5 dose and 10 dose .

22. An immunogenic composition according to any preceeding claim, wherein the effective dose of inactivated polio virus D antigen is reduced by atleast 5 fold.

23. An immunogenic composition according to claim 22, wherein the effective dose of inactivated polio virus D antigen is reduced preferably by 10 fold.