WO2016012385A1

WO2016012385A1 - Vaccine composition comprising ipv and cyclodextrins

Info

Publication number: WO2016012385A1
Application number: PCT/EP2015/066511
Authority: WO
Inventors: Isabelle Chacornac; Alain Francon; Pascal Vacus
Original assignee: Sanofi Pasteur
Priority date: 2014-07-21
Filing date: 2015-07-20
Publication date: 2016-01-28
Also published as: AR101256A1

Abstract

The present invention is directed to an immunogenic composition comprising at least one inactivated poliovirus (IPV) serotype and at least one cyclodextrin or derivative thereof, especially beta-cyclodextrin, gamma-cyclodextrin or 2-hydroxypropyl gamma-cyclodextrin, and optionally additional antigens, said composition being protected against IPV titer loss induced by thiomersal. The invention also concerns said immunogenic composition for use as a vaccine as well as the use of a cyclodextrin, or a derivative thereoffor preserving the IPV immunogenicity in presence of thiomersal. The invention is also directed to a method of preparing a vaccine composition and to a method of immunizing a host against poliomyelitis.

Description

VACCINE COMPOSITION COMPRISING IPV AND CYCLODEXTRINS

Field of the invention:

The present invention relates to the field of vaccines for protecting against poliovirus infection, and is in particular directed to vaccine compositions comprising inactivated poliovirus in combination with cyclodextrin(s), or derivatives thereof, wherein the cyclodextrin(s) or derivative(s) thereof protects and preserves the antigenicity and / or immunogenicity of inactivated poliovirus, especially in presence of thiomersal. Background to the invention:

Poliomyelitis, also called polio, is an acute, viral, infectious disease spread from person to person, primarily via the fecal-oral route. Although approximately 90% of polio infections cause no symptoms at all, affected individuals can exhibit a range of symptoms if the virus enters the blood stream. In about 1 % of cases, the virus enters the central nervous system, preferentially infecting and destroying motor neurons, leading to muscle weakness and acute flaccid paralysis.

Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and member of the family of Picornaviridae.

Poliovirus is composed of an RNA genome and a protein capsid. The genome is a single- stranded positive-sense RNA genome that is about 7500 nucleotides long. The viral particle is about 30 nanometres in diameter with icosahedral symmetry.

Two types of poliomyelitis vaccine exist, which are made up of three virus serotypes (1 , 2 and 3). The oral vaccine (OPV - Oral Polio Vaccine), uses three live attenuated strains, and the inactivated vaccine (IPV - Inactivated Polio Vaccine or Virus), uses three inactivated strains. The use of the inactivated polio vaccine has been given priority in most of countries, in view of the risks of poliovirus dissemination involved by the use of an oral vaccine containing a live attenuated virus having the possibility of reversion during intestinal transit of the latter. Although poliomyelitis is almost eradicated in many countries, the global effort to eradicate polio is facing serious setbacks, especially in Africa, Middle-East and Asia. New cases have indeed recently been reported in different countries, which had been largely free of cases for several years. It is thus of outmost importance to continue the global efforts to wipe out the disease and to intensify the vaccination programs in the exposed areas.

Thiomersal, also known as thimerosal, is an organomercurial derivative of ethyl mercury. This compound is a well-established antiseptic and antifungal agent. It has been used widely, and for a very long time, as a preservative in vaccines in their final bulk formulations. Its primary purpose has been to prevent microbial growth in the product during use. It has also been used during vaccine production both to inactivate certain organisms, such as whole cell Pertussis, and toxins and to maintain a sterile production line. Thiomersal is thus likely to be present in a vaccine further to its addition at different steps, namely either as a preservative to protect the production line, or as at inactivating agent, and /or as a preservative at the final stage of vaccine production.

It has however been observed that there is a strong incompatibility between IPV and thiomersal. There is indeed a degradation mechanism, which is not completely understood but leads to a loss of D-antigen titer of a composition comprising IPV and thiomersal, and consequently to a loss in capacity for raising a protective immune response in case of vaccination. This titer loss has been observed, in particular by the inventors, to be dramatically accelerated and amplified in presence of aluminum, and in particular in presence of aluminum gel. The titer losses on IPV are thus associated with an unacceptable lowering of immunogenicity of the composition comprising IPV and thiomersal.

Whereas IPV solutions are formulated without thiomersal, there are however situations where simultaneous presence of IPV and thiomersal cannot be avoided, as for example extemporaneous preparation of compositions for injection.

One of these situations is related to combination vaccines. Combination vaccines, which provide protection against more than one pathogen, are indeed very desirable in order to minimize the number of injections required to confer protection against multiple pathogens, to lower the costs associated with vaccinations, and to mitigate the unpleasantness due to the multiplicity of injections, which in turn allows improvement of vaccine coverage of a population. In this context, IPV is generally intended to be mixed with additional antigens, inter alia Diphtheria toxoid, Tetanus toxoid and acellular or whole cell Pertussis, known as DTP-IPV vaccines. Some of these additional antigens, and more specifically bacterial antigens, such as whole cell Pertussis, are however available most of the time in conjunction with thiomersal. In these conditions, combination vaccines combining IPV, extemporaneously or not, with for example thiomersal-killed whole cell Pertussis is unlikely to be effective in protecting against poliomyelitis.

Sawyer et al. (Vaccine, 1994, 12:851 ) have shown that the mixing of an enhanced IPV vaccine with a diphtheria-tetanus-pertussis vaccine containing thimerosal leads to a substantial loss in IPV D-antigen titers. As it arises from the examples presented hereafter, the loss in D-antigen titer resulting from the deleterious effect of thimerosal on IPV is further aggravated and accelerated by the presence of aluminum, and in particular by an aluminum gel in or according to the pH of the thiomersal-containing composition to which IPV is mixed. In such a case, the extemporaneously prepared combined vaccine composition cannot maintain a satisfactorily immunogenic property with regard to IPV.

The other frequent situation where IPV and thiomersal are likely to be combined, for example in an extemporaneous manner, is related to multidose vaccines, in which thiomersal is added in varying concentrations (generally 10 to 50 μg per dose) as a preservative to prevent contamination with microorganisms during the subsequent use of the multi-doses vial. Multidose presentation for antigens vaccines is indeed frequently preferred to single-dose vials, which would require significantly larger cold storage space as well as increased transport needs. This is currently not feasible for the majority of countries. For some vaccines, it is thus more cost effective to use multi-dose vials. Such vaccines include antigens against diphtheria, tetanus and pertussis (DTP), and DTP with Haemophilus influenzae type b (Hib). In order to diminish any risk of contamination associated with the mutlidose vial, preservatives are generally added. Thiomersal is a preservative of choice in the context of multidose vaccine. Therefore, IPV may extemporaneously encounter thiomersal, either after blending an IPV vaccine with a single-dose vaccine containing thiomersal-inactivated antigen, for example, thiomersal-inactivated whole cell Pertussis, or after blending an IPV vaccine with a multi-dose vaccine containing thiomersal as preservative to ensure the absence of microorganisms contaminations during the use of the multi-dose vial (which could result from the repeated use).

In the situations described above, thiomersal suppression is a challenge, especially when IPV is to be combined with already available vaccines, comprising thiomersal. Indeed, thiomersal suppression in this case would imply to rework the antigen process or to replace the preservative in the multidose presentation vaccine, and register the corresponding variations. Moreover, whereas the primary role of thiomersal in vaccines has been considered to be that of a preservative, data indicate that there are other effects of this additive on vaccine antigens which need to be taken into account if consideration is being given to its elimination, reduction or replacement. Indeed, in some production processes thiomersal is used in the inactivation of vaccine antigen along with heat, for example in the case of whole cell pertussis vaccine. Elimination, reduction, removal or replacement of thiomersal in vaccines, is likely to affect not only the subsequent ability of microbial contaminants to grow in vaccine preparations, but also vaccine quality, safety, costs and efficacy. Indeed, experience shows that eliminating or reducing thiomersal from an existing product can have some unexpected effects on vaccine, especially on safety and efficacy. Effects on vaccine stability might also be expected. There is thus no evidence that a vaccine where the thiomersal content has been altered will be as safe and efficacious as the already licensed product and such that making changes to the thiomersal content of vaccines already licensed with this preservative is a particularly complex issue. Finally, it is also to be stressed that the World Health Organization (WHO) still recommends the presence of thiomersal as conservative in some vaccines, such that its complete removal from vaccines is not expected in near future.

There is thus a need to find an alternative solution, rendering unnecessary the elimination, reduction, removal or replacement of thiomersal in vaccines, especially in cases of already licensed products, when they are to be combined, in particular extemporaneously, with IPV. There is especially a need to obtain a formulation of IPV which would be protected against the deleterious effect of thiomersal and thus to find a compound capable of protecting IPV from degradation by thiomersal and which is approved for vaccine application.

There is also especially a need to obtain a formulation of IPV which would be protected against the deleterious effect of thiomersal, in particular in presence of aluminum, notably in presence of a gel of aluminum.

There is further a need for improving the stability of IPV in presence of thimerosal in a vaccine formulation comprising an aluminum gel, such as an aluminum phosphate gel (also known as aluminum hydroxide phosphate) or an aluminum hydroxide (also known as aluminum oxyhydroxide) gel.

In particular, there is a need to improve the stability of an IPV vaccine, when blending the IPV vaccine with another vaccine preparation containing thiomersal, with regard to the deleterious effect of thiomersal for at least 6 hours, at a temperature ranging from 5°C to 25°C.

More particularly, there is a need to improve the stability of an IPV vaccine, when blending the IPV vaccine with another vaccine preparation containing thiomersal and aluminum, for instance a gel of aluminum, for at least 6 hours, at a temperature ranging from 5°C to 25°C.

The present inventors have unexpectedly found that cyclodextrin(s), or derivative(s) thereof, are capable of minimizing the degradation of IPV in presence of thiomersal, in particular in the situations described above. Whereas cyclodextrins have a wide range of applications in different areas of drug delivery and pharmaceutical industry, they have never been reported as protectant of IPV antigenicity and/or immunogenicity.

More particularly, the inventors have observed that cyclodextrin (s), or derivative(s) thereof, are capable of minimizing the loss of D-antigen titer of IPV blended to a composition comprising thiomersal and aluminum, in particular an aluminum gel. Summary of the invention:

The present invention is thus directed, in a first aspect, to an immunogenic composition comprising at least one inactivated poliovirus (IPV) serotype and at least one cyclodextrin, or derivative thereof.

In another aspect of the invention, there is provided an immunogenic composition according to the first aspect, for use as a vaccine for human beings or animals, preferably in combination with a second distinct immunogenic composition comprising thiomersal.

In a further aspect, there is provided a use of cyclodextrin, or derivative thereof, in combination with at least one inactivated poliovirus (IPV) serotype for preserving the IPV immunogenicity in presence of thiomersal, in particular in presence of thiomersal and aluminum, for instance a gel of aluminum.

In a further aspect, there is provided a method for preparing a vaccine comprising mixing a first immunogenic composition comprising at least one inactivated poliovirus (IPV) serotype and cyclodextrin with a 2^nd composition, the 2^nd composition comprising in particular thiomersal, and optionally aluminum. The present invention is also directed to a kit comprising a first immunogenic composition according to the 1^st aspect of the invention and a 2^nd immunogenic composition comprising thiomersal, and optionally aluminum.

The invention also concerns a method of immunizing a host again poliomyelitis, comprising administering to the host in need thereof, an immunoeffective amount of the immunogenic composition or vaccine as provided herein.

The invention also concerns a method of immunizing a host again poliomyelitis further comprising a step of observing an immunizing response.

In still another aspect, there is provided uses of cyclodextrin(s), or derivative(s) thereof, in combination with at least one inactivated poliovirus (IPV) serotype for preserving the IPV immunogenicity in presence of thiomersal, and in particular in presence of thiomersal and aluminum, such as a gel of aluminum.

Detailed description of the invention:

The present inventors have demonstrated, as detailed in the present experimental section, that the titer losses on IPV induced by addition of thiomersal, are reduced and/or delayed in presence of cyclodextrin(s) or a derivative thereof, for the three known poliovirus serotypes, namely types 1 , 2 and 3. Addition of cyclodextrin(s) or derivative(s) thereof to a composition comprising IPV thus allows minimizing and delaying IPV titer losses, induced by thiomersal or likely to be induced by thiomersal in case of extemporaneous blending of such composition with a composition comprising thiomersal.

An immunogenic composition according to the 1^st aspect of the invention comprises at least one inactivated poliovirus serotype, namely type 1 , type 2 and/or type 3; but preferably comprises at least two IPV serotypes, namely types 1 and 2, types 1 and 3 or types 2 and 3. Most preferably a composition of the invention comprises a mixture of the three serotypes 1 , 2 and 3, as this is standard in vaccination. IPV is used in the following to refer to any IPV type 1 , 2 and/or 3, present in the composition.

Inactivated poliovirus type 1 is preferably from the Mahoney or Brunhilde strain. Inactivated poliovirus type 2 is preferably from the MEF-1 strain. Inactivated poliovirus type 3 is preferably from the Saukett strain. The preparation of the IPV entering into the composition of the invention is for example made as described in US 4,525,349 or in Industrial Biotechnology - Bioprocess, Bioseparation, and Cell Technology, Wiley & Sons, 2010: 4789-4808.

Cyclodextrins and derivatives thereof

An immunogenic composition of the invention comprises cyclodextrin(s) or derivative(s) thereof, as protectant of IPV antigenicity against degradation induced by thiomersal. Cyclodextrins are a family of cyclic oligosaccharides with a hydrophilic outer surface and a less hydrophilic (or lipophilic) central cavity. Cyclodextrins, also called cycloa my loses, are composed of 5 or more a-D-glucopyranoside units linked 1->4. Typical cyclodextrins contain a number of glucose monomers ranging from six to eight units in a ring; the cyclic part thus creates a conical shape cavity that exhibit an electron rich less hydrophilic interior. The cavity shape is a truncated cone due to the asymmetry of hydroxyl groups, the secondary hydroxyl groups extending from the wider edge and the primary groups from the narrow edge. The cavity size depends on the number of forming units.

The naturally occurring cyclodextrins are:

a (alpha)-cyclodextrin: 6-membered sugar ring molecule,

β (beta)-cyclodextrin: 7-membered sugar ring molecule and

Y (gamma)-cyclodextrin: 8-membered sugar ring molecule.

Due to their cone shape, cyclodextrins are widely uses as "molecular cages" in the pharmaceutical industry, where they are used as complexing agents to increase aqueous solubility of poorly soluble drugs and to increase their bioavailability and stability. Indeed, in aqueous solutions, cyclodextrins are able to form inclusion complexes with many drugs by taking up the drug molecule or some lipophilic moiety of the molecule, into the central cavity. The cavity size is the major determinant factor for complexation specificity. The a-cyclodextrins (smallest size) cannot accept many molecules, the β-cyclodextrins (medium size) are adapted to most molecules (hormones, vitamins...) and frequently used as complexing agents for their solubilization, the γ-cyclodextrins have cavities much larger than many molecules, then being not as effective as smaller ones to facilitate their appropriate complexation. The γ-cyclodextrins were used, for example, for complexing the antibiotic natamycin (Cevher et ai, J Pharm Sci, 2008, 97:4319).

The incorporation into the cavity of cyclodextrins of hydrophobic molecules occurs by water displacement, i.e. a reaction favored by the repulsion of the molecule by water.

After final dilution in a much larger aqueous volume, and also possible competitive complexation by other compounds of the environment, the complexation equilibrium is reversed, releasing the compounds initially attached to the cyclodextrins.

Naturally occurring cyclodextrins exhibit good solubility in water; it is however significantly different versus size or additional lateral chain

Various derivatives of cyclodextrins have been designed including hydroxypropyl cyclodextrins, methylated cyclodextrins, sulfo ether cyclodextrins, etc...

According to the present invention, a cyclodextrin derivative is preferably a molecule deriving from a cyclodextrin as defined above, by attachment of one or more glycosyl and/or hydroalkyl moiety to one or more of the glucopyranoside units of the cyclodextrin. Cyclodextrin derivatives according to the invention are thus glycosylated and/ or hydroxy alkylated derivatives of cyclodextrins, presenting the same cyclic structure as cyclodextrins.

Usually, cyclodextrins are used to complex and protect small organic molecules. Surprisingly, the present inventors have now shown that cyclodextrins are also capable of protecting IPV from antigen degradation in presence of thiomersal. Given the respective size of IPV and cyclodextrins, and without being bound by theory, the protection allowed by the cyclodextrins according to the invention is not related to the formation of inclusion complexes between IPV and cyclodextrins, contrary to the classical role of cyclodextrins.

According to the present invention, the composition comprises at least one type of cyclodextrin, preferably the composition comprises a cyclodextrin comprising 6, 7 or 8 glucopyranose units and most preferably 7 or 8 units, i.e. the composition preferably comprises γ-cyclodextrin or β- cyclodextrin, or derivartive(s) thereof, and more preferably comprises a γ-cyclodextrin, or derivative(s) thereof. Alternatively, the composition may comprise a mixture of γ-cyclodextrin and β-cyclodextrin.

According to another embodiment of the invention, the cyclodextrin or derivative thereof comprised in the composition as protectant is a γ-cyclodextrin or a β-cyclodextrin derivative. γ-cyclodextrin or β-cyclodextrin derivatives useful for the invention may be C1-C3 hydroxyalkyi derivatives of γ-cyclodextrin or β-cyclodextrin, or oside derivatives of γ-cyclodextrin or β- cyclodextrin, or a mixture thereof.

A C1-C3 hydroxyalkyi derivative of γ-cyclodextrin or β-cyclodextrin useful for the invention comprises at least one C1-C3 hydroxyalkyi group. According to one embodiment, a C1-C3 hydroxyalkyi derivative of γ-cyclodextrin or β-cyclodextrin may comprise one C1-C3 hydroxyalkyi group per glucopyranose unit. According to one embodiment, a C1-C3 hydroxyalkyi derivative of β-cyclodextrin may comprise from one to 7, preferably from 1 to 6, more preferably from 1 to 5 C1-C3 hydroxyalkyi groups. According to one embodiment, a C1-C3 hydroxyalkyi derivative of γ-cyclodextrin may comprise from one to 8, preferably from 1 to 7, preferably from 1 to 6, more preferably from 1 to 5 C1-C3 hydroxyalkyi groups. According to one embodiment, a C1-C3 hydroxyalkyi derivative of γ-cyclodextrin or β-cyclodextrin may comprise from one to 6, preferably from 1 to 5, more preferably from 2 to 5, and more preferably 5 C1-C3 hydroxyalkyi groups.

When a plurality of C1-C3 hydroxyalkyi groups is present in cyclodextrin derivatives useful for the invention, the C1-C3 hydroxyalkyi groups may be identical or different from each other, and preferably are identical.

A C1-C3 hydroxyalkyi group may contain one or more hydroxyl groups, and preferably one or two hydroxyl groups, as for example 2-hydroxypropyl, 3-hydroxypropyl, and dihydroxypropyl. Preferably, a C1-C3 hydroxyalkyi group comprises one hydroxyl group.

An alkyl group of a C1-C3 hydroxyalkyi group may be a methyl, an ethyl or a propyl group, preferably an ethyl or a propyl group, and more preferably a propyl group.

According to one embodiment, a C1-C3 hydroxyalkyi group may be a hydroxypropyl or a hydroxyethyl group. More preferably, a C1-C3 hydroxyalkyi cyclodextrins derivative useful for the invention may be a hydroxypropyl γ-cyclodextrin, and more preferably a 2-hydroxypropyl v- cyclodextrin.

According to a preferred embodiment a C1-C3 hydroxyalkyi derivative of a cyclodextrin is a Cr C3 hydroxyalkyi derivative of γ-cyclodextrin. Preferably, a C1-C3 hydroxyalkyi derivative of γ- cyclodextrin is a hydroxypropyl derivative of γ-cyclodextrin. More preferably, a hydroxypropyl derivative of γ-cyclodextrin is 2-hydroxypropyl γ-cyclodextrin. More preferably, a 2- hydroxypropyl γ-cyclodextrin comprises from 1 to 6, more preferably from 2 to 5, and more preferably 5 hydroxypropyl groups.

According to another embodiment, a γ-cyclodextrin or β-cyclodextrin derivative may be an oside derivative of γ-cyclodextrin or β-cyclodextrin.

An oside derivative of γ-cyclodextrin or β-cyclodextrin useful for the invention may comprise at least one glycoside group. According to one embodiment, an glycoside derivative of v- cyclodextrin or β-cyclodextrin may comprise one glycoside group per glucopyranose unit. According to one embodiment, a glycoside derivative of β-cyclodextrin may comprise from one to 7, preferably from 1 to 6, more preferably from 1 to 5 glycoside groups. According to one embodiment, a glycoside derivative of γ-cyclodextrin may comprise from one to 8, preferably from 1 to 7, preferably from 1 to 6, more preferably from 1 to 5 glycoside groups. According to one embodiment, a glycoside derivative of γ-cyclodextrin or β-cyclodextrin may comprise from one to 6, preferably from 1 to 5, more preferably from 2 to 5, and more preferably 5 glycoside groups.

When a plurality of glycoside groups is present in cyclodextrin derivatives useful for the invention, the glycoside groups may be identical or different from each other, and preferably are identical.

According to one embodiment a glycoside group may be comprised of at least one ose unit (monosaccharide), preferably 2, more preferably 3, more preferably 4 ose units, more preferably 5 ose units, and more preferably 6 ose units (polysaccharide).

An ose unit may be a glucose or a galactose unit, and preferably is a glucose unit. A glycoside group comprised of two ose units may be maltose. A glycoside group comprised of three ose units may be a maltotriose. A glycoside group comprised of 4 ose units may be a dimaltose. Preferred glycoside groups for glycoside derivative of γ-cyclodextrin or β-cyclodextrin are glucosyl, maltosyl or maltotriosyl groups. According to a preferred embodiment a glycoside derivative of a cyclodextrin is a glycoside derivative of γ-cyclodextrin. Preferably, a glycoside derivative of γ-cyclodextrin is a glucosyl or a maltosyl derivative of γ-cyclodextrin. Preferably, a glucosyl or maltosyl γ-cyclodextrin comprises from 1 to 6, more preferably from 2 to 5, and more preferably 5 glucosyl or maltosyl units.

Preferred derivatives in the context of the invention are inter alia hydroxypropyl, hydroxyethyl, glucosyl, maltosyl or maltotriosyl derivatives of γ-cyclodextrin or β-cyclodextrin, or a mixture thereof.

A γ-cyclodextrin or β-cyclodextrin, or derivative thereof, according to this embodiment preferably has a molecular weight in the range of 1000 g/mol to 6000 g/mol, preferably in the range of 1 100 g/mol to 5500 g/mol, preferably in the range of 1 100 g/mol to 5000 g/mol, preferably in the range of 1 100 g/mol to 4000 g/mol, preferably in the range of 1 100 g/mol to 3500 g/mol, preferably in the range of 1200 g/mol to 3000 g/mol, preferably in the range of 1300 g/mol to 2500 g/mol, preferably in the range of 1300 g/mol to 2000 g/mol, and more preferably in the range of 1300 g/mol to 1900 g/mol. Preferably, the molecular weight of such derivatives does not exceed about 1800 g/mol.

Preferably, a γ-cyclodextrin or β-cyclodextrin, or derivatives thereof, has a molecular weight in the range of 1000 to 1700 g/mol, preferably in the range of 1 100 to 1600 g/mol.

The preferred cyclodextrin(s), or derivative(s) thereof, to be included in a composition in accordance with the invention are β-clyclodextrin, γ-cyclodextrin, an hydroxyethyl or hydroxypropyl-cyclodextrin, and in particular a 2-hydroxypropyl-cyclodextrin, or a mixture thereof.

Preferred cyclodextrin(s), or derivative(s) thereof, to be included in a composition in accordance with the invention are γ-cyclodextrin, an hydroxyethyl or hydroxypropyl- cyclodextrin, and in particular a 2-hydroxypropyl-cyclodextrin, or a mixture thereof.

The most preferred cyclodextrins or derivative(s) thereof to be included in a composition according to the invention are γ-cyclodextrin, 2-hydroxypropyl γ-cyclodextrin, preferably comprising five 2-hydroxypropyl units, or a mixture thereof.

Inactivated poliovirus

IPV is usually used without need for an adjuvant. However, IPV may be contacted with adjuvants when combined with others adjuvanted antigens. In such case an adsorption of the IPV on the adjuvant(s) may occur.

According to one embodiment of the composition according to the invention, the IPV is at least partially adsorbed. The adsorption may be on any inorganic adjuvant present in the composition, preferably on an aluminum gel, more preferably on aluminum hydroxide gel (also known as aluminum oxyhydroxide) or aluminum phosphate gel (also known as aluminum hydroxide phosphate), and more preferably on aluminum phosphate gel. According to another embodiment, the composition of the invention comprises unadsorbed IPV. The IPV protection by cyclodextrins according to the invention seems to be independent on adsorption or not of IPV.

An immunogenic composition according to the invention is preferably to be understood as a composition capable of generating an immune response, and preferably a protective immune response against poliovirus type 1 , 2 or 3, in a host after inoculation, preferably in a human host.

Respective amounts of each IPV serotypes to be included in a human dose of vaccine are known to the skilled person and specific amounts are recommended by the World Health Organization (WHO).

As models useable for evaluating the immunogenicity of a composition of the invention, one may cite the models mentioned in the European Pharmacopeia. In this respect, it is to be noted that the content of IPV in a vaccine or solution is generally expressed specifically for each serotype as D-antigen units. A corresponding enzyme immunoassay has been developed for measuring D-antigen content and the potency of vaccines now is conventionally expressed with regard to D-antigen references clinically 5 validated. A clinical correlation has been established between titers in D-antigen (antigenicity) and titers in antibodies (immunogenicity, or capacity to induce an immune response).

Methods for measuring D-antigen units are well known to the skilled person and are also detailed for example in the European Pharmacopeia. One such method is ELISA test, with standard antibodies against each serotype. Example 1 or Sawyer et al. (Biologicals, 1993, 21 : 10 169-177) provide details of such a method to assay D-antigen unit of a composition.

The D-antigen titer may be determined according to mathematical method: the sigmoid method and the parallel lines method (Brownlee, Statistical Theory and Methodology in Science and Engineering, Wiley & Sons (New York), 1965:352-358).

The D-antigen units given with regard to a composition of the invention are preferably 15 determined according to the ELISA principle detailed in Sawyer et al. (Biologicals, 1993, 21 :

169-177) or detailed in the Examples presented thereafter with the sigmoid method as the mathematical method. Preferably, the ELISA is performed with validated polyclonal antibodies. It must be noted that the D-antigen titers of an IPV serotype are given independently of the volume of liquid used to carry the IPV. Otherwise said the D-antigen titers are given per dose 20 of IPV or vaccine to be administered.

Examples of immunogenic doses of IPV contained in a vaccine are 40 D-antigen units of IPV type 1 , 8 D-antigen units of IPV type 2 and 32 D-antigen units of IPV type 3 (e.g. Pentavac, Hexavac) per dose (doses determined according to the sigmoid method). Typically, immunogenic doses of IPV are provided in 0.5 ml dose. An IPV vaccine may otherwise be 25 formulated in volumes ranging from 0.1 mL to 1 mL. But, the immunogenic doses stay the same whatever the volume used.

Some recent publications seem however to indicate that lower doses of IPV are still capable of inducing an acceptable protective response (see EP 2 097 102 and Quiambao et al, 2012). Whereas the doses proposed in these publications are lower than the standard doses, the

30 respective proportions of each serotype with respect to the others remain identical.

The respective immunogenic doses of IPV types 1 , 2 and 3 in a composition of the invention are preferably:

10 to 320 D-antigen units of poliovirus type 1 ;

2 to 32 D-antigen units of poliovirus type 2; and

35 8 to 64 D-antigen units of poliovirus type 3.

According to one embodiment, a suitable immunogenic dose of IPV type 1 for the invention may range from 10 to 320 D-antigen units, preferably from 10 to 100, preferably from 15 to 80, more preferably from 20 to 60, more preferably from 20 to 43, and more preferably is about 29 D-antigen units. According to one embodiment, a suitable immunogenic dose of IPV type 2 for the invention may range from 2 to 32 D-antigen units, preferably from 2 to 20, preferably from 4 to 15, more preferably from 5 to 10, more preferably from 5 to 9, and more preferably is about 7 D-antigen units.

According to one embodiment, a suitable immunogenic dose of IPV type 3 for the invention may range from 8 to 64 D-antigen units, preferably from 10 to 64, preferably from 10 to 60, more preferably from 15 to 40, more preferably from 16 to 36, and more preferably is about 26 D-antigen units.

As previously, mentioned an immunogenic dose of IPV may be formulated in a volume ranging from 0.1 imL to 1 imL, preferably from 0.2 ml. to 0.8 imL, and more preferably is about 0.5 mL. It is however noted that the invention also concerns bulk concentrate, comprising higher titer dose of IPV. The respective proportions with respect to each serotype is however to be maintained in such a bulk concentrate.

Moreover, the invention also concerns multidose compositions, i.e. compositions comprising "x" doses of IPV suitable for "x" injections, for example 10 doses of IPV or less, and most preferably 5 doses of IPV. In this case, multidose compositions of the invention comprise "x" times the immunogenic doses mentioned above. For example, a composition corresponding to 5 immunogenic doses of IPV comprises:

50 to 1600 D-antigen units of poliovirus type 1 ; and/or

10 to 160 D-antigen units of poliovirus type 2; and/or

40 to 320 D-antigen units of poliovirus type 3.

According to a preferred embodiment, a suitable amount of cyclodextrin(s), or derivative(s) thereof, relative to an immunogenic dose of IPV, may range from 0.03 μιηοΙ of clyclodextrin(s), or derivative(s) thereof, per dose of IPV to 1 μιηοΙ (micromole), preferably from 0.06 μιηοΙ to 1 μιηοΙ, preferably from 0.08 to 0.8 μιηοΙ, preferably from 0.1 to 0.6 μιηοΙ, preferably from 0.12 to 0.5 μιηοΙ, preferably from 0.125 to 0.4 μιτιοΙ, and more preferably from 0.125 to 0.25 μιηοΙ of cyclodextrin(s), or derivative(s) thereof, per dose of IPV. A dose of IPV is as defined above; it can be a dose of monovalent IPV (only one serotype); a dose of bivalent IPV (2 different serotypes) or a dose of trivalent IPV (the 3 different serotypes).

According to a preferred embodiment, a suitable amount of cyclodextrin(s), or derivative(s) thereof, may range from 0.03 to 1 μιηοΙ, preferably from 0.06 μιηοΙ to 1 μιηοΙ, and more preferably from 0.125 to 0.25 μιηοΙ for a mixture of IPV types 1 , 2 and 3 comprising from 10 to 320 D-antigen units of poliovirus type 1 , from 2 to 32 D-antigen units of poliovirus type 2, and from 8 to 64 D-antigen units of poliovirus type 3.

As specified above, the invention also encompasses bulk concentrate solution and multidose compositions. In such a case, the IPV titer is "x" times the titers per dose given above and the cyclodextrin amount, or derivative thereof, is thus also "x" times the range given above of 0.03 μιηοΙ to 1 μιτιοΙ. For example, a composition corresponding to 5 immunogenic doses of IPV comprises preferably from 0.15 μιηοΙ to 5 μιηοΙ of cylcodextrin(s) or derivative(s) thereof. It is also to be noted that, when a combination of different cyclodextrins or derivatives thereof are included in the immunogenic composition, the cyclodextrin amount is preferably the combined amount of all cyclodextrins or derivatives thereof present in the composition. Thiomersal-containing compositions

A composition according to the invention may either comprise or not thiomersal. The deleterious effects on IPV titer can be highlighted as shown in examples 2 and 3 of the present application. As demonstrated in these examples, the IPV titer losses are preferably determined in a suspension of aluminum hydroxide. Alternatively, IPV titer loss in presence of thiomersal may also be determined in formulated vaccine, as demonstrated by the inventors with the Shan5 vaccine.

As indicated, an immunogenic composition of the invention may comprise thiomersal.

According to one embodiment, an immunogenic composition according to the invention may be blended with a thiomersal-containing composition, optionally containing aluminum, preferably under the form of a gel of aluminum, most preferably containing aluminum phosphate gel.

A thiomersal-containing composition may be an aqueous thiomersal-containing solution or suspension or a dried thiomersal-containing composition.

A composition according to the invention obtained after blending with a thiomersal-containing composition, optionally containing aluminum, preferably under the form of a gel of aluminum, is still capable of generating an immune response against a poliovirus.

When an immunogenic composition of the invention is blended with an aqueous thiomersal- containing solution or suspension, the composition of the invention may be formulated as a liquid composition or as a dried-composition.

When an immunogenic composition of the invention is blended with a dried thiomersal- containing composition, the composition of the invention is preferably formulated as a liquid composition.

An immunogenic composition of the invention is such that after blending with a thiomersal- containing composition, optionally containing aluminum, preferably under the form of a gel of aluminum, the so obtained composition has a level of antigenicity of IPV relative to the level of antigenicity of a composition of the invention devoid of thiomersal which differs by less than 50% after a period of time ranging from 0.5 to 8 hours after contacting IPV with thiomersal, and preferably after a period of time ranging from 1 to 6 hours, and at a temperature ranging from 5°C to 35°C, preferably from 5°C to 25°C, preferably from 5°C to 10°C, and more preferably at about 5°C.

A composition according to the invention comprising IPV, cyclodextrin(s) or derivative(s) thereof, and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, is preferably an extemporaneous solution or suspension to be used in the following hours if intended for vaccination, most preferably in less than 8 hours, preferably in less than 6 hours, preferably in less than 4 hours, preferably in less than 2 hours, preferably less than 1 hour, preferably less than half an hour, or even less than 15 minutes after its reconstitution, namely after IPV and thiomersal have been brought into contact with each other. According to one embodiment, a composition according to the invention comprising IPV, cyclodextrin(s) or derivative thereof, and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, is used after a period of time ranging from 0.5 to 8 hours after contacting IPV with thiomersal, and preferably after a period of time ranging from 1 to 6 hours, and more preferably after a period of time ranging from 2 to 4 hours.

A composition according to the invention comprising IPV, cyclodextrin(s) and thiomersal may be a single dose or a multidose composition.

According to one embodiment, when a composition of the invention comprising IPV, cyclodextrin(s) or derivative(s) thereof, and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, inter alia aluminum phosphate gel, is a single dose composition, it is preferably used for administration to an individual immediately upon contacting IPV with thiomersal or up to half an hour after contacting IPV with thiomersal.

According to another embodiment, when a composition of the invention comprising IPV, cyclodextrin(s) or derivative(s) thereof, and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, is a multidose composition, it is preferably used for administration to an individual immediately upon contacting IPV with thiomersal or up to 8 hours after contacting IPV with thiomersal, and more preferably up to 6 hours.

According to one embodiment, a composition according to the invention comprising IPV, cyclodextrin(s) or derivative(s) thereof, and thiomersal, optionally containing aluminum, preferably under the form of a gel of aluminum, is kept at a temperature ranging from 5°C to 35°C, preferably from 5°C to 25°C, preferably from 5°C to 10°C, and more preferably at about 5°C after contacting IPV with thiomersal.

According to one embodiment, a composition according to the invention comprising IPV, cyclodextrin(s) or derivative(s) thereof, and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, has a level of antigenicity of IPV relative to the level of antigenicity of a composition of the invention devoid of thiomersal, which differs by less than 50%, preferably by less than 40%, more preferably by less than 30%, more preferably by less than 20%, and more preferably by less than 10%, after contacting IPV with thiomersal.

According to a preferred embodiment, an immunogenic composition according to the invention, comprising IPV, cyclodextrin(s) or derivative(s) thereof, and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, has a level of antigenicity (or D-antigen titer) of IPV which is not impaired, or not substantially impaired, with respect to the level of antigenicity (or D-antigen titer) of IPV in an identical composition but devoid of thiomersal. As used herein, "antigenicity" (or D-antigen titer) is the ability to be recognized by a specific antibody. Preferably, this means that the level of antigenicity (or D-antigen titer) of IPV of a composition comprising IPV, cyclodextrin(s) or derivative(s) thereof and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, differs by less than 50%, and even preferably by less than 30%, preferably by less than 25%, or even by less than 20%, from the same composition but without thiomersal, after a period of time from 0.5, or from 1 to 8 hours, after contacting IPV with thiomersal, and at a temperature ranging from 5°C to 35°C or below. Where different IPV serotypes are present in the composition, the maximal reduction of the level of antigenicity, i.e. 50% reduction, is applicable individually to each serotype. In the context of the present invention, the level of antigenicity of IPV is used interchangeably with IPV titer in D-antigen unit, and is measured as indicated above and as detailed in the experimental section, with specific well defined antibodies.

According to an embodiment, an immunogenic composition comprising IPV, cyclodextrin(s) or derivatives thereof and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, has a level of immunogenicity which is not impaired, or not substantially impaired, with respect to the level of immunogenicity of IPV in an identical composition but devoid of thiomersal. As used herein, "immunogenicity" refers to the ability of a compound to raise an immune response in vivo. Where different IPV serotypes are present in the composition, the maximal reduction of the antibody titer, i.e. 50% reduction, is applicable individually to each serotype. Immunogenicity of a composition is measured in vivo, by administration of the composition to a host, and by determining the titer of the antibodies raised by said administration.

The preferred maximal content or concentration of thiomersal in a composition of the invention is dependent on the content of IPV. It is preferred that the thiomersal concentration does not exceed 200 μg/mL, i.e. 100 μg per 0.5 imL, and more preferably does not exceed 150 μg/mL, for a composition comprising the following IPV doses or titers:

10 to 320 D-antigen units of poliovirus type 1 ; and/or

2 to 32 D-antigen units of poliovirus type 2;and/or

8 to 64 D-antigen units of poliovirus type 3.

Preferably, the thiomersal concentration does not exceed 150 or 100 μg/mL in a composition having the IPV doses mentioned above. Preferably, the thiomersal concentration may range from 4 to 200 μg/mL, preferably from 10 to 200 μg/mL, preferably from 20 to 150 μg/mL, more preferably from 30 to 1 10 μg/mL, and preferably from 60 to 100 μg/mL.

In another preferred embodiment, the thiomersal concentration does not exceed 75 or 50 μg per dose in a composition having the IPV titer doses mentioned above. Preferably the thiomersal concentration may range from 2 to 100 μg per dose, preferably from 5 to 100 μg per dose, preferably from 10 to 75 μg per dose, more preferably from 15 to 55 μg per dose, more preferably from 15 to 50 μg/mL, and preferably from 30 to 50 μg per dose.

According to one embodiment, an immunogenic composition of the invention may comprise thiomersal in a concentration ranging from 10 to 1 10 μg/mL or more preferably from 50 to 100 μg/mL, and IPV type 1 in a dose ranging from 10 to 43, and more preferably of about 29 D- antigen units, IPV type 2 in a dose ranging from 2 to 9, and more preferably of about 7 D- antigen units, and IPV type 3 in a dose ranging from 8 to 36, and more preferably of about 26 D-antigen units. Alternatively, a composition according to the invention may also be free of thiomersal. In such a case, the composition of the invention is however protected against the potential addition of thiomersal at a later stage of the preparation of the vaccine or of the vaccination process. As detailed in the introductive section, thiomersal may indeed be added simultaneously with addition of some antigens, and namely when adding whole cell Pertussis, inactivated by thiomersal, as exemplified in the experimental section. Thiomersal may also be added to multidose formulations as a preservative. A composition according to the invention is thus protected against IPV titer losses induced by potential further addition of thiomersal.

The composition according to the invention may also advantageously comprise one or more additional antigens, especially antigens capable of inducing a protective immune response against infectious disease. It is noted that, where such additional antigens are inactivated or otherwise produced or formulated in association with thiomersal a composition according to the invention may comprise such an additional antigen preferably only extemporaneously, only for a short period of time before administration or inoculation, preferably only for less than 8 hours, preferably less than 6 hours, preferably less than 4 hours, less than 2 hours, less than 1 hour, or even less than half an hour. Ideally, thiomersal and protected IPV should be contacted less than 6 hours, preferably less than 2 hours, more preferably less than 1 hour, preferably less than half an hour, or even less than 15 minutes, before administration. During this period of time before administration or inoculation, the composition is preferably stored at a temperature ranging from 5°C to 35°C or below, for example between 5°C and 25°C, and preferably at 5°C.

Particularly preferred additional antigens to be included in a composition according to the invention are the following ones:

Diphtheria antigens:

Diphtheria is an acute infection caused by the bacteria Corynebacterium diphtheriae.

The diphtheria antigen present in vaccine is generally a diphtheria toxoid (DT). The preparation of such diphtheria toxoids (DT) is well known to the skilled person. For instance, DT may be produced by purification of the toxin from a culture of Corynebacterium diphtheriae and then by chemical detoxification, or may be obtained by recombinant technology or by genetically detoxified analogue of the toxin.

In one embodiment, the diphtheria toxoid used in the context of the present invention may be pre-adsorbed, for example on an aluminum salt such as aluminum hydroxide or aluminum phosphate or a mixture thereof. Tetanus antigens:

Tetanus is an acute infection caused by Clostridium tetani. C. tetani exists as a nonpathogenic organism in the gut of humans and animals. The organism is also found in soil contaminated by feces and may survive in soil for years as infectious spores. Tetanus results from the anaerobic growth of C. tetani and neurotoxin production in contaminated wounds. Infection is caused by the introduction of materials contaminated by organisms or spores into tissue.

The tetanus antigen to be used in a vaccine of the invention is generally a tetanus toxoid (TT). Methods of preparing tetanus toxoids are well known to the skilled person. For example, TT may be obtained by purification of the toxoid from a culture of Clostridium tetani and then by chemical detoxification, or may be obtained by recombinant technology or by genetically detoxified analogue of the toxin.

Pertussis components

Whooping cough or pertussis is a severe, highly contagious upper respiratory tract infection caused by Bordetella pertussis.

The pertussis component used in vaccines may be either killed whole-cell (wcP) Pertussis vaccine, where chemically- and heat-inactivated whole cell Pertussis is used as the Pertussis component, or acellular (acP) Pertussis antigen, where purified defined pertussis antigens are used.

Chemically- and heat-inactivation methods are well known to the skilled reader. Such methods may include heat (e.g. 55-65°C for at least several minutes), formaldehyde, glutaraldehyde, acetone inactivation. Examples of inactivation methods for killed whole cell components are disclosed in WO 93/24148.

Thiomersal is frequently used in the inactivation method of whole-cell Bordetella pertussis. As detailed in the present description, presence of thiomersal is now proven as detrimental to IPV of combination vaccines.

More recently, defined component pertussis vaccines have been developed, known as acellular Pa vaccines. They generally include one of the following Bordetella pertussis antigens: Pertussis Toxin (PT), Filamentous haemagglutinin (FHA), the 69kDa outer membrane protein (pertactin or PRN) and fimbrial agglutinogens (FIM), as disclosed in WO 98/00167. PT and FHA are advantageously included in the formulation of the present invention. Hepatitis B antigens:

Hepatitis B is an infectious illness of the liver caused by the hepatitis B virus (HBV) that affects hominoidea, including humans. The acute illness causes liver inflammation, vomiting, jaundice, and, rarely, death. Chronic hepatitis B may eventually cause cirrhosis and liver cancer. The Hepatitis B antigen to be used in vaccine is one of the viral envelope proteins, Hepatitis B surface antigen (HBsAg). This antigen is preferably produced recombinantly into Saccharomyces cerevisiae yeast cells, where it is grown, harvested and purified. Such a procedure, as well as alternative processes for the production of HBsAg, is well known to the skilled person.

In one embodiment, the HBsAg used in the context of the present invention may be pre- adsorbed, for example on an aluminum salt such as aluminum hydroxide or aluminum phosphate or a mixture thereof. Haemophilus influenzae type b antigens:

Prior to the availability of effective vaccines, Haemophilus influenza type b (Hib) was a major cause of meningitis invasive bloodborne infections in young children and was the main cause of meningitis in the first 2 years of life.

Immunization against Haemophilus influenza began with a polysaccharide vaccine, namely polyribose ribitol phosphate [PRP] capsular polysaccharide from Haemophilus influenzae type b. Because of the immunological shortcomings of the polysaccharide vaccines, conjugate vaccines are now produced, for example, a vaccine consisting of PRP conjugated to diphtheria toxoid (PRP-D). Another PRP conjugates such as PRP conjugated with tetanus toxoid (PRP-T) are also used as vaccine.

According to a preferred embodiment, the Haemophilus influenzae b (Hib) antigen is a capsular polysaccharide or oligosaccharide antigen, optionally conjugated to a carrier protein.

Meningococcal Meningitis and Neisseria meningitidis types A, C, W or Y antigens

Meningococcal meningitis is a rare but serious infection. It causes the membranes that cover the brain and spinal cord to become inflamed. The bacterium Neisseria meningitidis, also called meningococcus, is the causative agent of meningococcal meningitis.

Disease-causing strains are classified according to the antigenic structure of their polysaccharide capsule. Serotype distribution varies markedly around the world, with type A being most prevalent in Africa and Asia but practically absent in North America. This distribution has hindered the development of a universal vaccine for meningococcal disease. Among the 13 capsular types of N. meningitidis that have been identified, six of these (A, B, C, W135, X, and Y) account for most disease cases worldwide.

The immunogenic composition according to the invention may advantageously comprise one or more of the N. meningitidis type B antigens disclosed above, either alone or in association with one or more of N. meningitidis type A, C, Y or W-135 capsular polysaccharide.

In one embodiment, the N. meningitidis antigen(s), either conjugated to a protein carrier or not, used in the context of the present invention may be pre-asdorbed, for example on an aluminum salt such as aluminum hydroxide or aluminum phosphate or a mixture thereof. In another embodiment, the N. meningitidis antigen(s) is (are) not adsorbed. Other additional bacterial polysaccharides or bacterial oligosaccharides which may be included in the composition of the invention are inter alia Streptococcus pneumoniae component, preferably serotypes 1 , 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F, group A streptococci component, group B streptococci component, Staphylococcus aureus or Staphylococcus epidermis component.

For example, according to one embodiment of the present invention, the composition comprises at least one bacterial polysaccharide or one bacterial oligosaccharide, preferably conjugated to a carrier protein as detailed above. These bacterial polysaccharides or oligosaccharides comprise capsular polysaccharides from any bacterium, for example one or more from Neisseria meningitidis (for example, capsular polysaccharides derived from one or more serogroups A, C, W-135 and Y), from Haemophilus injluenzae b, from Streptococcus pneumoniae (preferably serotypes 1 , 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F), group A streptococci, group B streptococci, Staphylococcus aureus or Staphylococcus epidermis.

In addition to or in place of the above mentioned bacterial polysaccharides or oligosaccharides, a composition according to the invention may also advantageously comprises one or more of the components of the group consisting of the following components: Botulinum neurotoxin (BoNT), Bacillus anthracis antigen, Staphylococcal enterotoxin, Yersinia pestis antigen, Salmonella typhi antigen(s), Hepatitis A antigen and Malarial antigen(s).

Any other antigenic components likely to be useful in vaccination may also be included in an immunogenic composition according to the present invention.

Combination Vaccines

Although there are many actual and potential benefits of vaccines that combine antigens to confer protection against multiple pathogens, it must be checked that these combinations do not have a detrimental effect on the immunogenicity of the individual components. Combinations of diphtheria and tetanus toxoids with whole cell pertussis vaccine (DTP) have been available for over 50 years and the antibody response to the combination is superior to that of the individual components, perhaps as a result of the adjuvant effect of the whole cell pertussis vaccine. DTaP combinations that include inactivated poliovirus vaccine and acellular Pertussis are also available and give good results. The effects of combining DTP vaccines with Hib conjugate vaccine have also been studied and satisfactory combinations are now available.

In combining different antigens as previously described, it is important to select appropriate relative amounts for the included antigens, in order for such combination vaccines to be effective at achieving the criterion of seroprotection for each individual antigenic component. Vaccines are available on the market combining up to 10 separate antigens designed to elicit seroprotection for up to seven different infectious diseases. Preferred combinations of additional antigens in a composition of the invention, i.e. in addition to IPV and cyclodextrin(s) or derivative(s) thereof, are a) diphtheria toxoid and tetanus toxoid; b) diphtheria toxoid, tetanus toxoid and acellular Pertussis component; c) diphtheria toxoid, tetanus toxoid, acellular Pertussis component and Hepatitis B surface antigen; d) diphtheria toxoid, tetanus toxoid and PRP capsular polysaccharide from Haemophilus influenzae type b conjugated to a protein carrier, e) diphtheria toxoid, tetanus toxoid, acellular Pertussis component and PRP capsular polysaccharide from Haemophilus influenzae type b conjugated to a protein carrier and f) diphtheria toxoid, tetanus toxoid, acellular Pertussis component, PRP capsular polysaccharide from Haemophilus influenzae type b conjugated to a protein carrier and Hepatitis B surface antigen. A more preferred combination is composition f).

Further preferred combinations of additional antigens in a composition of the invention, i.e. in addition to IPV and cyclodextrin(s) or derivative(s) thereof, are g) diphtheria toxoid, tetanus toxoid and whole cells Pertussis component; h) diphtheria toxoid, tetanus toxoid, whole cell Pertussis component and Hepatitis B surface antigen; i) diphtheria toxoid, tetanus toxoid, whole cell Pertussis component and PRP capsular polysaccharide from Haemophilus influenzae type b conjugated to a protein carrier and j) diphtheria toxoid, tetanus toxoid, whole cell Pertussis component, PRP capsular polysaccharide from Haemophilus influenzae type b conjugated to a protein carrier and Hepatitis B surface antigen. A more preferred combination is composition j).

When several conjugated bacterial oligosaccharides or polysaccharides are present in the composition according to the present invention, the oligosaccharides or polysaccharides can be conjugated to the same carrier protein or to different carrier proteins, preferably while adhering to the teaching of application WO 98/51339 (AU 748716B) regarding the maximum load (amount) of carrier proteins in one dose.

According to an embodiment of the present invention, the immunogenic composition described above comprising IPV and cyclodextrin(s), is an aqueous or liquid solution or suspension. The immunogenic composition is thus formulated such that it can directly be inoculated, potentially after dilution or mixture with additional antigens or with other components. The liquid formulations are also suitable for reconstituting other vaccines from a lyophilized form.

The pH of a liquid composition according to the invention is preferably in the range of 5.5 to 8.5, but variations are acceptable, provided the structure of the antigens present in the composition are not impaired by the pH. Preferably, the pH of the composition is a physiological pH, in the range of 7.3 to 7.5.

According to another embodiment of the present invention, the immunogenic composition is formulated as a dried formulation or as a highly viscous liquid formulation, preferably as a powder, microparticles, or micropellets formulation. The immunogenic composition is thus formulated such that it needs to be reconstituted in a liquid solution or suspension before being inoculated to a host. The antigenicity of the IPV in a final liquid composition, obtained either by combination of liquid or dried immunogenic composition with a 2^nd composition, liquid or dried, containing thiomersal, may be expressed either with respect to the antigenicity of the antigen in the immunogenic composition, but taking into account the dilution effect due to the mix, or with regard to the antigenicity of the antigen in a similar final composition but devoid of thiomersal. In the context of the present invention, the term "dry" or "dried" denotes a product which is characterized by a residual water content of less than 6%, and more preferably less than 3% (measured by the method according to Karl Fischer) and which is solid.

Indeed, in order to stabilize fragile, heat-sensitive products, a drying technique known as freeze-drying is generally used. Freeze-drying is a technique which uses successively freezing and then sublimation in order to dry and stabilize fragile products. Whereas such a process is sometimes accompanied by a reasonably large loss of titer or of activity, there are now methods of preparing dry vaccine composition comprising at least one of the three inactivated poliovirus (IPV) serotypes which enable the IPV polio antigen to be dried, for example by freeze-drying, without a large loss of titer during the drying process and which enable the resulting composition to be stored for at least 7 days at 37°C with a loss of titer which is less than anything which has been disclosed to date. Such a freeze-drying process, preserving the IPV titer, is for example disclosed in the application WO 2012/028315.

According to different embodiments of the present invention, the immunogenic dry composition is obtained or obtainable from an initially liquid composition, for example by freeze drying, spray-freeze drying, spray-drying or prilling and freeze-drying of an originally liquid solution. According to a preferred embodiment of the invention, the composition is obtained or likely to be obtained by freeze drying of an initially liquid composition.

When the composition of the invention is a dried composition, the different amounts of IPV, cyclodextrins or additional antigens are preferably expressed by reference to the initially liquid composition, before the drying step.

A dry composition according to an embodiment of the invention is preferably intended for reconstitution in an aqueous solution or suspension, in conditions allowing the titers of the antigen components of the composition not to be impaired by the drying and reconstitution steps. In this respect, the immunogenic composition, after reconstitution in a similar volume as the initial one, has an IPV titer which is preferably less than 50% inferior to the titer of the initially liquid composition, preferably less than 50% or 30% or 25% or 20% inferior, in a period of time from 0.5 or 1 to 8 or 6 hours, and at a temperature ranging from 5°C to 35°C or 25°C. According to a preferred embodiment of the invention, the dried immunogenic composition is intended for reconstitution in an aqueous thiomersal-containing solution or suspension. In this case, thanks to the presence of the cyclodextrin(s) or derivatives thereof in the composition of the invention, the reconstitution will not generate IPV titer loss, at least in the hours following reconstitution with the aqueous thiomersal-containing solution or suspension, and at least not in an extent liable to be detrimental to the obtaining of an immunizing response. A dried immunogenic composition is thus suitable for reconstitution in a solution or suspension comprising antigen(s) inactivated or stored in presence of thiomersal, inter alia whole cell Pertussis or multidose formulation. After reconstitution in an aqueous thiomersal-containing solution or suspension, a dried composition according to the invention may indeed still be capable of generating an immune response against polio virus, due to protection of IPV titer by cyclodextrins.

According to an embodiment, the immunogenic composition, after drying and reconstitution step in an aqueous thiomersal-containing solution or suspension, has an IPV titer relative to the IPV titer of the original liquid solution or suspension which still differs by less than 50%, preferably by less than 30% or 25% or 20%, in a period of time from 0.5 or 1 to 8 or 6 hours, and at a temperature ranging from 5°C to 35°C or 25°C, when reconstitution is operated in the same volume as the initial volume of the composition, before drying. It is reminded that IPV titer can be measured as detailed in the experimental section with the level of antigenicity of IPV with respect to reference antibodies.

Preferably, the reconstituted IPV composition, reconstituted in an aqueous thiomersal- containing solution or suspension, has an immunogenicity which is not impaired, or at least not substantially impaired, with respect to the immunogenicity of the originally liquid solution. Immunogenicity of a composition can be measured as the antibody titer generated in vivo upon administration of the composition to a host.

According to a preferred embodiment, an immunogenic composition of the invention to be dried may be formulated, in particular with regard to the antigen titer, so as the reconstituted composition can contain the target titer in. The freeze-drying process may result in some antigen losses which may range, depending on the antigen and the freeze-drying process, from 0 to 50%. For instance, with regard to serotype 1 of IPV, the loss due to lyophilization may be about 30% of the initial titer.

Therefore, before being freeze-dried, an immunogenic composition of the invention may be formulated with an antigen "overage", namely additional amount of antigen, so as to offset the freeze-drying loss. This overhead may be, depending on the antigen and the lyophilization process, up to 50% of target titer to be reach in the reconstituted composition. For instance, regarding IPV serotype 1 , it may be added 30% more of antigen in the composition to be lyophilized than the target titer in the reconstituted composition. With regard to serotypes 2 and 3 of IPV, the antigen "overage" may be respectively 0 and 50%.

In addition, before being freeze-dried an immunogenic composition of the invention may be concentrated.

After reconstitution with a second liquid composition containing thiomersal, the antigen titer of IPV in the reconstituted composition is preferably less than 50% inferior to a titer obtained by reconstituting a same immunogenic composition of the invention with a second composition devoid of thiomersal, preferably less than 30% or 25% or 20%, or 10% inferior. If one wants to express the antigen titer of IPV in the reconstituted composition with respect to the antigen titer of the immunogenic composition before freeze-drying, it would be necessary to deduct from the antigen titer in the immunogenic composition to be freeze-dried the antigen overage, and to take into account a possible concentration of the composition so as to have 5 comparable antigen titer.

Alternatively, an immunogenic composition of the invention comprising IPV and cyclodextrin(s) or derivative(s) thereof may be a liquid composition, preferably an aqueous solution or suspension, and may be used to rehydrate a dried composition comprising other antigen(s) 10 and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum. Such a dried composition may be prepared as above-described for the dried IPV compositions.

In this case, thanks to the presence of the cyclodextrin(s) or derivative(s) thereof in the composition of the invention, the reconstitution will not generate IPV titer loss, at least in the

15 hours following reconstitution, and at least not in an extent liable to be detrimental to the obtaining of an immunizing response. A dried immunogenic composition comprising antigen(s) inactivated or stored in presence of thiomersal is thus suitable for reconstitution in a solution or suspension comprising IPV and cyclodextrin(s). After blending with a dry thiomersal-containing composition, a liquid composition according to the invention may indeed still be capable of

20 generating an immune response against polio virus, due to protection of IPV titer by cyclodextrin(s) or derivative(s) thereof.

According to an embodiment, the liquid immunogenic composition, after reconstitution step of a dry thiomersal-containing composition, has an IPV titer relative to the IPV titer of the original liquid solution or suspension which still differs by less than 50%, preferably by less than 30% or

25 25% or 20%, in a period of time from 0.5 or 1 to 8 or 6 hours, and at a temperature ranging from 5°C to 35°C or 25°C. It is reminded that IPV titer can be measured as detailed in the experimental section with the level of antigenicity of IPV with respect to reference antibodies. Preferably, the reconstituted IPV composition, reconstituting a dry thiomersal-containing composition, has an immunogenicity which is not impaired, or at least not substantially

30 impaired, with respect to the immunogenicity of the originally liquid solution.

According to still another embodiment, an immunogenic composition of the invention comprising IPV and cyclodextrin(s) or derivative(s) thereof, is a liquid composition, preferably an aqueous solution or suspension, and is to be combined or mixed with a second liquid composition comprising other antigen(s) and thiomersal, and optionally containing aluminum,

35 preferably under the form of a gel of aluminum. This second composition may be an aqueous solution or suspension. Thanks to the presence of the cyclodextrin(s) or derivative(s) thereof in the composition of the invention, the combination of both liquid compositions will not generate IPV titer loss, at least in the hours following mixture, and at least not in an extent liable to be detrimental to the obtaining of an immunizing response. Preferably, the liquid immunogenic composition, after combination with the thiomersal-containing composition, has an IPV titer relative to the IPV titer of the original liquid solution or suspension which still differs by less than 50%, preferably by less than 30% or 25% or 20%, in a period of time from 0.5 or 1 to 8 or 6 hours, and at a temperature ranging from 5°C to 35°C or 25°C. Preferably, the final IPV composition, after combination with the liquid thiomersal-containing composition, has an immunogenicity which is not impaired, or at least not substantially impaired, with respect to the immunogenicity of the liquid solution of the invention, comprising IPV and cyclodextrin(s) or derivative(s) thereof. Immunogenic compositions of the invention including vaccines may be prepared inter alia as injectables, as liquid solutions or suspensions, as powder to be reconstituted before injection or as bulk concentrate solution.

According to further embodiments of the invention, the composition as detailed above, either liquid or dried, may comprise further compounds or agents, which are not antigenic components, or immunogens.

The antigenic components, including IPV and potential additional antigens, and cyclodextrin(s), may be mixed with pharmaceutically acceptable excipients which are compatible with the antigenic components. Such excipients may include inter alia water, saline, dextrose, glycerol, ethanol, and combinations thereof. The immunogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the immunogenic effectiveness.

The buffering agent or system which can be present in a composition of the invention is advantageously selected from the group consisting of Hepes, Tris, acetate, succinate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate, bicarbonate, and phosphate. According to different embodiments, the buffering agent is selected from ammonium acetate, ammonium formate, ammonium carbonate, ammonium bicarbonate, triethylammonium acetate, triethylammonium formate, triethylammonium carbonate, trimethylamine acetate trimethylamine formate, trimethylamine carbonate, pyridinal acetate and pyridinal formate. Suitable buffers for use in the invention are phosphate buffer and tris-sucrose buffer.

Moreover, immunogenicity of IPV and of additional further antigenic components present in the composition can be significantly improved if the antigens are coadministered with adjuvants, commonly used as 0.005 to 0.5 percent suspension in phosphate buffered saline. Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvants to be added to the composition of the invention are preferably aluminum oxyhydroxide and/or aluminum hydroxide phosphate (collectively commonly referred to as alum). Alternative adjuvants which can be cited are salts of calcium, iron or zinc, insoluble suspension of acylated tyrosine, acylated sugars, and muramyl peptides (e.g., N-acetyl- muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme (norMDP), N-acetylmuramyl-L-alanyl-D-isogl uatminyl-L-alanine-2-(1 '-2'-dipalmitoyl-sn-glycero- 3-hydroxyphosphory-loxy)-ethylamine (MTP-PE), etc.).

In particular, it may be desirable to improve the TH-1 component of the immune response and to incorporate a TH-1 adjuvant in the vaccine composition according to the invention. TH-1 immune response is usually considered as the cellular component of the immune response and involves cytotoxic T lymphocytes, and natural killer cell responses. High levels of TH1-type cytokines tend to favor the induction of cell mediated immune responses to the given antigen, whilst high levels of TH2-type cytokines tend to favor the induction of humoral immune responses to the antigen. Adjuvants useful for the invention are, for example, described in WO 94/00153 and WO 95/17209. 3 de-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant, known from GB222021 1. A preferred form of 3 de-O-acylated monophosphoryllipid A is disclosed in EP 0 689 454. Usually, the particles of 3D-MPL are small enough to be sterile filtered through a 0.22 micron membrane (as described in EP 0 689 454). 3D-MPL is present in the range of 10 μg-100 μg, more particularly within the range of 25-50 μg whereas the antigen is usually present in a range 5-100 μg per vaccine dose. A 3D-MPL derivative, named RC-529, is described in US 6,1 13,918.

Others useful adjuvants are represented by TLR4 agonists. Preferably the TLR4 agonist is chosen from the group consisting of the chemical compounds identified and exemplified in US 2003/0153532 under the names ER803022, ER803058, ER803732, ER803789, ER804053, ER804057, ER804058, ER804059, ER804442, ER804764, ER1 1 1232, ER1 12022, ER1 12048, ER1 12065, ER1 12066, ER1 13651 , ER1 18989, ER1 19327 and ER1 19328.

Others examples of adjuvant comprises QS21 , an HPLC purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina. Optionally this may be admixed with 3 de-O- acylated monophosphoryllipid A (3D-MPL) or the like. The method of production of QS21 is disclosed in US 5,057,540. A combination of QS21 with cholesterol or a derivative thereof is a useful combination as it decreases the side effects of QS21 (WO 96/33739) and may be also be used as adjuvant.

Further adjuvants include immunomodulatory oligonucleotides, for example unmethylated CpG sequences as disclosed in WO 96/02555 or TH-1 cytokines such as IFNy, IL-2, IL-12, IL-18. Combinations of different adjuvants, such as those mentioned hereinabove, are also contemplated. 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 optimal synergy is 2.5: 1 to 1 :1 3D-MPL:QS21.

The immunogenic composition of the invention may also comprise a surfactant, for example a detergent such a Tween 80. Preferably, the presence of detergent is to be minimized. If intended to be lyophilized, a composition of the invention may also comprises one or several stabilizing excipients such as sucrose, glucose, lactose, trehalose, maltose or a sugar alcohol, such as sorbitol, mannitol or inositol, or dextran, or a mixture of two or more different of these before mentioned stabilizers, such as mixtures of sucrose and trehalose.

Advantageously, the concentration of stabilizing excipients ranges from 2% (w/v) to limit of solubility in the formulated liquid product. In general, the concentration of stabilizing excipients ranges between 5% (w/v) and 40% (w/v), 5% (w/v) and 20% (w/v) or 20% (w/v) and 40% (w/v). Other supplemental components may also be added to a composition of the invention, including a buffer, urea, a reducing or non-reducing disaccharide, a gelling polymer, amino acids, a preservative, an antiseptic or an antifungal agent.

The invention is not limited with respect to the additional components, provided these components are pharmaceutically acceptable.

An immunogenic composition according to the invention can be packaged in unit dose or in multiple dose form, wherein a dose preferably corresponds to a volume of 0.5 imL The immunogenic composition may be packaged inter alia in ready-filled syringes, in vial, or in any other suitable container.

According to a preferred embodiment, the immunogenic composition according to the invention is indeed intended for use as a vaccine for animals, preferably for mammals and especially for human beings. According to another preferred embodiment, the composition is to be used for vaccinating human children, especially newborns, infants and / or toddlers. The composition is advantageously also suitable for vaccinating adults.

The composition is thus intended to be used for the treatment or the prevention of disease caused by infection by poliovirus, or to be used for generating a protective immune response against infection by poliovirus, i.e. poliomyelitis, or to be used for immunizing a host against infection by poliovirus.

Preferably, depending on the further antigens present in the composition, it is also intended to be used for the treatment or the prevention of the diseases caused by the corresponding bacterial or viral pathogens, inter alia for the prevention of diphtheria, tetanus, whooping cough, meningitis, hepatitis B, hepatitis A, etc...

According to this embodiment, the immunogenic composition is thus formulated as a vaccine for in vivo administration to the host wherein the individual antigenic components of the composition are formulated such that the immunogenicity of individual components is not impaired.

Immunogenic compositions and vaccines may be administered parenterally, by injection subcutaneously or intramuscularly. The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation and vaccination course, and in such amount as will be therapeutically effective, immunogenic and protective. A preferred vaccination regimen for human babies is 2 or 3 doses in the first months of life, given one to two months apart, with a booster dose, preferably in the second year of life.

According to another aspect of the invention, the subject immunogenic composition is for use as a vaccine for human beings or animals, preferably mammals, in combination with a second composition. By second composition, it is meant a composition which is distinct from the first immunogenic composition according to the invention. This aspect of the invention relates to a specific combined use of the immunogenic composition according to the 1^st aspect detailed previously. According to the combined use, the 1^st immunogenic composition of the invention is to be used in combination with a further composition, or with further compositions, i.e. one or more than one further composition(s).

In an embodiment, the intended combined use is a combined, simultaneous, sequential or separate administration of both the 1^st and the 2^nd compositions. A simultaneous, sequential or separate administration is likely to occur in cases where the second composition is for example an adjuvant, aiming at reinforcing the immune response elicited by the first immunogenic composition according to the invention. Another case is the administration of an anesthetic composition or a composition aiming at reducing the side effects of the immune response against the first immunogenic composition.

Preferably, such a second or further composition is an immunogenic composition, comprising at least an antigenic compound capable of generating an immune response.

According to one embodiment of the combined use, both compositions are to be mixed together before administration. In this case, the 2 compositions are thus to be administered as a single composition, resulting from the blending of both.

According to a preferred embodiment of this aspect of the invention, the second composition to be used in combination with the immunogenic composition of the invention is a composition comprising thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum.

Such a situation is likely to occur when adding, as a second composition, for example a composition comprising whole cell Pertussis, inactivated with thiomersal, or a liquid multidose preparation.

In the context of the invention, it is particularly preferred that the immunogenicity of the IPV contained in the first immunogenic composition, according to the invention, is not impaired when combined with the second or further composition, especially when said second composition is a composition comprising thiomersal. It is considered that the immunogenicity of the IPV is not impaired if the mixture of both compositions is still a composition capable of generating an immune response, and preferably a protective immune response against poliovirus type 1 , 2 and/or 3, in a human recipient after inoculation. The immunogenic composition according to the invention is indeed advantageously to be used as vaccine after mixture with the second or further composition, comprising thiomersal or not. Preferably, the relative IPV titer loss of the combined composition, after mixture of the first composition with the second composition comprising thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, is less than 50%, preferably less than 30% or 25%, or 20% or 10%, in a period of time ranging from 0.5 or 1 hour to 8 or 6 hours, and at a temperature ranging from 5°C to 35°C, or 25°C.

Preferably, the relative IPV titer loss of the combined composition, after mixture of the first composition with the second composition comprising thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, is less than 30%, preferably less than 25%, preferably less than 20%, in a period of time ranging from 0.5 hour to 6 hours, and at a temperature ranging from 5°C to 35°C, or 25°C.

Preferably, the relative IPV titer loss of the combined composition, after mixture of the first composition with the second composition comprising thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, is less than 25%, preferably less than 20%, in a period of time ranging from 1 hour to 4 hours, and at a temperature ranging from 5°C to 35°C, or 25°C.

According to an embodiment of the combined use, the first immunogenic composition intended for combined therapeutic or prophylactic use with a second or further composition, is formulated as a liquid mixture of inactivated poliovirus types 1 , 2 and 3 in the doses of:

- about 10 to about 160 D-antigen units of poliovirus type 1 , and more preferably of about 29 D-antigen units; and / or

- about 2 to about 16 D-antigen units of poliovirus type 2, and more preferably of about 7 D-antigen units; and / or

- about 8 to about 27 D-antigen units of poliovirus type 3, and more preferably of about 26 D-antigen units,

for 0.5 imL corresponding to a single human dose, before any mixture.

As already disclosed, in a preferred embodiment, the 3 IPV serotypes are present in a composition of the invention.

According to a preferred embodiment, the first immunogenic composition according to the invention is to be combined with the second or further composition, potentially comprising thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, before administration to the host as a vaccine. The host is preferentially a human host.

When the immunogenic composition is to be used in combination with a composition comprising thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, the adverse effects of thiomersal on IPV, will be delayed and/or reduced by the presence of cyclodextrins in the immunogenic composition. It is however highly preferred that the combination takes place less than 24 hours before administration as a vaccine, and even preferably less than 8 hours. A preferred schedule is a mixture of both compositions less than 2 hours before administration, for example less than one hour before administration, or less than an half of hour, or even preferably less than 15 minutes.

In this respect, it must be borne in mind that the ambient temperature greatly influences the available time before administration. The more elevated is the ambient temperature, the more quickly is the vaccine to be administered after mixture of both compositions.

According to a preferred embodiment of the invention, the first immunogenic and/or second composition(s) is/are formulated for intranasal, intradermal, subcutaneous or intramuscular administration, either alone or in combination with each other. Whereas alternative routes of administration are indeed explored for IPV, inter alia subcutaneous administration, intramuscular injection remains the preferred route of administration, especially when combined with additional antigenic components, either present in the first composition, or in the second or further one(s).

According to a further aspect, the invention is also directed to different methods of vaccine preparation.

According to a specific embodiment of this aspect, the invention is directed to a method for preparing a vaccine comprising mixing a first immunogenic composition comprising at least one inactivated poliovirus (IPV) serotype and cyclodextrin(s) or derivative(s) thereof with a second or further composition.

The first immunogenic composition, comprising IPV and cyclodextrin(s) or derivative(s) thereof, is an immunogenic composition as defined with respect to the first aspect of the invention. All the detailed and preferred embodiments specifically disclosed with respect to this first aspect of the invention are applicable to the first immunogenic composition to be used in the frame of the methods of the invention.

The method is not limited to the mixing of a 1^st immunogenic composition with a 2^nd composition, preferably distinct, but also encompasses the mixing of further compositions. The method is also not limited with respect to additional steps, either previous or subsequent steps to the mixing step.

According to a preferred embodiment of a method of the invention, the first immunogenic composition, comprising IPV and cyclodextrin(s) or derivative(s) thereof, is formulated as a dried composition and the second composition, which is to be mixed with the first one, is formulated as an aqueous solution or suspension. According to this embodiment, the mixture of both compositions will thus be a reconstitution of the first dried composition in the second one. In this embodiment, the second composition may be for example a multidose vaccine or single dose vaccine. The volume of the second composition is preferably to be adjusted such that the reconstitution of the first dried composition gives rise to a composition comprising a single vaccine human dose, which is generally of 0.5 imL Alternatively, the volume of the second composition is preferably to be adjusted such that the reconstitution of the first dried composition gives rise to a vaccine human multidose, suitable for the administration of 10, and preferably of 5 doses.

According to another preferred embodiment of a method of the invention, the first immunogenic composition, comprising IPV and cyclodextrin(s) or derivative(s) thereof, is formulated as an aqueous composition and the second composition, which is to be mixed with the first one, is formulated as dry composition. According to this embodiment, the mixture of both compositions will thus be a reconstitution of the second dried composition in the first one. The volume of the first composition is preferably to be adjusted such that the reconstitution of the second dried composition gives rise to a vaccine human dose, which is generally of 0.5 imL Alternatively, the volume of the first composition is preferably to be adjusted such that the reconstitution of the second dried composition gives rise to a vaccine human multidose, suitable for the administration of 10, and preferably of 5 doses.

According to another embodiment, the first immunogenic composition, comprising IPV and cyclodextrin(s) or derivative(s) thereof, and the second composition are formulated as aqueous compositions. Preferably, the first immunogenic composition comprising IPV and cyclodextrin(s) or derivative(s) thereof is a liquid composition, and the second composition is also a liquid composition comprising other antigen(s), distinct from IPV, and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum.

The second composition as used in the methods of the invention is to be understood as the same 2^nd composition detailed in the context to the combined use of the invention.

Namely, the second composition is preferably a composition comprising thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum.

The second composition as used in the methods of the invention is preferably an immunogenic composition. The definition of immunogenic composition has been detailed with respect to the other aspects of the invention. Such an immunogenic composition may or may not comprise thiomersal. A suitable second composition according to this embodiment is a composition comprising inter alia inactivated whole cell Pertussis, which may have been inactivated in presence of thiomersal. The second composition may advantageously comprise additional antigens, which have also been inactivated by thiomersal or which are conveniently stored in presence of thiomersal, as for example Hepatitis B surface antigen.

The second composition according to an embodiment of the method is an immunogenic composition formulated as an aqueous solution and comprising inactivated whole cell Pertussis. According to a specific embodiment, the second immunogenic composition comprises whole cell Pertussis, and either Diphtheria toxoid or Tetanus toxoid, or both Diphtheria and Tetanus toxoids. In such a case, the second composition is thus an immunogenic composition known as DTP or preferably DTwcP.

The second composition as defined above, comprising wcP and possibly Diphtheria toxoid and/or Tetanus toxoid, may also comprise Hepatitis B surface antigen. The different antigens of the second composition are preferably those detailed with respect to the first aspect of the invention, including whole cell Pertussis, Diphtheria toxoid, Tetanus toxoid, Hepatitis B surface antigen, Hib capsular polysaccharide ....

In a preferred embodiment, the first and second compositions to be used in the methods of the invention comprise distinct antigens. It is however also envisaged that some antigens are simultaneously present in both compositions.

In the methods of the invention, the second composition, which is preferably an immunogenic composition, may also comprise an adjuvant. Preferred adjuvants according to the invention have already been detailed; particularly preferred adjuvants are aluminum-based adjuvants, especially aluminum phosphate or aluminum hydroxide.

According to a preferred embodiment, a second composition of the invention comprises an aluminum-based adjuvant, preferably an aluminum phosphate or an aluminum hydroxide- based adjuvant, and preferably under the form of a gel, most preferably an aluminum phosphate gel or an aluminum hydroxide gel.

According to a preferred embodiment of the methods of the invention, the mixing of the first composition with the second composition is carried out less than 8 hours, most preferably less than 6 hours, most preferably less than 4 hours, most preferably less than 2 hours, or even preferably less than 1 hour, preferably less than a half hour, or less than 15 minutes. Moreover, the mixing is preferably carried out at a temperature which is not exceeding 35°C, and preferably less than 30°C or 25°C, and for example is between 5°C and 35°C. As already emphasized, the ambient temperature greatly influences the available time before administration. The more elevated is the ambient temperature, the more quickly is the vaccine to be administered after mixture of both compositions. The methods of vaccine preparation according to the invention are thus preferably methods for preparing extemporaneous vaccines.

According to a further aspect, the present invention is also directed to different kits, referred to herewith as kits of the invention. Indeed, an immunogenic composition comprising IPV, cyclodextrin(s) or derivative(s) thereof, and thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, as encompassed by the present invention, is preferably prepared extemporaneously, preferably just before administration to the human hosts, or a few hours before. Thus, the invention provides kits including the various components ready for mixing.

A kit of the invention comprises at least two components, namely a first immunogenic composition, comprising IPV and cyclodextrin(s) or derivative(s) thereof, and devoid of thiomersal, and a 2^nd composition, wherein the second composition preferably comprises thiomersal, and optionally containing aluminum, preferably under the form of a gel of aluminum, the two components being ready for mixing. With regard to the first immunogenic composition, comprising IPV and cyclodextrin(s) or derivative(s) thereof, it corresponds to a composition according to the invention, as detailed with respect to the first aspect of the invention, but devoid of thiomersal. The second composition is as detailed with respect to the combined use of the invention as well as the method of preparation of a vaccine. All the embodiments described with respect to these other aspects of the invention are applicable to the kits of the invention. Especially, the kits are not limited to kits of two elements, and may comprise more than two elements, i.e. a first immunogenic composition as detailed above, and one, or more than one further compositions.

The kit allows the 1^st composition, especially the IPV of this composition, and thiomersal to be kept separately until the time of use.

The components are physically separate from each other within the kit, and this separation can be achieved in various ways. For instance, the 1^st composition comprising IPV and the thiomersal-comprising second composition may be in two separate containers, such as vials. The contents of the two vials can then be mixed, e.g., by removing the content of one vial and adding it to the other vial, or by separately removing the contents of both vials and mixing them in a third container. In a preferred arrangement, one of the kit components is in a syringe and the other is in a container such as a vial. The syringe can be used (e.g., with a needle) to insert its contents into the second container for mixing, and the mixture can then be withdrawn into the syringe. The mixed contents of the syringe can then be administered to a patient, typically through a new sterile needle. Packing one component in a syringe eliminates the need for using a separate syringe for patient administration. In another preferred arrangement, the two kit components are held together but separately in the same syringe, e.g., a dual-chamber syringe. When the syringe is actuated (e.g., during administration to a patient) the contents of the two chambers are mixed. This arrangement avoids the need for a separate mixing step at the time of use. The kit components will generally be in aqueous form.

In some alternative embodiments, a component, and preferably the first component comprising IPV, is in dry form (e.g., in a lyophilized form), with the other component being in aqueous form. Alternatively, the first component comprising IPV, is in aqueous form, with the other component being in dry form (e.g., in a lyophilized form). The two components can be mixed in order to reactivate the dry component and give an aqueous composition for administration to a patient. A lyophilized component can be located within a vial or in a syringe. Dried components may include stabilizers such as mannitol, sucrose, or dodecyl maltoside, as well as mixtures thereof e.g. lactose/sucrose mixtures, sucrose/mannitol mixtures, etc. Specific embodiments in connection with a dried composition of IPV and cyclodextrins have been detailed above.

According to an embodiment of this aspect of the invention, the second component of the kit comprising thiomersal, also comprises whole cell Pertussis antigens. According to a specific embodiment, the second component of the kit comprises whole cell Pertussis antigens, and either Diphtheria toxoid or Tetanus toxoid, or both Diphtheria and Tetanus toxoids. In such a case, the second component is thus an immunogenic composition known as DTP or preferably DTwcP. The second component as defined above, comprising wcP and possibly Diphtheria toxoid and/or Tetanus toxoid, may also comprise Hepatitis B surface antigen.

The different antigens present in the second or further component are preferably as detailed with respect to the first aspect of the invention.

Specifically, a second component of the kit of the invention may advantageously comprise one or several of the antigens disclosed with respect to the first aspect of the invention, including whole cell Pertussis, Diphtheria toxoid, Tetanus toxoid, Hepatitis B surface antigen, Hib capsular polysaccharide, etc... It is also envisaged that the further antigens are comprised in distinct further components of the kit.

In a preferred embodiment, the first and second or further components of the kit comprise distinct antigens; it is however also envisaged that some antigens are simultaneously present in both components.

According to a further embodiment of the kit, the second component also comprises an adjuvant. Preferred adjuvants according to the invention have already been detailed; particularly preferred adjuvants are aluminum-based adjuvant, especially aluminum phosphate or aluminum hydroxide.

According to a preferred embodiment, a second composition of a kit according to the invention comprises an aluminum-based adjuvant, preferably an aluminum phosphate or an aluminum hydroxide-based adjuvant, and preferably under the form of a gel.

According to a different embodiment of the invention, the kit comprises three or more distinct components ready for mixing, a first component as defined above, comprising IPV and cyclodextrins and devoid of thiomersal, and two or more additional components, at least one of them comprising thiomersal. The two or more additional components may comprise any further antigens intended for vaccination. Some of them have already been detailed in the context of previous aspects of the invention.

According to another embodiment, the 1^st component of the kit comprises IPV, however without cyclodextrins and without thiomersal, a second component comprises cyclodextrin(s) or derivative(s) thereof, and the 3^rd component comprises thiomersal, for example with whole cell Pertussis. In such a case, the kit preferably comprises instructions such that the first two components are to be mixed before mixing with the 3^rd one.

A kit according to the invention is preferably intended for vaccination.

In such a case, the kit advantageously comprises also instructions for use, especially instructions regarding extemporaneous mixing of the components, i.e. less than 8 hours, preferably less than 6, or 4, or 2, or 1 or half hour(s), preferably less than 15 minutes before administration, at a temperature not exceeding 35°C, preferably in the range 5°C-25°C.

In a further aspect of the invention, there is also provided a method of immunizing a host against poliomyelitis, preferably a human or mammal host, comprising administering to the host an immunogenic composition or vaccine according to the first aspect of the invention, or a vaccine prepared by the methods for preparing a vaccine of the invention, or a vaccine prepared from the kits of the invention. The method is preferably a method for raising an immune response in a host, preferably a human or mammal, preferably a protective immune response. The method may also raise a booster response. The method is preferably a prophylactic method, used to prevent infection by poliovirus, but may also be used to treat an already infected mammal.

For any vaccine comprising further antigens, in addition to IPV, also intended for vaccination, the method according to the invention is also for simultaneously immunizing the host against the diseases corresponding to these antigens. According to an embodiment, a preferred method is thus a method for immunizing a host, preferably a human host, against poliomyelitis, Diphtheria, tetanus and whooping cough or pertussis.

According to another aspect, the invention is also directed to different uses of cyclodextrins. The invention indeed covers the use of a cyclodextrin in combination with at least one inactivated poliovirus (IPV) serotype for preserving the IPV antigenicity and/or immunogenicity in presence of thiomersal, inter alia for reducing and/or delaying the loss of IPV antigenicity or immunogenicity.

The meaning of IPV, cyclodextrins, thiomersal have already been defined above with respect to the other aspects of the invention; said definitions are applicable to this aspect of the invention.

As for the other aspects of the invention, preferred cyclodextrins are for example chosen amongst γ-cyclodextrin, β-cyclodextrin, C1-C3 hydroxyalkyl derivatives of γ-cyclodextrin or β- cyclodextrin, oside derivatives of γ-cyclodextrin or β-cyclodextrin, or a mixture thereof.

By preserving the IPV immunogenicity, it is to be understood limiting the IPV titer loss occurring when IPV is combined with thiomersal, and optionally with aluminum, preferably under the form of a gel of aluminum, in the absence of cyclodextrin(s) or derivative(s) thereof, either by reducing or cancelling said titer loss, or by delaying said titer loss. Preferably, the use according to the invention is to protect IPV titer such that the IPV titer loss due to addition of thiomersal, optionally with aluminum, preferably under the form of a gel of aluminum, is less than 50%, preferably less than 30%, or less than 20%, in a period of time ranging from 0.5 or 1 hour to 8 or 6 hours after addition, and at a temperature ranging from 5°C to 35°C, or 25°C. Alternatively, the use of cyclodextrin(s) or derivative(s) thereof according to the invention may be as a protectant against degradation of antigenic structures of IPV, when combined with thiomersal, and optionally with aluminum, preferably under the form of a gel of aluminum. The use of cyclodextrin(s) or derivative(s) thereof according to the invention may be a preventive use, in case of further addition of thiomersal, and optionally with aluminum, preferably under the form of a gel of aluminum, to a composition comprising IPV.

The relative proportion of cyclodextrin(s) or derivative(s) thereof, to be added to composition comprising IPV in order to protect said IPV against degradation by thiomersal, is as defined in the previous aspect of the invention. The maximal amount of thiomersal to be added relative to the IPV content and cyclodextrin(s) or derivative(s) thereof concentration is also as disclosed with respect to the other aspects of the invention.

Uses according to the invention encompass the use of cyclodextrin(s) or derivative(s) thereof for protecting each IPV serotype in bulk concentrate solution.

Any one of the previously disclosed embodiments can be combined with the different aspects of the invention.

The examples thereafter are presented only for illustrative purposes of the invention.

Examples:

Example 1 : Material and methods. The preparation of the IPV solution is preferably made as described in US 4,525,349 or in Industrial Biotechnology - Bioprocess, Bioseparation, and Cell Technology, Wiley & Sons, 2010: 4789-4808. This process entails separately, for each of the 3 types of poliomyelitic virus, the stages consisting in multiplying by successive passages the VERO strain by culturing on microcarriers in suspension, into a suitable nutritive medium, drawing off the liquid medium at the end of the final passage and replacing it by a new liquid medium containing no serum, inoculating the biogenerator of the last cells passage, withdrawing the liquid suspension after virus culture, filtering the suspension drawn off, clarifying, purifying and concentrating the filtered suspension, diluting the concentrated suspension obtained with a serum-free medium, inactivating the suspension thus diluted and purified, preferably after a filtration, and then mixing the three suspensions of the respective types 1 , 2 and 3.

In the following examples, IPV of the compositions or vaccine is prepared from a Polio trivalent 5xC solution, comprising the three IPV serotypes. "5xC" means that the Polio trivalent 5xC solution has to be diluted 5 times to provide "1 HD", i.e. 1 human dose, for injection of 0.5 imL 1 human dose corresponds to 40 D-antigen units of IPV type 1 , 8 D-antigen units of IPV type 2 and 32 D-antigen units of IPV type 3, for a volume of 0.5 imL This is the human dose recommended by WHO for IPV.

The target formulation of polio trivalent 5xC used in the following examples is the 40 UD for type 1 , 8 UD for type 2 and 32 UD of type 3 for a theoretical dose volume of 0.1 imL

When diluted solutions are used in the examples, the polio trivalent 5xC solution is diluted with Medium M199 (without Phenol red - Invitrogen). Polio D-antigen titration:

The determination of the polio D-antigen titer is carried out by ELISA.

If the solution to be assayed comprises adsorbed IPV, for example in case of formulated vaccines, a preliminary step of desorption of the IPV from the aluminum gel is generally recommended, although such a step has not been carried out in the following examples, for the reasons detailed below. Such a desorption step is preferably carried out according to the following method:

- centrifugation of the solution to be assayed, and removal of the surpernatant,

- addition of a desorption phosphate buffer to the pellet, mixing and incubation at least 4 hours at 37°C,

The harvest of the desorption step is diluted with phosphate buffered saline.

After desorption (if such a step is needed), the D-antigen content of the composition is quantified by ELISA.

However, in the following examples, D-antigen titration has been carried out on supernatant after centrifugation but without desorption steps. The titration after desorption has not been determined. Indeed, as detailed above, the desorption from gel requires a long heating step at 37°C after addition of phosphate, which would amplify the IPV titer losses induced by the presence of thiomersal in the titrated solutions, and thus would bias the results on samples with thiomersal.

D-antigen titration by Elisa:

In this respect, microtiter plates are coated with specific validated polyclonal anti-polio virus (type 1 , 2 or 3) IgG, generally rabbit IgG, diluted with carbonate/ bicarbonate buffer (pH 9.6), and incubated overnight at 4° C. After washing, the saturating solution (phosphate buffered saline without Ca and Mg + 1 % Bovine serum albumin) is added. Blanks (PBS) and serial dilutions of vaccine samples and in-house unadsorbed standard are added in duplicate. The in house trivalent standard preparation contains calibrated type 1 , 2 and 3 antigens.

The calibrator is the European Pharmacopoeia Biological reference (EPBRP).

For all following steps, the microtiter plates are incubated during 1 h30 at 37°C and washed. Rabbit, validated mono or polyclonal, anti-polio virus (type 1 , 2 or 3) IgG conjugated to peroxydase, diluted with phosphate buffer (w/o Ca and Mg + Tween 20) containing BSA, is added. The substrate solution, containing the tetramethylbenzidine dissolved in dimethyl sulfoxyde (DMSO) and diluted in acetate buffer containing 0.003% H2O2, is added, followed by incubation in the dark. The blocking solution, containing H₂S0₄, is then added. Within one hour, the optical density (O.D.) of each well is read using a photometer set at 450 nm with a reference at 620 nm.

The D-antigen concentration in test samples is calculated from the standard curve obtained by plotting the O.D. values against the standard antigen concentrations. Variability of the method: in the following examples, different batches of in-house polio trivalent 5xC solution and of in-house reference antigens have been used, such that the measured D- antigen units may deviate from the 40/8/32 content of IPV1/2/3 corresponding to 1 human dose. In order to eliminate the variability due to these different batches, in all the following examples, a control solution has also been assayed for comparison, corresponding to the same concentrate bulk IPV solution and titrated with the same antigens as the tested compositions.

Variability of the method applied to formulated vaccines: The determination of IPV titer applied without desorption to formulated vaccines combined with IPV, is measured with an uncertainty on the result which has been estimated by the inventors to lie between 15% and 20%.

Shan5:

Shan5 is a pentavalent vaccine as detailed in table 1.

Table 1 : Shan5 composition.

Method for obtaining freeze-dried IPV composition:

A suitable process for obtaining freeze-dried IPV composition is inter alia disclosed in WO 2012/028315.

Example 2: IPV degradation by different amounts of thiomersal.

Tests were carried out in order to quantify the IPV degradation in presence of different amounts of thiomersal. The solution under test is a formulation DTwCP, comprising the diphtheria and tetanus toxoids, and the whole cell Pertussis antigen, completed by IPV, at standard doses, i.e. 40 D-antigen units for type 1 , 8 D-antigen units for type 2 and 32 D-antigen units for type 3, per vaccine dose of 0.5 imL Different concentrations of thiomersal were tested, ranging from 0.5 to 40.0 μg per vaccine dose. After addition of thiomersal, the mixtures remained 13 days at 5°C before assaying D- antigen titer. The results are reported in table 2.

Table 2: D-antigen titer at different concentrations of thiomersal. Example 3: Quantification of IPV degradation in different formulations.

Tests were carried out in order to quantify the IPV degradation, in presence of thiomersal, in different formulations, with or without adsorption of the IPV on aluminum, as a function of time.

3.1. Compatibility study of the ALW//I vaccine in presence of thiomersal.

The goal of this study is to compare the stability at 25°C of the ALW//I vaccine, with and without thiomersal, in order to evaluate the effect of thiomersal on the polio valence of the ALW//I vaccine (extemporaneous blending of ALW//I with IPV). The polio valence is assayed by measuring D-antigen titer with Elisa test, as detailed in example 1 , without preliminary desorption.

To this end, the polio D-antigen content of the ALW//I vaccine is assayed after addition of thiomersal (43.75 μg of thiomersal per dose, after dilution) or without thiomersal, at T₀ and after 6 and 12 hours storage hours at 25°C. The exact compositions of the two tested formulations are given in table 3 Component Quantity / dose

With thiomersal Without thiomersal

Aluminum hydroxide gel 0.3 mg Al 0.3 mg Al

Thiomersal 43.75 pg 0

Polio 1xC 1xC

Hepatitis B 10 pg 10 pg

Diphtheria 25 Lf 25 Lf

Tetanus 10 Lf 10 Lf

Pertussis whole cell 15 OU (containing thiomersal) 19 OU (without thiomersal)

PRP Conjugate 1.008 pg phosphorus 1.008 pg phosphorus

Phosphate buffer 60 mM 60 mM

tris-sucrose buffer 2.5 mM 2.5 mM

Tween 0 0.05%

Table 3: ALW//I vaccine composition, with and without thiomersal.

The pH of the formulation is in the range 6.5-7. Thiomersal is added during the formulation to adjust the final concentration of thiomersal to the targeted value of 43.75 pg/dose.

The results are detailed in table 4:

Table 4 : stability at 25 °C, at T₀ and after 6 and 12 hours of ALW//I, with and without thiomersal.

Conclusion For the formulation ALW//I comprising IPV, when thiomersal is added, the statistical analysis shows that: - There is a significant decrease of the type 1 and type 2 titers after 6 hours at 25°C: a decrease of respectively 9.1 D-antigen unit/dose, corresponding to 29% and 1.4 D- antigen unit/dose corresponding to 21 %.

- The titer loss for the type 3 titer at 25°C, in the first 6 hours, is less important than for types 1 and 2.

D-antigen titers of serotypes 1 and 2, and of serotype 3 to a lesser extent, do not remain stable at 25°C in presence of thiomersal. Thiomersal has thus a deleterious effect on the stability of the polio virus serotypes.

This study also shows that the 3 serotypes remain mainly unadsorbed in presence of Aluminum hydroxide gel, under the tested conditions (48 hours at 25°C), as the D-antigen titers have been determined without desorption step.

3.2. Compatibility study of different IPV formulations in presence of thiomersal.

The goal of this study is to determine and quantify the incompatibility between an existing formulated pentavalent vaccine, namely Shan5, comprising thiomersal, and added polio valence.

To this end, the Shan5 vaccine is combined with IPV, under different forms.

Shan 5 is a pentavalent vaccine containing 5 bulk concentrates: 3 pre-adsorbed (Diphtheria, Tetanus, Hepatitis B) and 2 additional (whole cell Pertusis and Haemophilus influenzae B conjugate (PRP-TT)) in saline NaCI 0.9 %. pH: 6.4 - 6.6 at 5 °C (adjustment if required) The details of the Shan5 vaccine are given in table 1.

Moreover, insofar as Shan5 contains Aluminum phosphate, on which IPV tends to adsorb, a comparative formulation is made with aluminum phosphate gel mixed with IPV, in order to evaluate the fraction of IPV titer loss which is due to adsorption on Aluminum phosphate. It is indeed reminded that the IPV titration has been carried out without desorption step, such that any adsorbed IPV will not be measured and will thus participate to the IPV titer loss.

Details of the formulations:

A) formulations comprising Shan5 and IPV:

Formula 1 : "Shan5 / IPV (2,5 / 1 )" comprising 0.5 ml Shan5 and 0.2 ml IPV 2.5xC (predicted liquid IPV from Polio trivalent 5xC solution, see example 1 ),

Formula 2 : "Shan5 / IPV (1 / 1 )" comprising 0.5 ml Shan5 and 0.5 ml IPV 1xC (pre-diluted liquid IPV from Polio trivalent 5xC solution, see example 1 )

Formula 4: freeze dried IPV (corresponding to 1 Human dose) rehydrated with 0.5 imL Shan5.

B) control formulations:

Control 1 : IPV 1xC liquid (26. 1/6.7/22.7 for IPV 1 , 2, 3 respectively) Control 2 : freeze dried IPV (corresponding to 1 Human dose) rehydrated with water (32.1/6.9/22 for IPV 1 , 2, 3 respectively).

C) Formulations comprising IPV and aluminum phosphate gel:

In order to evaluate the adsorption of IPV on aluminum phosphate gel, the following formulation has also been tested :

Formula 3: 1 ml IPV 1 xC + 1 ml AIP0₄ diluted with NaCI 9/1000 buffer, such that its concentration is reduced from 5.1 mg Al/ml to 1 .2 mg/ml. pH Aluminum phosphate gel is 6,3.

The concentrations in gel and in thiomersal for formulas 1 to 4 are given below in table 5:

* : HD = human dose

Table 5 : Details of the formulas 1 -4 composition.

The concentrations in gel and in thiomersal for formulas 1 to 4 correspond to the following quantities for one human dose (table 6) :

Table 6 : compositions of formulas 1 to 4, for a volume corresponding to one human dose of IPV The thiomersal present in the Shan5 vaccine is exclusively brought by the presence of Pertussis Whole Cell ; there is no further addition of thiomersal in the formulation.

D-antigen titrations have been carried out at T₀ and after 3 hours and 6 hours at room temperature, as detailed in example 1 , and without desorption for the reasons detailed above.

Results :

Results a Tn:

The results of the D-antigen titration in the supernatant, for one human dose of IPV, are presented in table 7. The titer is expressed as D-antigen unit per dose, for each serotype.

The diminutions of titer at T₀ of formulas 1 , 2 and 3 with respect to the control 1 , and the diminution of titer at T₀ of formula 4 with respect to freeze-dried IPV rehydrated in water are detailed in table 8, for each series, expressed in D-antigen unit per dose and percentage.

Table 7 : Composition and D-antigen titers of different formulations and controls, immediately after mixture (T₀). Formula 1 Formula 2 Formula 4 Formule 3

TD** TD TD TD TD TD TD TD

(DU dose) (%) (DU/dose) (%) (DU/dose) (%) (DU/dose) (%)

Type

12.4 47% 9.8 37% 20.75 65% 6.95 26% 1

titer Type

2.65 40% 2.05 31 % 4.35 63% 5.3 79% 2

Type

4.9 22% 2.3 10% 8.2 37% 0.45 2% 3

DU stands for D-antigen unit ^** TD stands for titer diminution

Table 8: diminution of D-antigen titer of the different formulas, with respect to appropriate controls

Key observations:

For the formulations Shan5 + liquid IPV, titer reductions are observed at T₀ for all 3 serotypes. With respect to the controls comprising neither thiomersal nor gel, the titer difference varies between 10 to 13 D-antigen unit /dose, i.e. 37-47% for type 1 , 2 to 3 D-antigen unit /dose, i.e. 31-40% for type 2 and 2 to 5 D-antigen unit /dose, i.e. 10-22% for type 3.

There is no significant difference observed between Formulas 1 & 2, but Freeze-dried IPV (formula 4) gives rise to significantly higher losses.

The larger diminution observed for Shan5 + freeze-dried IPV (formula 4) may be linked to the rehydratation step. A dilution factor may also explain the results (the formula 2 being the most diluted formulation and formula 4 being the least diluted).

The formula 3, with AIP04 gel and IPV reveals that serotypes 1 and 2 are adsorbed on the gel. The extent of adsorption is particularly important for type 2. The serotype 3 is almost not adsorbed on the gel, under the tested conditions.

From these results, it is thus deduced that titer reductions are linked to 2 separate causes

Adsorptions on gel which intervene on short times;

Large D-antigen degradation due to thiomersal which intervenes quickly and lasts over long period of times. Results of Titer losses over time:

The evolution of the D-antigen titer over time for the formulations detailed above has been studied, with respect to one human dose.

The results of the study obtained for formulas 1 and 2 have been compiled with those obtained for ALWI/I with and without thiomersal (see example 3.1 and table 4), after 6 hours at room temperature; these results are presented in table 9:

Table 9: D-antigen titer losses after 6 hours, for different formulations.

The titer reductions in D-antigen unit /dose or percentage, are expressed with respect to the D- antigen unit titer measured at T₀ (see table 7). The results show an important titer reduction for types 1 and 2 in presence of thiomersal, in case of adsorption (formulas 1 and 2) or without adsorption (ALW//I environment). The comparison shows that, for the three serotypes, the reduction in D-antigen titer over 6 hours is similar for the formula ALW//I with thiomersal and for the 2 formulas Shan5/IPV, i.e. 8 to 9 D- antigen unit /dose for type 1 ; 2 to 3 D-antigen unit /dose for type 2, and 1 to 5 D-antigen unit /dose for type 3.

The losses over 48 hours were also studied for the above mentioned formulations. Whatever types 1 , 2 or 3, both conditions, i.e. contact with ALW//I and thiomersal or with Shan 5, show essentially similar linear decrease trends over time.

The factors underlying the reductions observed in formulations combining Shan5 and liquid IPV are the adsorption of the serotypes on the Aluminum phosphate gel and incompatibility with thiomersal, and can be summarized as follows: Factors underlying the titer Factors underlying the titer reduction reduction in the supernatant at in the supernatant after 6 hours at

To 22°C

Incompatibility Yes for serotypes 1 , 2 and 3

yes

with thiomersal

Yes, dominating factor for No, because the loss after 6 hours is serotypes 1 and 2, negligible for comparable with the loss with the

Adsorption on the serotype 3 assay ALW//I where there is not gel (see table 8) adsorption of the polio on the aluminum phosphate gel

(see table 9)

CONCLUSIONS:

The results of this study demonstrate that

- The presence of thiomersal has a deleterious effect on IPV titer, which can be observed immediately after addition of thiomersal to a composition comprising IPV.

- Even in case of adsorption of IPV on aluminum phosphate, there is still a fraction of IPV which is degraded in presence of thiomersal.

In conclusion, the results show the impossibility of making a mixture, even extemporaneous, of IPV and a composition comprising thiomersal, for example the formulated vaccine Shan5, due to the incompatibility between IPV and thiomersal.

Example 4: Use of cyclodextrins for protecting IPV.

This example demonstrates that a significant protective effect against deleterious effect of thiomersal can be obtained when cyclodextrins are added to IPV.

In this study, different cyclodextrins have been investigated, namely α-, β-, γ- & 2-hydroxy propyl-γ- cyclodextrins, in order to determine the best candidates for protecting polio valence. In a first phase, the absence of interference of cyclodextrins with D-antigen titration was checked.

The conditions for the addition of cyclodextrins have then been optimized with respect to the following parameters:

•Impact of addition sequence to get the final mix

•Impact of Cyclodextrin Concentration

Finally, the degradation of IPV antigens in refrigerated and accelerated stability conditions (5 or 37 40°C) has been evaluated, in presence of cyclodextrins. Material and methods:

Reference « x1 » with respect to the concentration of Cyclodextrin refers to equimolarity versus Thiomersal (target thiomersal concentration is 50 μg/dose or 0,25mM). Cyclodextrins (CD) α & γ have been added to

- either the Shan5 vaccine (see example 1 and table 1 for composition) in the following volumes: 0.5 ml Shan5 with 50 μΙ of CD;

- IPV3,3xc (control) in the following volumes :150 μΙ IPV 3.3xC with 50 μΙ de CD.

The contact time was arbitrarily set to 32 hours, before mixing Shan5 and IPV. After mixing, the D-antigen content has been measured in the surpernatants by Elisa (as detailed in example 1 ), either immediately or after 16 hours at 5°C.

Results:

Phase 1 : Evaluation of the impact of the presence of CD on titration method.

In this test, the D-antigen content is measured by Elisa, before and after addition of γ CD (50μΙ_), for each serotype. The results are reported in table 10.

Table 10: influence of CD on the D-antigen titration method.

No significant impact on IPV titration method has been observed in case of addition of γ- cyclodextrin. The results with addition of a-CD are comparable. It is thus to be concluded that the addition of CD does not interfere with IPV titer and titration method. Phase 2: comparison of aCD vs vCD and influence of addition order

The D-antigen content has been measured immediately after mixture and after 16 hours at 5°C. The CD were either mixed with Shan5, before contacting with IPV (assays 1 and 2), or mixed with IPV, before contacting with Shan 5 (assays 3 and 4). The results with aCD (assays 1 and 3) and YCD (assays 2 and 4) were compared.

The results are reported in the following table (table 1 1 ). D Ag titer per ml Losses in 16 hours at

Assay Mixtures CD TO T 16h 5 °C ( % )

Type 1 12 8.2 32

Shan5 + CD

1 Alpha Type 2 3.1 1.8 42

then IPV

Type 3 25.5 25.3 1

Type 1 13.3 8.2 38

Shan5 + CD

2 Gamma Type 2 3.3 1.9 42

then IPV

Type 3 26.1 23.2 1 1

Type 1 14.2 1 1.7 18

IPV + CD

3 Alpha Type 2 3.6 2.7 25

Then Shan5

Type 3 24.2 26.7 0

Type 1 16.5 17.4 Q

IPV + CD

4 Gamma Type 2 4.5 4.9 Q

Then Shan5

Type 3 26.3 28.3 Q

Table 1 1 : Losses in D-antigen titer of IPV preparation in presence of Shan5, in presence of different CD.

Preliminary contact between cyclodextrins and IPV before mixing them with Shan 5 gives better results than those obtained in contacting first Shan5 with CD.

The Y cyclodextrins appear more efficient than a cyclodextrins in protecting IPV from titer losses in presence of thiomersal (brought by Shan5). Phase 3: comparison of PCD vs yCD and cyclodextrins concentration effect :

The D-antigen content has been measured after mixture and after 40 hours at 5°C. The CD were mixed with IPV, before contacting with Shan5. The results with $CD and YCD were compared, at different concentrations.

The results are reported in table 12.

Addition of YCD is more efficient than addition of &CD for protecting IPV. Reduced concentration of cyclodextrin, i.e. less than equimolarity with thiomersal, is preferable.

The difference observed in the losses after 16 hours (table 1 1 ) and after 40 hours (table 12) implies that extemporaneous use after mixing is probably to be preferred in order to retain IPV immunogenicity. D Ag titer (D-antigen unit/ml) Losses in

Cyclo - 40 h at

Mixtures TO T 40h

dextrin 5 °C ( % )

Type 1 10.7 3.8 64

Shan 5 +

none Type 2 3.1 0.6 81

IPV

Type 3 18.1 15.8 13

IPV + CD Type 1 13.9 8.4 40

Gamma

Then Type 2 3.9 2.5 36

1xC

Shan5 Type 3 27.1 24.4 10

IPV + CD Type 1 13.4 9.8 27

Gamma

Then Type 2 3,6 2.9 19

½ xC

Shan5 Type 3 25.3 24.2 4

IPV + CD Type 1 10.8 5.7 47

Beta

Then Type 2 2.9 1.1 62

1xC

Shan5 Type 3 25.6 23.5 8

IPV + CD Type 1 10.8 6.9 36

Beta

Then Type 2 2.9 1.9 34

½ xC

Shan5 Type 3 25.6 24 6

Table 12: impact of different CD, at two different concentrations

Phase 4: study of longer contact and temperature influence

Accelerated aging of premixed IPV+y-cyclodextrin was tested: IPV and yCD were contacted during 24 h at 37°C or 30 h at 5°C or 25°C, prior to contact with Shan 5.

The inventors have observed that there is no benefit of longer contact between IPV and cyclodextrin prior to mixing with Shan 5.

They have also observed that there is no significant effect of temperature during initial contact of IPV and cyclodextrin prior to mixing with Shan 5.

In view of the results of Example 4, one may conclude that cyclodextrins, and in particular β- cyclodextrin, γ-cyclodextrin and 2-hydroxypropyl-y-cyclodextrin protect IPV from the detrimental effects of the thiomersal. References:

Aunins "Viral vaccine production in cell culture" in Industrial Biotechnology - Bioprocess, Bioseparation, and Cell Technology, Wiley & Sons, 2010: 4789-4808

Brownlee, Statistical Theory and Methodology in Science and Engineering, Wiley & Sons (New York), 1965:352-358

Cevher et al., "Preparation and characterisation of natamycin: γ-cyclodextrin inclusion complex and its evaluation in vaginal mucoadhesive formulations", J Pharm Sci, 2008, 97(10): 4319- 4335

Quiambao et al, "A randomized, dose-ranging assessment of the immunogenicity and safety of a booster dose of a combined diphtheria-tetanus-whole cell pertussis-hepatitis B-inactivated poliovirus-Hemophilus influenzae type b (DTPw-HBV-IPV/Hib) vaccine vs. co-administration of DTPw-HBV/Hib and IPV vaccines in 12 to 24 months old Filipino toddlers." Hum Vaccin Immunother. 2012 Mar;8(3):347-54.

Sawyer et al. "Deleterious effect of thimerosal on the potency of inactivated poliovirus vaccine", Vaccine, 1994, 12(9): 851-56

Sawyer et al. "Quantitation of D Antigen Content in inactivated Poliovirus Vaccine Derived from

Wild-type or Sabin Strains", Biologicals, 1993, 21 : 169-177

GB222021 1

EP 0 689 454

EP 2 097 102

US 4,525,349

US 5,057,540

US 6,1 13,918

US 2003/0153532

WO 94/00153

WO 95/17209

WO 96/02555

WO 96/33739

WO 98/51339

WO 2012/028315

Claims

Claims:

1. An immunogenic composition comprising at least one inactivated poliovirus (IPV) serotype and at least one cyclodextrin or derivative thereof.

2. The immunogenic composition according to claim 1 , wherein the cyclodextrin is chosen amongst γ-cyclodextrin, β-cyclodextrin, C1-C3 hydroxyalkyl derivatives of γ-cyclodextrin or β-cyclodextrin, glycoside derivatives of γ-cyclodextrin or β-cyclodextrin, and a mixture thereof, and preferably amongst γ-cyclodextrin, 2- hydroxypropyl γ-cyclodextrin and a mixture thereof.

3. The composition according to any one of claim 1 and 2, wherein said composition comprises one or more additional antigens, preferably chosen from the group comprising Diphtheria toxoid, Tetanus toxoid, Pertussis antigen, Neisseria meningitidis antigen, Hepatitis B surface antigen and Haemophilus influenzae b (Hib) antigen.

4. The immunogenic composition according to any one of claims 1 to 3, being formulated as an aqueous solution or suspension.

5. The immunogenic composition according to any one of claims 1 to 4, further comprising thiomersal.

6. The immunogenic composition according to any one of claims 1 to 4, being intended for blending with an aqueous thiomersal-containing solution or suspension or with a dried thiomersal-containing composition, wherein the obtained composition after blending is capable of generating an immune response against a poliovirus.

7. The immunogenic composition according to claim 5 or the obtained composition after blending according to claim 6, wherein said composition has a level of antigenicity of IPV relative to the level of antigenicity of a composition according to any one of claims

1-4 and devoid of thiomersal, which differs by less than 50% after a period of time ranging from 0.5 to 8 hours after contact with the thiomersal, and at a temperature ranging from 5°C to 35°C, preferably from 5°C to 25°C.

8. The immunogenic composition according to any one of claims 1 to 3, formulated as a dried formulation, preferably as a powder, microparticles, or micropellets formulation.

9. The immunogenic composition according to claim 8, being intended for reconstitution in an aqueous thiomersal-containing solution or suspension, wherein said composition after reconstitution is capable of generating an immune response against polio virus.

5 10. An immunogenic composition according to any one of claims 1 to 9, for use as a vaccine for human beings or animals.

1 1. An immunogenic composition according to any one of claims 1 to 4, 6 and 8 to 10, for use as a vaccine for human beings or animals, in combination with a second distinct

10 immunogenic composition comprising thiomersal.

12. Use of one cyclodextrin or a derivative thereof in combination with at least one inactivated poliovirus (IPV) serotype for preserving the IPV immunogenicity in presence of thiomersal.

15

13. A method for preparing a vaccine comprising mixing a first immunogenic composition comprising at least one inactivated poliovirus (IPV) serotype and one cyclodextrin or a derivative thereof, with a 2^nd composition.

20 14. The method according to claim 13, wherein the first immunogenic composition is formulated as a dried composition and the second composition is formulated as an aqueous solution or suspension for reconstitution, or wherein the first immunogenic composition is formulated as an aqueous solution or suspension and the second composition is formulated as an aqueous solution or suspension or a dried

25 composition.

15. A kit comprising a first immunogenic composition according to any one of claims 1 to 4, 6 and 8 to 10, and a 2^nd immunogenic composition comprising thiomersal.

30 16. The kit according to claim 15, for use as a vaccine.