WO2020043874A1

WO2020043874A1 - Conjugated haemophilus influenzae vaccine using bordetella outer membrane vesicle

Info

Publication number: WO2020043874A1
Application number: PCT/EP2019/073195
Authority: WO
Inventors: Roberto ADAMO; Christiane Marie-Paule Simone Jeanne FERON
Original assignee: Glaxosmithkline Biologicals Sa
Priority date: 2018-08-31
Filing date: 2019-08-30
Publication date: 2020-03-05

Abstract

The present invention relates to conjugates of outer membrane vesicles (OMV), particularly conjugates comprising capsular polysaccharides from H. influenzae and their use in vaccines.

Description

CONJUGATED HAEMOPHILUS INFLUENZAE VACCINE USING

BORDETELLA OUTER MEMBRANE VESICLE

TECHNICAL FIELD

This invention is in the field of antigen conjugates and their use in combination vaccines.

BACKGROUND ART

The combination of diphtheria (D), tetanus (T), and pertussis (P) vaccines into a single product has been central to the protection of the paediatric population over the past 50 years. The addition of inactivated polio, Haemophilus influenzae, and Hepatitis B vaccines into the combination has facilitated the introduction of these combination vaccines into recommended immunization schedules by reducing the number of injections required and has therefore increased immunization compliance (1 ). However, the development of these combinations has encountered numerous challenges, the most common of which is the immune interference in DTP combination vaccines comprising acellular Pertussis antigens (aP), with a reported reduction in antibody titres to the Haemophilus influenzae type b component of the vaccine polyribosylribitol phosphate antigen (PRP). The interference has not been reported to the same extent for DTP combination vaccines comprising whole-cell pertussis (wP), possibly due to the adjuvant effect of the wP component. However, the DTwP-based combination vaccines can generate a number of adverse reactions, mostly caused by lipooligosaccharide (LOS) (2).

On the other hand, a number of countries have experienced substantial increases in the number of reported cases of pertussis in the last few years. Several hypotheses have been postulated, including the evolution of pertussis strains and the decreased duration of protection from aP compared to wP vaccines. One of the fields of research aimed at controlling the re- emergence of pertussis is directed towards the development of new vaccines; there are several approaches to new pertussis vaccines, including the development of (i) less reactogenic DTwP vaccines, (ii) new DTaP vaccines with different adjuvants and (iii) live-attenuated pertussis vaccines.

Therefore, there is still the need to provide new immunogenic compositions which overcome the problems of the prior art, and that are achievable by an easy and convenient procedure.

SUMMARY OF THE INVENTION

The Applicant has found that when native or non-native outer membrane vesicles (OMVs) from Bordetella pertussis are conjugated to the capsular polysaccharide of Hemophilus influenzae type b (PRP), the conjugates are not only immunogenic, but also possess physical properties that may make them more amenable for use in liquid compositions than conjugates of the prior art. In a first aspect, the invention relates to a conjugate comprising an OMV conjugated to a capsular polysaccharide from Hemophilus influenzae type b, wherein the OMV is obtained from Bordetella. Particularly, the conjugate is an immunogenic conjugate.

The OMV of the invention may be obtained from any of the designated species of Bordetella, particularly from Bordetella pertussis, Bordetella parapertussis or Bordetella bronchiseptica. In a preferred embodiment, the OMV is obtained from Bordetella pertussis. In certain embodiments, the OMV is obtained from a B. pertussis strain expressing a genetically detoxified pertussis toxoid. In certain embodiments, the OMV is obtained from B. pertussis Tohama I strain. In certain embodiments, the Tohama strain is ATCC^® Number: BAA-589.

In some embodiments, the OMV is a native OMV. The OMV may be produced from wild type bacteria or from a genetically-modified bacterial strain that has been mutated, for example, to enhance vesicle production, to remove or modify specific genetic features, or to over-express homologous antigens or express antigens from other organisms.“Homologous” as used herein means the two or more referenced molecules or structures are derived from organisms of the same species, for example, Bordetella pertussis.

In other embodiments, the OMV is a non-native OMV such as a detergent derived OMV (dOMV). The dOMV may be produced from wild type bacteria or from a genetically-modified bacterial strain. Particularly, the dOMV may be prepared from frozen bacterial pellets using techniques comprising tangential flow filtration and/or ultracentrifugation.

The conjugate comprises a capsular polysaccharide of Hemophilus influenzae type b. Particularly, the capsular polysaccharide has a molecular weight of approximately from about 1 to 100 KDa. More particularly, the capsular polysaccharide has a molecular weight of approximately from about 5 to 75 KDa. Yet more particularly, the capsular polysaccharide has a molecular weight of approximately from about 9 to 65 KDa.

In a second aspect, the invention relates to an immunogenic composition comprising the conjugate of the first aspect and at least one pharmaceutically acceptable carrier or excipient.

Particularly the immunogenic composition is a liquid composition, more particularly a fully liquid immunogenic composition, yet more particularly a fully liquid stable immunogenic composition. The term“fully liquid” as used herein refers to a composition in which all of the components of the composition are provided in a combined form in a liquid state such that none of the components needs to be reconstituted prior to administration. Reference to the term“stable” is understood to refer to compositions that maintain their physicochemical properties through standard vaccine manufacturing and storage processes. More particularly, use of the term “stable” in the context of the present invention, means that there is no substantial loss of immunogenicity of the components during storage for a prolonged period. Particularly a composition, for example a liquid immunogenic composition, comprising the conjugate of the invention is stable if the conjugate is capable of inducing an anti-PRP antibody concentration of > 0.15 pg/ml after storage at about 4° C for at least one, at least two, at least six, at least twelve or at least 24 months. Particularly a composition, for example a liquid immunogenic composition, comprising the conjugate of the invention is stable if the conjugate is capable of inducing an anti-PRP antibody concentration of > 1 pg/ml after storage at about 4° C for at least one, at least two, at least six, at least twelve or at least 24 months. Yet more particularly, each antigen in the fully liquid immunogenic composition maintains immunogenicity at or above an established threshold and/or for example, is capable of inducing a protective immune response after storage at about 4° C for at least one, at least two, at least six, at least twelve or at least 24 months. Stability of compositions comprising polysaccharide conjugates may also be assessed by reference to rate of increase of free polysaccharide over time. Methods of determining the amount of free (unconjugated) polysaccharide are known in the art and may be based, for example, on precipitation with deoxycholate followed by analysis with orcinol dye. Particularly, the rate of increase of free polysaccharide may be less than 5%, less than 10%, less than 15%, less than 20% or less than 25% at the end of shelf life compared with the amount of free polysaccharide at release. Accelerated stability studies may be performed at higher temperatures, for example at 40°C. Particularly, the rate of increase of free polysaccharide may be less than 5%, less than 10%, less than 15%, less than 20% or less than 25% after incubation at 40°C for at least three weeks, for example, up to 12 weeks.

Yet more particularly, the immunogenic composition is a vaccine, particularly a combination vaccine. Still yet more particularly, the immunogenic composition is a combination vaccine comprising the conjugate of the invention and at least one antigen selected from the group consisting of: Diphtheria toxoid (DT), Tetanus toxoid (TT), acellular pertussis (aP), Hepatitis B (HepB) and Inactivated Poliovirus (I PV) antigens. The DT may be chemically detoxified DT or CRM197. Particularly, the aP is selected from the group consisting of pertussis toxoid, genetically detoxified pertussis toxoid, FHA, pertactin, Fim 2 and Fim 3.

In a third aspect of the invention, the conjugate or composition is for use in inducing an immune response in a vertebrate, preferably a mammal. In more preferred embodiments, the conjugate or the composition is used in prophylaxis or as a vaccine.

Particularly administration of the conjugate generates an antibody response against Hib. More particularly, the antibody response is a protective antibody response. Yet more particularly, the antibody response is an antibody response resulting in an anti-PRP antibody concentration of > 0.15 pg/ml. Yet more particularly, the antibody response is an antibody response resulting in an anti-PRP antibody concentration of > 1 pg/ml. Even yet more particularly, the antibody response is a protective antibody response resulting an anti-PRP antibody concentration of > 0.15 pg/ml. Particularly, the antibody response is a protective antibody response resulting an anti-PRP antibody concentration of > 1 pg/ml. Yet even more particularly, the conjugates of the invention also generate an antibody response against B. pertussis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Flocculation assays: The level of flocculation following mixing of INFANRIX Penta with each of three Hib-dOMV conjugates (each comprising different mW of polysaccharide) was observed with an Olympus 1X51 optical microscope. The framed area in the left panel is magnified in the right panel: (1 ) Infanrix Penta; (2) Infanrix + Hib-TT; (3) Infanrix + Hib-OMV.

Figure 2. Adsorption assays: Antigen adsorption following mixing of INFANRIX Penta and each of the three different Hib-dOMV conjugates was evaluated by SDS-PAGE.

Figure 3. Immunogenicity of Hib-dOMV conjugates: The immunogenicity of the conjugates was evaluated in infant rats immunized intramuscularly at two different site administrations with compositions comprising INFANRIX Penta and each of the three Hib-dOMV conjugates. Rat serum was tested for the presence of Haemophilus influenzae type b polyribosyl- ribitol- phosphate (PRP) specific IgG antibodies. (^* For logistic reasons, males and females were bled at 6 and 7 days post dose II, respectively)

Figure 4. Bactericidal dilution titers against B. pertussis measured by serum bactericidal test. Higher SBA titers against B. pertussis were observed in all groups that received Hib-dOMV conjugates co-administered with Infanrix Penta as compared to Hiberix or unconjugated Hib co-administered with Infanrix Penta.

Figure 5. Anti-Hib (anti-PRR’P) IgG was measured on day 21 and day 35. The immunogenicity of the Hib-dOMV conjugates was confirmed in the adult rat model. Figure 6. Evaluation of SBA against pertussis. Higher SBA titers against B. pertussis were observed in all groups that received Hib-dOMV conjugates in co-adminstration with Infanrix Penta as compared to Hiberix co-administered with Infanrix Penta.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to immunogenic conjugates comprising an OMV obtained from Bordetella and a capsular polysaccharide of Hemophilus influenzae type b. Some polysaccharide vaccine antigens may be prone to hydrolytic degradation reactions; for example, the Hib capsular polysaccharide conjugate is generally unstable, particularly in the presence of aluminium adjuvants. As a result, many commercial vaccines comprise a Hib conjugate component in lyophilized form. Surprisingly, the present inventors have discovered that conjugating the Hib PRP to an OMV enables the conjugate to be formulated in liquid form without the need for lyophilisation. After 15 minutes, Hib-TT conjugates show high levels of aggregation/flocculation following mixing with multivalent compositions. Surprisingly, such aggregation/flocculation is not observed when using the Hib-OMV conjugates of the invention (see Examples). Advantageously, the use of OMVs from Bordetella, for example Bordetella pertussis, provides additional antigens that may also enhance the immune response against Bordetella.

Outer Membrane Vesicles

OMVs are non-replicative vesicles that are naturally produced by Gram-negative bacteria and contain excellent intrinsic immunostimulatory properties based on their particulate nature and composition (3). The vesicles comprise phospholipids, LPS, outer membrane proteins and entrapped periplasmic components, and are ascribed many biological functions such as cell to cell communication, surface modifications and the expulsion of components.

OMVs combine antigen presentation with optimal physicochemical adjuvant properties, making them highly suitable as a vaccine platform. Antigens may be present inside the OMV or displayed on the OMV surface. Antigens may be produced by the bacterium itself or introduced in a separate process step.

Genetic engineering of OMV-producing bacteria can be used to improve and expand their usefulness as vaccine; OMVs can be modified, for example, to reduce lipopolysaccharide reactogenicity. The overexpression of antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of the range of applications of OMVs. As used herein,“heterologous” refers to two or more referenced molecules or structures, such as an antigen, that are derived from organisms of different species. Bacteria may be genetically modified to increase release of OMVs, sometimes called ‘hyperblebbing’. The use of genetically modified bacteria in combination with specific production processes may be used to obtain high amounts of well-defined, stable and uniform OMVs.

In some embodiments of the invention, the OMV is a native OMV (nOMV). The term nOMV indicates vesicles spontaneously released into and isolated from the medium; they are intact membrane vesicles not exposed to detergents or denaturing agents i.e. not detergent extracted. The nOMV used in the invention may present outer membrane proteins (OMP) and lipopolysaccharide (LPS) in their native conformation and correct orientation in the natural membrane environment, and usually lack the cytoplasmatic components.

Native OMVs can be obtained e.g. by culturing bacteria in broth culture medium, separating whole cells from the smaller nOMVs in the broth culture medium (e.g. by filtration or by low- speed centrifugation to pellet only the cells and not the smaller vesicles), and then collecting the nOMVs from the cell-depleted medium (e.g. by filtration, by differential precipitation or aggregation, by high-speed centrifugation to pellet the vesicles). Strains for use in production of nOMVs can generally be selected on the basis of the amount of nOMVs produced in culture. The nOMVs of the invention may be isolated substantially without or completely without the use of detergents.

The nOMV used in the present invention may be produced from wild type bacteria or from genetically-modified bacterial strains that are mutated to enhance vesicle production, and optionally also to remove or modify antigens (e.g. lipid A) and/or to over-express homologous antigens or antigens from other organisms. Such nOMVs are also known as Generalized Modules of Membrane Antigens (GMMA) as described for example in (4).

Enhanced spontaneous generation of vesicles can be achieved, for example, by targeted deletion of proteins involved in maintenance of membrane integrity. It has been observed that the outer surface of nOMVs substantially corresponds to the outer surface of the bacterium from which they are derived, preserving the membrane antigens (including e.g. lipopolysaccharides, lipooligosaccharides and lipoproteins) in the context of the membrane. The nOMVs used in the invention retain these outer membrane components in their native conformation and correct orientation, better preserving immunogenicity against the bacterial strain from which they are derived. nOMVs may be characterized by a defined size distribution (typically in the range 20-250 nm) typically measured by Dynamic Light Scattering DLS technique.

In some embodiments of the invention, the OMV is a non-native OMV such as a detergent extracted OMV (dOMV). The term“dOMV” encompasses a variety of proteoliposomic vesicles obtained by disruption of the outer membrane of a Gram-negative bacterium typically by a detergent extraction process to form vesicles therefrom. The detergent extraction process may reduce or remove LPS, phospholipids and lipoproteins. dOMVs may have a different size distribution, for example between from about 40 to about 5500 nm as measured by Dynamic Light Scattering DLS technique. Particularly, the dOMVs of the invention may have a size between from about 40 to about 500 nm. More particularly, the dOMVs may have a size between from about 40 to about 100 nm. Even more particularly, the DOMVs may have a size of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nm. Particularly, the detergent is deoxycholate.

The dOMV used in the present invention may be produced from wild type bacterial strains or from genetically-modified bacterial strains. Particularly, the dOMV may be purified from frozen bacterial pellets using techniques comprising tangential flow filtration and/or ultracentrifugation.

According to prior art methodologies, both dOMVs and nOMVs may be analysed and described in terms of size, shape and overall appearance of impurities or contaminating non-OMV materials (like vesicle aggregates or detergent residues in case of dOMVs) using T ransmission Electron Microscopy (TEM). For detailed references regarding the differences between dOMVs and nOMVs see e.g. (5). .

Bordetella

The OMV of the invention may be obtained from any of the designated species of Bordetella. The genus Bordetella contains nine designated species, three of which are referred to as the "classical Bordetella": Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Although highly similar at the DNA sequence level, they vary in host specificity, severity of diseases, and their ability to cause acute versus chronic infection. Bordetella bronchiseptica causes infections ranging from lethal pneumonia to asymptomatic respiratory carriage and chronically colonizes the respiratory tracts of various mammalian hosts, with some lineages primarily isolated from humans. Bordetella pertussis and Bordetella parapertussis are causative agents of whooping cough in humans. The OMV of the invention is preferably obtained from Bordetella pertussis, Bordetella parapertussis or Bordetella bronchiseptica.

In preferred embodiments of the invention, the OMV is obtained from Bordetella pertussis.

Pertussis, caused by Bordetella pertussis, is a highly contagious airway infection. Acute infection can cause severe illness characterized by severe respiratory failure, pulmonary hypertension, leucocytosis, and death. Pertussis is a vaccine-preventable disease, with either acellular pertussis vaccines or whole-cell vaccines being used worldwide. However, the disease has persisted in vaccinated populations, and epidemiological data has reported a worldwide increase in pertussis incidence among children during the past years. The resurgence of pertussis may be due to missing booster immunizations among adolescents and adults, low vaccine coverages in some geographic areas, and genetic changes of different B. pertussis strains.

The production of Bordetella OMVs and their use in vaccines has been described elsewhere (see for example (6)).

Hib-PRP

Haemophilus influenzae is a gram-negative bacterium which can exist in two forms: encapsulated and non-encapsulated (non-typeable). The non-typeable forms can cause non- invasive respiratory tract infections and middle ear infection. The encapsulated forms have a polysaccharide capsule and can be classified into six serotypes (a-f) based on antigenic differences. These forms can cause more serious invasive conditions, including bacteraemia, pneumonia and meningitis.

Haemophilus influenzae type b is the most virulent and, prior to routine vaccination, was responsible for the vast majority of invasive H. influenzae infections, particularly meningitis in young children. Hib is more pathogenic than other H. influenzae because its capsule consists of a repeating polymer of ribosyl and ribitol phosphate (polyribosyl-ribitol-phosphate, PRP), which enables the organism to effectively evade complement-mediated killing and avoid splenic clearance. Hib conjugate vaccines contain PRP conjugated to a carrier protein are very immunogenic, resulting in high concentrations of antibodies against the PRP capsule.

The term“capsular polysaccharide” (CPS) indicates those saccharides which can be found in the layer that lies outside the cell envelope of bacteria, thus being part of the outer envelope of the bacterial cell itself. CPSs are expressed on the outermost surface of a wide range of bacteria, and in some cases even in fungi. It will be appreciated that polysaccharide moieties can exist in open and closed (ring) form. Similarly, it will be appreciated that saccharide moieties can exist in pyranose and furanose forms and that, while pyranose forms are shown in structural formulae herein, furanose forms are also encompassed. Different anomeric forms of saccharide moieties are also encompassed.

The saccharide moiety of the conjugate may be used in its full-length natural form, comprising full-length PRP as prepared from Hib bacteria, or, as an alternative, it may be fragmented from its natural length; optionally, a size fraction of these fragments can also be used. In some embodiments the PRP may be a synthetic polysaccharide. Synthetic PRP (sPRP) is known in the art and used in, for example, the QUIMI-HIB vaccine. The fragments of the capsular polysaccharides of the invention may be obtained by different methods described in the art. Particularly, the fragments may be obtained by reacting native Hib PRP with sodium metaperiodate using different molar ratios Hib PRP repeating unit to periodate in order to obtain Hib oligosaccharide (OS) populations of different lengths. The polysaccharides may be purified using different techniques described in the art, including gel filtration and size exclusion chromatography.

The molecular weight of the capsular polysaccharides of the invention prior to conjugation is between from about 1 KDa to about 250 KDa. In some embodiments of the invention, the molecular weight of the capsular polysaccharides is between from about 5 KDa to about 100 KDa. Particularly, the molecular weight of the capsular polysaccharides may be between from about 5 to about 70 KDa. Therefore, the molecular weight of the capsular polysaccharides of the invention may be about 5, 8 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 KDa. In certain embodiments, the molecular weight of the capsular saccharides is between from about 4 to about 14 KDa, such as 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or 14 KDa. In other embodiments of the invention, the molecular weight of the capsular saccharides is between from about 23 to about 33 KDa, such as 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or 33 KDa. In other embodiments of the invention, the molecular weight of the capsular saccharides is between from about 58 to about 68 KDa, such as 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67 or 68 KDa. Any whole number integer within any of the above ranges may be contemplated.

The OMV used in the invention is conjugated to a capsular polysaccharide of Haemophilus influenza type b.

Hib-OMV conjugates

Unless otherwise provided, the term“conjugation” indicates the connection or linkage of the OMV and the selected capsular saccharides.

Saccharide antigens from H. influenzae type b and their preparation are well known (7-17). The Hib saccharide is usually conjugated to a carrier protein in order to enhance its immunogenicity, especially in children.

Various different carrier proteins have been used in Hib conjugates (18). For example, the PRP-D product uses a DT carrier protein and the HbOC product uses a CRM197 carrier protein. The PRP-T product uses a TT carrier protein; this is the conjugate present in the HIBERIX product. The PRP-OMPC product uses an outer membrane protein complex from serogroup B meningococcus as the carrier. The more recent DTaP5-HB-IPV-Hib vaccine (VAXELIS) contains the PRP conjugated to the outer membrane protein complex (OMPC) of Neisseria meningitidis (PRP-OMPC). In all cases, the typical amount of Hib saccharide per vaccine dose is 10 pg. The conjugates of the invention comprise a Hib PRP and/or PRP fragment conjugated to a nOMV or dOMV purified from Bordetella. Conjugates of the Hib PRP conjugated to such OMVs may be referred to as Hib-dOMV or Hib-nOMV. The conjugates may be obtained by standard conjugation methods, including but not limited to the use of sodium cyanoborohydride (see the Example section for further details). The conjugates may also be purified by standard protein purification methods, including but not limited to tangential flow filtration.

Conjugates with a saccharide:OMV ratio (w/w) between from about 0.05 to about 5 may be used within the invention. For example, the saccharide:OMV ratio (w/w) may be between from about 0.1 to about 3. In some embodiments of the invention, the saccharide:OMV ratio (w/w) is between from about 0.1 to about 2. Therefore, the saccharide:OMV ratio (w/w) may be 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1 .1 , 1.2, 1.3, 1 .4, 1 .5, 1.6, 1.7, 1.8, 1 .9 or 2. Particularly, the amount of unconjugated saccharide is no more than 25% of the total amount of saccharide in the composition as a whole. More particularly, the amount of unconjugated saccharide is no more than 10% of the total amount of saccharide in the composition as a whole. Even more particularly, the amount of unconjugated saccharide is no more than 8% of the total amount of saccharide in the composition as a whole. Yet even more particularly, the amount of unconjugated saccharide is no more than 6% of the total amount of saccharide in the composition as a whole. In some embodiments of the invention, the amount of unconjugated saccharide is 1 %, 2%, 3%, 4% 5% or 6% of the total amount of saccharide in the composition as a whole.

Hib conjugates according to the invention may be lyophilised prior to their use. Further components may also be added prior to freeze-drying e.g. as stabilizers. Preferred stabilizers for inclusion are lactose, sucrose and mannitol, as well as mixtures thereof e.g. lactose/sucrose mixtures, sucrose/mannitol mixtures, etc. The final immunogenic composition may thus contain lactose and/or sucrose. Using a sucrose/mannitol mixture can speed up the drying process.

Immunogenic compositions

The invention further relates to immunogenic compositions comprising the above-described conjugates. The conjugates and compositions of the invention are immunogenic. The conjugates may therefore be used as immunogenic agents, for example as an antigen.

In preferred embodiments of the invention, the conjugate or the immunogenic composition is used as a vaccine. The Hib-OMV conjugate or the immunogenic composition of the invention is capable of inducing an immune response not only against the conjugated antigen but also against the OMV component and are thus good candidates for use in the preparation of multivalent immunogenic compositions. The conjugates of the invention may be useful e.g. as bi-valent vaccines, with the OMV and the conjugated heterologous antigen both showing good immunogenicity. Thus, conjugates of the invention are capable of inducing an immune response against both Haemophilus influenzae Type B and Bordetella, particularly Bordetella pertussis.

Immunogenic compositions of the invention will generally comprise at least one pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers and excipient are known in the art, and include liquids such as water, saline, glycerol and ethanol.

Adjuvants

In addition to antigenic components, compositions of the invention will typically include at least one adjuvant, such as an aluminium salt adjuvant. The compositions can include both aluminium hydroxide and aluminium phosphate adjuvants. Where both are included, the weight ratio of the two adjuvants is approximately 1 : 1 e.g. an aluminium hydroxide: aluminium phosphate ratio of about 1.58: 1.6. Although aluminium adjuvants are typically referred to either as "aluminium hydroxide" or as "aluminium phosphate" adjuvants, these are names of convenience and not a precise description of the actual chemical compound which is present. The invention can use any of the "hydroxide" or "phosphate" adjuvants that are in general use as adjuvants.

The adjuvants known as "aluminium hydroxide" are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline. Aluminium oxyhydroxide, which can be represented by the formula AIO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide AI(OH)3, by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070 cm^-1 and a strong shoulder at 3090-3100 cm^-1 (19).

The adjuvants known as "aluminium phosphate" are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a P0₄/Al molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AIPO4 by the presence of hydroxyl groups. For example, an IR spectrum band at 3164 cm^-1 (e.g. when heated to 200°C) indicates the presence of structural hydroxyls (19). The adjuvants can take any suitable form (e.g. gel, crystalline, amorphous, etc.). The PO4/ AI3+ molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1 .2, preferably between 0.8 and 1.2, and more preferably 0.95±0.1. A typical adjuvant is amorphous aluminium hydroxyphosphate with PO4/AI molar ratio between 0.84 and 0.92, included at 0.6mg Al³7ml. The aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts. The aluminium phosphate will generally be particulate. Typical diameters of the particles are in the range between from 0.5-20 pm (e.g. about 5-I0 pm) after any antigen adsorption.

The point of zero charge (PZC) of aluminium phosphate is inversely related to the degree of substitution of phosphate for hydroxyl, and this degree of substitution can vary depending on reaction conditions and concentration of reactants used for preparing the salt by precipitation. PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate= more acidic PZC) or by adding a buffer such as a histidine buffer (makes PZC more basic). Aluminium phosphates used according to the invention will generally have a PZC of between from about 5.0 to about 7.0, more preferably between from about 5.5 to about 6.0 e.g. about 5.7.

The aluminium phosphate is preferably used in the form of an aqueous solution to which antigens are added (NB: it is common to refer to aqueous aluminium phosphate as a "solution" although, on a strict physicochemical view, the salt is insoluble and forms a suspension). It is preferred to dilute the aluminium phosphate to the required concentration and to ensure a homogenous solution before the addition of the antigenic components.

The concentration of Al³⁺ prior to addition of antigens is generally between from about 0.01 to about 10 mg/ml. A preferred concentration is between from about 2 to about 6 mg/ml.

An aluminium phosphate solution used to prepare a vaccine of the invention may contain a buffer (e.g. a phosphate or a histidine buffer), but this is not necessary. The aluminium phosphate solution is preferably sterile and pyrogen-free. The aluminium phosphate solution may include free aqueous phosphate ions e.g. present at a concentration between from about 1 to about 20 mM, preferably between from about 5 to about 15 mM, and more preferably about 10 mM. The aluminium phosphate solution may also comprise sodium chloride. The concentration of sodium chloride is preferably in the range of 0.1 to 100 mg/ml (e.g. 0.5-50 mg/ml, 1-20 mg/ml, 2-10 mg/ml) and is more preferably about 3±1 mg/ml. The presence of NaCI facilitates the correct measurement of pH prior to adsorption of antigens.

Compositions of the invention may include a TLR agonist i.e. a compound which can agonise a Toll-like receptor. Most preferably, a TLR agonist is an agonist of a human TLR. The TLR agonist can activate any of TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or TLR1 1 ; preferably it can activate human TLR4 or human TLR7.

A TLR agonist used with the invention ideally includes at least one adsorptive moiety. The inclusion of such moieties in TLR agonists allows them to adsorb to insoluble aluminium salts (e.g. by ligand exchange or any other suitable mechanism) and improves their immunological behaviour. Phosphorus-containing adsorptive moieties are particularly useful, and so an adsorptive moiety may comprise a phosphate, a phosphonate, a phosphinate, a phosphonite, a phosphinite, etc.

The TLR agonist may include at least one phosphonate group. In particular embodiments, a composition of the invention may include a TLR7 agonist which includes a phosphonate group. This phosphonate group can allow adsorption of the agonist to an insoluble aluminium salt. In some embodiments, the TLR agonist is 3-(5-amino-2-(2-methyl-4-(2-(2-(2- phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo [f]-[1 ,7]naphthyridin-8-yl)propanoic acid, as shown below.

Preferred TLR agonists are water-soluble. Thus they can form a homogenous solution when mixed in an aqueous buffer with water at pH 7 at 25°C and 1 atmosphere pressure to give a solution which has a concentration of at least 50 pg/ml. The term“water-soluble” thus excludes substances that are only sparingly soluble under these conditions.

A composition of the invention can include more than one TLR agonist. These two agonists are different from each other and they can target the same TLR or different TLRs. Both agonists can be adsorbed to an aluminium salt.

Where an antigen is described as being "adsorbed" to an adjuvant, it is preferred that at least 50% (by weight) of that antigen is adsorbed e.g. 50%, 60%, 70%, 80%, 90%, 95%, 98% or more. In some embodiments the DT and TT are both totally adsorbed i.e. none is detectable in supernatant. Total adsorption of HepB is also preferred.

If the vaccine of the invention contains an aluminium-based adjuvant, settling of components may occur during storage. The vaccine should therefore be shaken prior to administration to a patient. The shaken vaccine will be a turbid white suspension.

Further components of the vaccine

As well as containing antigen(s) and adjuvant(s), the combination vaccines of the invention may include further components. These components may have various sources. For example, they may be present in one of the antigenic components that is mixed during the process of the invention or may be added during the process separately from the antigenic components. To control tonicity of the final vaccine product, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCI) is preferred, which may be present in the final vaccine product at between from about 1 to about 20 mg/ml.

Due to the adsorbed nature of antigens, the final vaccine product may be a suspension with a cloudy appearance. This appearance means that microbial contamination is not readily visible, and so the vaccine may contain an antimicrobial agent. This is particularly important when the vaccine is packaged in multidose containers. Particular antimicrobials for inclusion are 2- phenoxyethanol (2-PE) and thimerosal. It is preferred, however, not to use mercurial preservatives (such as thimerosal).

Immunogenic compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration. Intramuscular administration is preferred e.g. to the thigh or the upper arm. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is about 0.5 ml. The invention may also be used to elicit systemic and/or mucosal immunity. Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4-16 weeks) and between priming and boosting can be routinely determined.

Infections affect various areas of the body and so the compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectable, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition be prepared for oral administration e.g. as a tablet or capsule, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. Compositions suitable for parenteral injection are most preferred. The composition is preferably sterile and it is preferably pyrogen-free. It may be buffered, for example, at between from pH 6 to pH 8, generally around pH 7. Compositions of the invention may be isotonic with respect to humans. Immunogenic compositions comprise an immunologically effective amount of a conjugate of the invention, as well as any other of other specified components, as needed. By“immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount can vary depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the composition, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses). The composition may be administered in conjunction with other immunoregulatory agents.

Combination Vaccines

The invention further relates to multi-valent combination vaccines comprising the immunogenic conjugate or composition and at least one antigen selected from the group consisting of DT, TT, aP, HepB and IPV antigens.

Diphtheria toxoid

Diphtheria is caused by Corynebacterium diphtheriae, a Gram-positive non-sporing aerobic bacterium. This organism expresses a prophage-encoded ADP-ribosylating exotoxin ('diphtheria toxin'), which can be treated (e.g. using formalin or formaldehyde) to give a toxoid that is no longer toxic but that remains antigenic and is able to stimulate the production of specific anti-toxin antibodies after injection. DTs are disclosed in more detail in (20). In some embodiments DT s are those prepared by formaldehyde treatment. The DT can be obtained by growing C. diphtheriae n growth medium (e.g. Fenton medium, or Linggoud & Fenton medium), which may be supplemented with bovine extract, followed by formaldehyde treatment, ultrafiltration and precipitation. Where bovine materials are used in the culture of C. diphtheriae, they should be obtained from sources that are free from bovine spongiform encephalopathy (BSE) or from other transmissible spongiform encaphalopathies (TSEs).The toxoided material may then be treated by a process comprising sterile filtration and/or dialysis.

In other embodiments, DT in the form of CRM197 may be used. The wild-type DT catalyzes the ADP-ribosylation of eucaryotic elongation factor-2 (eEF2) by using NAD, thus inactivating this protein. CRM197 has an alteration of 52nd Gly to Glu and it has no ADP ribosylation activity nor toxicity to cells. Therefore, it does not need to be chemically toxoided.

The DT may be adsorbed onto an adjuvant, for example, aluminium salts; it may also be formulated in saline and contain 2-phenoxyethanol.

Particularly, the DT component is substantially free from any mercurial preservatives. Quantities of DT can be expressed in international units (IU). For example, the NIBSC supplies the 'Diphtheria Toxoid Adsorbed Third International Standard 1999' (21 , 22), which contains 160 IU per ampoule. As an alternative to the IU system, the 'Lf unit ("flocculating units" or the "limes flocculating dose") is defined as the amount of toxoid which, when mixed with one International Unit of antitoxin, produces an optimally flocculating mixture (23). For example, the NIBSC supplies 'Diphtheria Toxoid, Plain' (24), which contains 300 LF per ampoule, and also supplies 'The 1 st International Reference Reagent For Diphtheria Toxoid For Flocculation Test' (25) which contains 900 LF per ampoule.

The amount of DT in vaccines of the invention is typically between from about 20 to about 100 lU/dose. Particularly, the amount of DT in vaccines of the invention is between from about 30 to about 90 lU/dose. Even more particularly, the amount of DT in vaccines of the invention is between from about 30 to about 80 lU/dose. Therefore, the amount of DT in vaccines of the invention may be 30, 40, 50, 60, 70 or 80 lU/dose.

Tetanus toxoid (TT)

Tetanus is caused by Clostridium tetani, a Gram-positive, spore-forming bacillus. This organism expresses an endopeptidase ('tetanus toxin'), which can be treated to give a toxoid that is no longer toxic but that remains antigenic and is able to stimulate the production of specific anti-toxin antibodies after injection.

Preferred TTs are those prepared by formaldehyde treatment. The TT can be obtained by growing C.tetani in growth medium (e.g. a Latham medium derived from bovine casein), followed by formaldehyde treatment, ultrafiltration and precipitation. The material may then be treated by a process comprising sterile filtration and/or dialysis.

The TT may be adsorbed onto an aluminium hydroxide adjuvant, but this is not necessary (e.g. adsorption of between from about 0 to about 10% of the total TT can be used).

Preferably, the TT component is substantially free from any mercurial preservatives.

Quantities of TT can be expressed in international units (IU). For example, the NIBSC supplies the 'Tetanus Toxoid Adsorbed Third International Standard 2000' (26, 27) which contains 469 IU per ampoule. As an alternative to the IU system, the 'LP unit ("flocculating units" or the "limes flocculating dose") is defined as the amount of toxoid which, when mixed with one International Unit of antitoxin, produces an optimally flocculating mixture (23). For example, the NIBSC supplies 'The 1 st International Reference Reagent for Tetanus Toxoid For Flocculation Test' (28) which contains 1000 LF per ampoule. Where bovine materials are used in the culture of C. tetani, they should be obtained from sources that are free from bovine spongiform encephalopathy (BSE) or from other transmissible spongiform encaphalopathies (TSEs).

The ratio of TT to DT in vaccines of the invention is usually between from about 1 :2 to about 1 :3 (measured in Lf units), preferably between from about 1 :2.4 to about 1 :2.6, and is more preferably 1 :2.5.

The amount of TT in vaccines of the invention is typically from about 40 to about 120 lU/dose. Particularly, the amount of TT in vaccines of the invention is between from about 50 to about 1 10 lU/dose. Even more particularly, the amount of DT in vaccines of the invention is between from about 50 to about 100 lU/dose. Therefore, the amount of DT in vaccines of the invention may be 50, 60, 70, 80, 90 or 100 lU/dose.

Pertussis antigens

Pertussis antigens may be cellular (whole cell) or acellular.

Immunogenic compositions of the invention may comprise aP antigens, including but not limited to chemically or genetically detoxified pertussis toxin, filamentous haemagglutinin (FHA), 69 kDa outer-membrane protein (also known as pertactin), fimbrial-2 and fimbrial-3 antigens (FIM).

The pertussis antigens of the invention may be obtained by extraction and purification from Bordetella pertussis cultures and may be followed by irreversible detoxification of the pertussis toxin and treatment of FHA and PRN. PT, FHA and PRN may be isolated separately from the supernatant culture medium, while FIM may be extracted and co-purified from the bacterial cells. The antigens may be purified by techniques well known to the person skilled in the art, for example sequential filtration, salt-precipitation, ultrafiltration or chromatography.

In most currently available vaccines, the detoxification of pertussis toxin is achieved by treatment with high quantities of chemical agents such as formaldehyde, glutaraldehyde or hydrogen peroxide. However, pertussis toxin may also be detoxified genetically through the substitution or deletion of residues necessary for its enzymatic activity, for example deletion of residues in subunits S1 (Trp26, His 35), S2 (Asn105) and S3 (Lys105, Tyr 102) or single amino acid substitutions in subunit S1 (Arg9, Arg13, Glu129) and S2 (Asn105). Thus, in some embodiments, immunogenic compositions of the invention comprise the genetically detoxified pertussis toxoid referred to as PT-9K/129G.

The pertussis antigens may be adsorbed onto or mixed with an aluminium phosphate adjuvant and/or 2-phenoxyethanol. The amount of detoxified PT in vaccines of the invention is typically from about 1 to about 50 mV^obb. Particularly, the amount of detoxified PT is between from about 2 to about 40 mV^obb. Even more particularly, the amount of DT in vaccines of the invention is between from about 2 to about 30 mV^obb. Therefore, the amount of DT in vaccines of the invention may be 2, 5, 10, 20 or 30 mV^obb.

The amount of FHA in vaccines of the invention is typically from about 1 to about 50 mV^obb. Particularly, the amount of FHA is between from about 2 to about 40 mV^obb. Even more particularly, the amount of FHA is between from about 5 to about 30 mV^obb. Therefore, the amount of FHA in vaccines of the invention may be 5, 10, 20 or 30 mV^obb.

The amount of PRN in vaccines of the invention is typically from about 1 to about 10 mV^obb. Particularly, the amount of PRN is between from about 2 to about 9 mV^obb. Even more particularly, the amount of PRN is between from about 2 to about 8 mV^obb. Therefore, the amount of PRN in vaccines of the invention may be 2, 3, 4, 5, 6, 7, 8, 9 or 10 mV^obb.

The amount of FIM in vaccines of the invention is typically from about 1 to about 10 mV^obb. Particularly, the amount of FIM is between from about 3 to about 9 mV^obb. Even more particularly, the amount of FIM is between from about 4 to about 8 mV^obb. Therefore, the amount of FIM in vaccines of the invention may be 4, 5, 6, 7 or 8 mV^obb.

Hepatitis B surface antigen

Hepatitis B virus (HBV) is one of the known agents which causes viral hepatitis. The HBV virion consists of an inner core surrounded by an outer protein coat or capsid. The viral core contains the viral DNA genome. The major component of the capsid is a protein known as HBV surface antigen or, more commonly, 'HBsAg', a 226-amino acid peptide with a molecular weight of approximately 24 kDa. All existing hepatitis B vaccines contain HBsAg, and when this antigen is administered to a vaccinee it stimulates the production of anti-HBsAg antibodies which protect against HBV infection.

For vaccine manufacture, HBsAg can be made in two ways. The first method involves purifying the antigen in particulate form from the plasma of chronic hepatitis B carriers, as large quantities of HBsAg are synthesized in the liver and released into the blood stream during an HBV infection. The second way involves expressing the protein by recombinant DNA methods.

HBsAg for use with the method of the invention may be prepared in either way, but it is preferred to use HBsAg which has been recombinantly expressed. In particular, it is preferred that the HBsAg is prepared by expression in a yeast, such as a Saccharomyces (such as S.cerevisiae) or a Hanensula (such as H. polymorpha), which carries the gene coding for the major surface antigen of the HBV. This HBsAg expressed in yeast cells may be purified by several physicochemical steps. The HBsAg assembles spontaneously, in the absence of chemical treatment, into spherical particles of 20 nm in average diameter containing non- glycosylated HBsAg polypeptide and a lipid matrix consisting mainly of phospholipids. Extensive tests have demonstrated that these particles display the characteristic properties of the natural HBsAg.

Yeast-expressed HBsAg particles may include phosphatidylinositol, which is not found in natural HBV virions. The particles may also include a non-toxic amount of LPS in order to stimulate the immune system (29). The HBsAg may be from HBV subtype adw2.

Although HBsAg may be adsorbed to an aluminium hydroxide adjuvant in the final vaccine (as in the well-known ENGERIX-B™ product), or may remain unadsorbed, it will generally be adsorbed to an aluminium phosphate adjuvant prior to being used in the process of the invention (30).

Quantities of HBsAg are typically expressed in micrograms. The amount of HBsAg in vaccines of the invention is typically between from about 1 to about 50 mg/dose. Particularly, the amount of HBsAg is between from about 5 to about 25 mg/dose. Even more particularly, the amount of HBsAg is between from about 5 to about 15 mg/dose. Therefore, the amount of HBsAg in vaccines of the invention may be 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 mg/dose.

Polio Virus

The inactivated polio virus component in the invention may be derived from any suitable strains, such as the Salk or Sabin strains. IPV may be produced on a Vero or Sabin cell line using poliovirus strains type 1 , 2 and/or 3 as seed materials. The poliovirus strains used in the invention include but are not limited to Mahoney (type 1 ), MEF-1 (type 2) and Saukett (type 3). The identity of the strains may be confirmed by seroneutralisation, infectivity measure or microbiological purity. The production of the vaccine may include the following steps: preparation of cell substrate, virus inoculation, virus harvest, virus purification, virus inactivation, sterile filtration and pool of the monovalent bulks to obtain a trivalent concentrate.

Purification yields a product from which proteins and VERO cell DNA are virtually eliminated. Inactivation using formaldehyde or any other chemical agents that allow for effective inactivation is consistently achieved.

Quantities of IPV are typically expressed in DU. The amount of type I poliovirus antigen in vaccines of the invention is typically between from about 10 to about 70 DU. Particularly, the amount of type I poliovirus antigen is between from about 20 to about 60 DU. Even more particularly, the amount of type I poliovirus antigen is between from about 30 to about 50 DU. Therefore, the amount of type I poliovirus antigen in vaccines of the invention may be 30, 35, 40 or 50 DU.

The amount of type 2 poliovirus antigen in vaccines of the invention is typically from about 1 to about 15 DU. Particularly, the amount of type 2 poliovirus antigen is between from about 3 to about 12 DU. Even more particularly, the amount of type 2 poliovirus antigen is between from about 5 to about 10 DU. Therefore, the amount of type 2 poliovirus antigen in vaccines of the invention may be 5, 6, 7, 8, 9 OR 10 DU.

The amount of type 3 poliovirus antigen in vaccines of the invention is typically from about 5 to about 60 DU. Particularly, the amount of type 3 poliovirus antigen is between from about 10 to about 50 DU. Even more particularly, the amount of type 3 poliovirus antigen is between from about 20 to about 40 DU. Therefore, the amount of type 2 poliovirus antigen in vaccines of the invention may be 20, 25, 30, 35 or 40 DU.

Standard vaccines comprising IPV may contain 40 DU of type 1 , 8 DU of type 2 and 32 DU of type 3 poliovirus antigen. However, in some embodiments the IPV dose may be a fractional IPV dose, for example, comprising 1/5 of the amount of IPV. Thus, compositions of the invention may comprise about 8 DU of type 1 , about 1.6 DU of type 2 and about 6.4 DU of type 3 poliovirus antigen.

When the conjugate or immunogenic composition of the invention is combined with other antigens to generate a multivalent vaccine, substances such as thimerosal and other residual components from the individual antigens may be present in trace amounts in the final vaccine produced by the process of the invention.

The presence of trace amounts of such components may be unavoidable if an antigen used during the process (e.g. HBsAg) has previously been treated with such a preservative. For safety, however, it is preferred that the final vaccine product contains less than about 25 ng/ml mercury. More preferably, the final vaccine product contains no detectable thimerosal. This will generally be achieved by removing the mercurial preservative from an antigen preparation prior to its addition in the process of the invention or by avoiding the use of thimerosal during the preparation of individual antigenic components.

If formaldehyde is used to prepare the toxoids of diphtheria, tetanus and pertussis then the final vaccine product may retain trace amounts of formaldehyde (e.g. less than 10 pg/ml, preferably <5pg/ml). Free amino acids (e.g. alanine, arginine, aspartate, cysteine and/or cystine, glutamate, glutamine, glycine, histidine, proline and/or hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine and/or valine), vitamins e-g-choline, ascorbate, etc.), disodium phosphate, monopotassium phosphate, calcium, glucose, adenine sulfate, phenol red, sodium acetate, potassium chloride, etc. may be retained in the final vaccine at < 100 ug/ml of each (e.g . £ 50pg/ml, < 10 mV/GhI). A further possible component of the final vaccine which originates in the antigen preparations arises from less-than-total purification of antigens. Small amounts of B.pertussis, C.diphtheriae, CJetani and S.cerevisiae proteins and/or genomic DNA may therefore be present. To minimize the amounts of these residual components, antigen preparations are preferably treated to remove them prior to the antigens being used in the process of the invention. Where aluminium salts are present within the final vaccine, the total amount of aluminium, expressed in terms of Al³⁺, is preferably < 2 mg/ml (e.g. between 1 .2-1 .5 mg/ml, or about 1.4 mg/ml; or between 0.4 and 0.8 mg/ml, or about 0.6 mg/ml).

Dilution of components to give desired final concentrations will usually be performed with WFI (water for injection). To prevent interference between antigens, particularly conjugate antigens, it is possible to include a polyanionic polymer, such as poly-L-glutamic acid.

Vaccine manufacturing

The invention provides the use of (i) DT, (ii) TT, (iii) aP, (iv) HbsAgand (v) IPV antigens and (vi) a Haemophilus influenzae type b capsular saccharide conjugated to an OMV in the manufacture of an immunogenic composition for use in therapy, as immunogenic compositions or as vaccines.

In typical use, the process of the invention will be used to provide bulk combination vaccine which is suitable for packaging, and then for distribution and administration. Concentrations mentioned above are typically concentrations in final packaged vaccine, and so concentrations in bulk vaccine may be higher (e.g. to be reduced to final concentrations by dilution).

The process of the invention may therefore comprise the further step of packaging the vaccine into containers for use. Suitable containers include vials and disposable syringes (preferably sterile ones). Where the vaccine is packaged into vials, these are preferably made of glass or of a plastic material. The vial is preferably sterilized before vaccine is added to it. To avoid problems with latex-sensitive patients, vials can be sealed with a latex-free stopper. The vial may include a single dose of vaccine, or it may include more than one dose (a 'multidose' vial) e.g. 10 doses. When using a multidose vial, each dose should be withdrawn with a sterile needle and syringe under strict aseptic conditions, taking care to avoid contaminating the vial contents. Preferred vials are made of colorless glass.

Where the vaccine is packaged into a syringe, the syringe will not normally have a needle attached to it, although a separate needle may be supplied with the syringe for assembly and use. Safety needles are preferred. 1 -inch 23-gauge, 1-inch 25-gauge and 5/8-inch 25-gauge needles are typical. Syringes may be provided with peel-off labels on which the lot number and expiration date of the contents may be printed, to facilitate record keeping. The plunger in the syringe preferably has a stopper to prevent the plunger from being accidentally removed during aspiration. The syringes may have a latex rubber cap and/or plunger. Disposable syringes contain a single dose of vaccine. The syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap is preferably made of butyl rubber. If the syringe and needle are packaged separately then the needle is preferable fitted with a butyl rubber shield. Grey butyl rubber is preferred. Preferred syringes are those marketed under the trade name "Tip-Lok"™.

Where a glass container (e.g. a syringe or a vial) is used, then it is preferred to use a container made from a borosilicate glass rather than from a soda lime glass. When contained separately, conjugate antigens will typically be freeze-dried (lyophilized) in a separate container, such that the packaged vaccine will contain at least two separate containers. Prior to administration to a patient, the freeze-dried material will be reconstituted and diluted with the liquid from the other container. Typically, the lyophilized conjugate container will be a vial and the second container will contain a liquid within a vial or a pre-filled syringe. The liquid contents of the second container will be transferred into the vial containing the freeze-dried conjugate antigen powder, thereby reconstituting the conjugate antigens for administration to a patient.

Alternatively, the conjugate container may be in a liquid state, contained in a vial or a pre-filled syringe. Alternatively, the conjugate and the antigens will be in a liquid state, contained in a single vial or pre-filled syringe.

The container for lyophilised conjugates is preferably a vial which has a cap (e.g. a Luer lock) adapted such that a pre-filled syringe can be inserted into the cap, the contents of the syringe can be expelled into the vial to reconstitute the freeze-dried material therein, and the contents of the vial can be removed back into the syringe. After removal of the syringe from the vial, a needle can then be attached, and the vaccine can be administered to a patient. The cap is preferably located inside a seal or cover, such that the seal or cover has to be removed before the cap can be accessed.

The combination vaccine of the invention is preferably administered to patients in 0.5 ml doses. The process of the invention may therefore comprise the step of extracting and packaging a 0.5 ml sample of the bulk vaccine into a container. For multidose situations, multiple dose amounts will be extracted and packaged together in a single container. Where a vaccine is presented as a kit with a lyophilised component then the final dose after reconstitution is preferably 0.5 ml. References to 0.5 ml doses herein should be taken to mean 0.5ml±0.05ml.

The container in which the vaccine is packaged will usually then be enclosed within a box for distribution e.g. inside a cardboard box, and the box will be labelled with details of the vaccine e.g. its trade name, a list of the antigens in the vaccine (e.g. 'Diphtheria, tetanus, inactivated whole cell pertussis and hepatitis B recombinant, adsorbed vaccine', etc.), the presentation container (e.g. 'Disposable Prefilled Tip-Lok Syringes' or Ί 0 x 0.5 ml Single-Dose Vials'), its dose (e.g. 'each containing one 0.5ml dose'), warnings (e.g. 'For Paediatric Use Only'), an expiration date, etc. Each box might contain more than one packaged vaccine e.g. five or ten packaged vaccines (particularly 20 for vials). If the vaccine is contained in a syringe then the package may show a picture of the syringe. The vaccine may be packaged together (e.g. in the same box) with a leaflet including details of the vaccine e.g. instructions for administration, details of the antigens within the vaccine, etc. The instructions may also contain warnings e.g. to keep a solution of adrenaline readily available in case of anaphylactic reaction following vaccination, etc.

The packaged vaccine materials are preferably sterile.

The packaged vaccine materials are preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose.

The packaged vaccine materials are preferably gluten free.

The pH of any aqueous packaged vaccine materials is preferably between from about 6 to about 8 e.g. between from about 6.5 to about 7.5. The process of the invention may therefore include a step of adjusting the pH of the bulk vaccine prior to packaging.

Any aqueous material within the packaged vaccine may be a turbid white suspension.

The packaged vaccine is preferably stored at between 2°C and 8°C. It should not be frozen.

Methods of administration

The conjugate or composition of the invention may be used to induce an immune response in a vertebrate, preferably a mammal, particularly a suitable mammal. The mammal is preferably a human; the immune response is preferably protective and preferably involves antibodies.

The invention further provides the use of DT, TT, aP, HepB and IPV antigens with a Haemophilus influenzae type b capsular saccharide conjugated to a OMV in the manufacture of a medicament for immunising a patient.

The patient is preferably a human and may be a child (e.g. a toddler or infant), a teenager or an adult. The final combination vaccines of the invention are particularly suitable for administration to children. Preferred patients are aged between 0-36 months e.g. between 0- 24 months, between 0-12 months, or between 0-6 months.

A typical dosage schedule for the vaccine, in order to have full efficacy, will involve administering more than one dose in a primary immunization schedule. A typical primary schedule will involve three doses, given at intervals of about 6 to 8 weeks, with the first dose being given to a child aged between 6 and 9 weeks of age. A 3-dose primary schedule at 6, 10 and 14 weeks of age is preferred, and this may be followed up with a fourth dose at 18 months.

These uses, methods and medicaments are preferably for immunisation against the pathogens stated within the present disclosure. Methods for checking the efficacy of the separate antigens are known in the art.

The vaccine may also be used to complete the primary immunization schedule of a different vaccine. Preferred sites for injection are the anterolateral thigh or the deltoid muscle of the upper arm. Vaccines of the invention may be administered at substantially the same time as an oral polio vaccine, such as a trivalent oral polio vaccine e.g. containing Type 1 poliovirus, Type 2 poliovirus and Type 3 poliovirus. A child receiving the vaccine of the invention for the first time may have previously received oral polio vaccine and/or Bacillus Calmette-Guerin (BCG) vaccine.

Thus preferred patient groups for immunisation include, but are not limited to: (a) children who have previously received oral polio vaccine; (b) children who have previously received BCG vaccine; (c) children who have previously received both oral polio and BCG vaccine; ( d) children in group (a), (b) or (c) who have not previously received any of D, T, wP/aP, HBsAg, Hib conjugates and at least one meningococcal conjugate; and (e) children who have previously received oral polio vaccine, BCG, D, T, wP/aP, HBsAg, Hib conjugate and at least one meningococcal conjugate. These children may be in any of the age groups specified above e.g. 0-36, 0-24, 0-12 or 0-6 months.

The method of administration may raise a booster response. The subject in which disease is prevented may not be the same as the subject that receives the conjugate of the invention. For instance, a conjugate may be administered to a female (before or during pregnancy) in order to protect offspring (so-called‘maternal immunisation’).

The invention provides the use of a Haemophilus influenzae type b capsular saccharide conjugated to an OMV in the manufacture of a medicament for immunising a patient, wherein the medicament is lyophilised and is administered after reconstitution by an aqueous vaccine comprising at least one further antigen.

The invention also provides the use of a Haemophilus influenzae type b capsular saccharide conjugated to an OMV in the manufacture of a medicament for immunising a patient, wherein the medicament is a fully liquid stable combination vaccine. The conjugated Hib polysaccharide is usually not stable in liquid vaccines, as discussed above. However, the unique physicochemical properties of the Hib-OMV conjugates conveniently allows for the addition of the Hib antigen to a fully liquid composition. In another embodiment of the invention, the immune response to the conjugate or composition or combination vaccine is enhanced with respect to the anti-Hib antibody response observed in the presence of an equivalent combination vaccine comprising the Haemophilus influenzae type b polysaccharide conjugated to TT as carrier protein.

Antigens with an associated antibody titre above which a host is considered to be seroconverted against the antigen are well known, and such titres are published by organisations such as WHO. Preferred conjugates of the invention can confer an antibody titre in a patient that is superior to that required for seroconversion. Preferably more than 80% of a statistically significant sample of subjects is seroconverted, more preferably more than 90%, still more preferably more than 93% and most preferably 96-100%.

Based on efficacy studies with unconjugated Haemophilus influenza type B PRP vaccine and data from passive antibody studies an anti-PRP level of 0.15 mg/mL has been accepted as a minimum protective level in the art. An anti-PRP level of 1 .0 mg//mL has been accepted in the art as predicting long-term (at least one year) protection. Anti-PRP antibodies may be measured by validated Enzyme Linked Immunosorbent Assays (ELISA) known in the art using reference serum from regulatory bodies such as the FDA. Particularly, administration of conjugates of the invention will induce anti-PRP Geometric Mean Concentrations (GMCs) of at least 0.15 mg//mL and more particularly of at least 1.0 mg//mL.

In another embodiment of the invention, the conjugate or composition in the fully liquid stable combination vaccine has a reduced or lower level of flocculation in comparison with an equivalent combination vaccine comprising Haemophilus influenzae type b polysaccharide conjugated to TT as carrier protein.

As stated above, the two main aluminium adjuvants for use in human vaccines are aluminum phosphate (AIP0₄) and aluminum hydroxide [AI(OH)3j. Vaccines containing either of these adjuvants are suspensions possessing a tendency for phase separation. Two main factors influence the properties of aluminum adjuvant suspensions: (1 ) the particle size and (2) the electrical charge of the dispersed particles.

As stated above, zeta potential is a measure of the charge on the surface of the aluminum particles. A high zeta potential indicates that the particles are fully dispersed in the medium due to the dominant repulsive forces that exist between the particles, and the suspension is classified as being in a deflocculated state. The deflocculated particles exist in suspension as separate entities and the sedimentation rate is often slow because of the wide distribution of particle size; the sediment formed in this manner is often closely packed and can be difficult to redisperse. The same particles tend to come together more closely if the zeta potential is lower; they form loose aggregates, or floes, due to the lowering of repulsive forces, and the system is said to be flocculated. The sediment formed in this manner is held loosely and typically allows for resuspension to occur easily.

In yet another embodiment of the invention, the conjugated derivative or composition in the fully liquid stable combination vaccine decreases the levels of desorption of the DT, TT, aP, HepB and IPV antigens observed in the presence of an equivalent combination vaccine comprising Haemophilus influenzae type b polysaccharide conjugated to TT as carrier protein.

As stated above, the antigenicity of Hib in vaccines may be stabilised by adsorbing it and other antigen components onto an aluminium-based adjuvant with a zero point charge of less than 7.2, for instance aluminium phosphate or aluminium hydroxide to which anions have been added. Insoluble aluminum salts, e.g., aluminum oxyhydroxide and aluminum hydroxyphosphate, have been widely used as human vaccine adjuvants for decades, and many currently licensed and commercially available vaccines, including those for diphtheria- tetanus-pertussis, hepatitis A and B, pneumococcal disease, anthrax, and rabies, contain aluminum salts as adjuvants.

When aluminium phosphate is used the approach can result in the increase in desorption of other antigens in the combination vaccine that work better when adsorbed onto aluminium hydroxide adjuvant, therefore resulting in a decrease in vaccine immunogenicity. Interestingly, the experimental results presented herein prove that all the antigens present in the composition are fully adsorbed in the presence of the Hib-OMV conjugates.

In another embodiment of the invention, the conjugate or composition in the fully liquid stable combination provides an enhanced or improved immune response against Bordetella pertussis, for example enhanced or improved T cell response, compared with that obtained with an equivalent combination vaccine comprising Haemophilus influenzae type b polysaccharide conjugated to TT as carrier protein.

Specific aspects of the invention

Further aspects of the disclosure are defined by the following clauses:

Embodiment 1 : An immunogenic conjugate comprising an outer membrane vesicle (OMV) and a capsular polysaccharide of Haemophilus influenza type b (Hib) wherein the OMV is obtained by detergent extraction from Bordetella pertussis and wherein the capsular polysaccharide has a MW between from about 55 kDa to about 65 kDa, for example about 63 kDa. Embodiment 2: An immunogenic conjugate comprising an outer membrane vesicle (OMV) and a capsular polysaccharide of Haemophilus influenza type b (Hib) wherein the OMV is obtained by detergent extraction from Bordetella pertussis and wherein the capsular polysaccharide has a MW between from about 25 kDa to about 35 kDa, for example about 28 kDa.

Embodiment 3: An immunogenic conjugate comprising an outer membrane vesicle (OMV) and a capsular polysaccharide of Haemophilus influenza type b (Hib) wherein the OMV is obtained by detergent extraction from Bordetella pertussis and wherein the capsular polysaccharide has a MW between from about 5 kDa to about 15 kDa, for example about 9 kDa.

Embodiment 4: An immunogenic conjugate comprising an outer membrane vesicle (OMV) and a capsular polysaccharide of Haemophilus influenza type b (Hib) wherein the OMV is obtained by detergent extraction from Bordetella pertussis and wherein the capsular polysaccharide has a MW between from about 55 kDa to about 65 kDa, for example about 63 kDa, and wherein the conjugate is obtained after reductive amination in the presence of sodium cyanoborohydride for 72 hours.

Embodiment 5: An immunogenic conjugate comprising an outer membrane vesicle (OMV) and a capsular polysaccharide of Haemophilus influenza type b (Hib) wherein the OMV is obtained by detergent extraction from Bordetella pertussis and wherein the capsular polysaccharide has a MW between from about 25 kDa to about 35 kDa, for example about 28 kDa, and wherein the conjugate is obtained after reductive amination in the presence of sodium cyanoborohydride for 72 hours.

Embodiment 6: An immunogenic conjugate comprising an outer membrane vesicle (OMV) and a capsular polysaccharide of Haemophilus influenza type b (Hib) wherein the OMV is obtained by detergent extraction from Bordetella pertussis and wherein the capsular polysaccharide has a MW between from about 5k Da to about 15 kDa, for example about 9 kDa, and wherein the conjugate is obtained after reductive amination in the presence of sodium cyanoborohydride for 72 hours.

Embodiment 7: A fully liquid stable immunogenic composition wherein one dose (0.5 ml) comprises (1 ) DT or CRM197, (2) Tetanus toxoid, (3) Hepatitis B surface antigen, (4) aP antigens comprising (4i) Pertussis toxoid, (4ii) Filamentous Haemagglutinin, (4iii) Pertactin, (5) Inactivated Poliovirus (IPV) comprising (5i) type 1 , (5ii) type 2, (5iii) type 3 and (6) a conjugate according to any one of Embodiments 1 to 6.

Embodiment 8: A fully liquid stable immunogenic composition wherein one dose (0.5 ml) comprises (1 ) DT or CRM197, (2) TT, (3) Hepatitis B surface antigen, (4) aP antigens comprising (4i) genetically detoxified Pertussis toxoid, (4ii) Filamentous Haemagglutinin, (4 i i i ) Pertactin, (5) IPV comprising (5i) type 1 , (5ii) type 2, (5iii) type 3 and (6) a conjugate according to any one of Embodiments 1 to 6.

Embodiment 9: A fully liquid stable immunogenic composition wherein one dose (0.5 ml) comprises (1 ) DT (not less than 30 IU), (2) TT (not less than 40 IU), (3) Hepatitis B surface antigen (about 10 micrograms), (4) aP antigens comprising (4i) Pertussis toxoid (about 25 micrograms), (4ii) Filamentous Haemagglutinin (25 micrograms), (4iii) Pertactin (8 micrograms), (5) IPV comprising (5i) type 1 (40 D-antigen units), (5ii) type 2 (8 D-antigen units), (5iii) type 3 (32 D-antigen units) and (6) a conjugate according to any one of Embodiments 1 to 6.

Embodiment 10: A fully liquid stable immunogenic composition wherein one dose (0.5 ml) comprises (1 ) DT (not less than 30 IU), (2) TT (not less than 40 IU), (3) Hepatitis B surface antigen (about 10 micrograms), (4) aP antigens comprising (4i) Pertussis toxoid (about 25 micrograms), (4ii) Filamentous Haemagglutinin (25 micrograms), (4iii) Pertactin (8 micrograms), (5) IPV comprising (5i) type 1 (8 D-antigen units), (5ii) type 2 (1 .6 D-antigen units), (5iii) type 3 (6.4 D-antigen units) and (6) a conjugate according to any one of Embodiments 1 to 6.

Embodiment 11 : A kit comprising a fully liquid component and a lyophilised component, wherein the fully liquid component comprises (1 ) DT or CRM197, (2) TT, (3) Hepatitis B surface antigen, (4) aP antigens comprising (4i) Pertussis toxoid, (4ii) Filamentous Haemagglutinin, (4 i i i ) Pertactin, (5) IPV comprising (5i) type 1 , (5ii) type 2, (5iii) type 3 and wherein the lyophilised component comprises (6) a conjugate according to any one of Embodiments 1 to 6.

Embodiment 12: A kit comprising a fully liquid component and a lyophilised component, wherein the fully liquid component comprises (1 ) DT (not less than 30 IU), (2) TT (not less than 40 IU), (3) Hepatitis B surface antigen (about 10 micrograms), (4) aP antigens comprising (4i) Pertussis toxoid (about 25 micrograms), (4ii) Filamentous Haemagglutinin (25 micrograms), (4 i i i ) Pertactin (8 micrograms), (5) IPV comprising (5i) type 1 (40 D-antigen units), (5ii) type 2 (8 D-antigen units), (5iii) type 3 (32 D-antigen units) and wherein the lyophilised component comprises (6) a conjugate according to any one of Embodiments 1 to 6.

Embodiment 13: A kit comprising a fully liquid component and a lyophilised component, wherein the fully liquid component comprises (1 ) DT (not less than 30 IU), (2) TT (not less than 40 IU), (3) Hepatitis B surface antigen (about 10 micrograms), (4) aP antigens comprising (4i) Pertussis toxoid (about 25 micrograms), (4ii) Filamentous Haemagglutinin (25 micrograms), (4 i i i ) Pertactin (8 micrograms), (5) IPV comprising (5i) type 1 (8 D-antigen units), (5ii) type 2 (1.6 D-antigen units), (5iii) type 3 (6.4 D-antigen units) and wherein the lyophilised component comprises (6) a conjugate according to any one of Embodiments 1 to 6.

Embodiment 14: A kit comprising a first fully liquid component and a second fully liquid component, wherein the first fully liquid component comprises (1 ) DT or CRM197, (2) TT, (3) Hepatitis B surface antigen, (4) aP antigens comprising (4i) Pertussis toxoid, (4ii) Filamentous Haemagglutinin, (4 i i i ) Pertactin, (5) IPV comprising (5i) type 1 , (5ii) type 2, (5iii) type 3 and wherein the second fully liquid component comprises (6) a conjugate according to any one of Embodiments 1 to 6.

Embodiment 15: A kit comprising a first fully liquid component and a second fully liquid component, wherein the first fully liquid component comprises (1 ) DT (not less than 30 IU), (2) TT (not less than 40 IU), (3) Hepatitis B surface antigen (about 10 micrograms), (4) aP antigens comprising (4i) Pertussis toxoid (about 25 micrograms), (4ii) Filamentous Haemagglutinin (25 micrograms), (4iii) Pertactin (8 micrograms), (5) IPV comprising (5i) type 1 (40 D-antigen units), (5ii) type 2 (8 D-antigen units), (5iii) type 3 (32 D-antigen units) and wherein the second fully liquid component comprises (6) a conjugate according to any one of Embodiments 1 to 6.

Embodiment 16: A kit comprising a fully liquid component and a lyophilised component, wherein the fully liquid component comprises (1 ) DT (not less than 30 IU), (2) TT (not less than 40 IU), (3) Hepatitis B surface antigen (about 10 micrograms), (4) aP antigens comprising (4i) Pertussis toxoid (about 25 micrograms), (4ii) Filamentous Haemagglutinin (25 micrograms), (4iii) Pertactin (8 micrograms), (5) IPV comprising (5i) type 1 (8 D-antigen units), (5ii) type 2 (1 .6 D-antigen units), (5iii) type 3 (6.4 D-antigen units) and wherein the second fully liquid component comprises (6) a conjugate according to any one of Embodiments 1 to 6.

Definitions

To facilitate an understanding of the present invention, a number of terms and phrases are defined below. Art-recognized synonyms or alternatives of the following terms and phrases (including past, present, etc. tenses), even if not specifically described, are contemplated.

As used in the present disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise; i.e., "a" means "one or more" unless indicated otherwise.

The terms“about” or“approximately” mean roughly, around, or in the regions of. The terms “about” or“approximately” further mean within an acceptable contextual error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. the limitations of the measurement system or the degree of precision required for a particular purpose, e.g. the amount of a nutrient within a feeding formulation. When the terms "about" or "approximately" are used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. For example, "between 0.2 and 5.0 mg/ml" means the boundaries of the numerical range extend below 0.2 and above 5.0 so that the particular value in question achieves the same functional result as within the range. For example, “about” and "approximately" can mean within 1 or more than 1 standard deviation as per the practice in the art. Alternatively,“about” and "approximately" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1 % of a given value.

The term "and/or" as used in a phrase such as "A and/or B" is intended to include "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless specified otherwise, all of the designations "A%-B%," "A-B%," "A% to B%," "A to B%," "A%-B," "A% to B" are given their ordinary and customary meaning. In some embodiments, these designations are synonyms.

The terms “substantially” or "substantial" mean that the condition described or claimed functions in all important aspects as the standard described. Thus, "substantially free" is meant to encompass conditions that function in all important aspects as free conditions, even if the numerical values indicate the presence of some impurities or substances. "Substantial" generally means a value greater than 90%, preferably greater than 95%, most preferably greater than 99%. Where particular values are used in the specification and in the claims, unless otherwise stated, the term“substantially” means with an acceptable error range for the particular value.

The invention will be now described by the following experiment part, without posing any limitation to its scope.

EXPERIMENTAL PART

Purification and characterization of B. pertussis DOC 0.1%-extracted dOMV B. pertussis Tohama I PTg strain (B2696), containing genetically detoxified PT (R9K/E129G), was pre-cultured in shake flasks and batch-fermented in a 20L fermenter using commercial medium (FCO001AA). After fermentation, the cell pellets were harvested by centrifugation of the entire cell broth and stored at -20°C.

Frozen pellets were homogenized in 20mM TrisHCI 2mM EDTA pH 8.6 complemented with 0.1 % DOC (final concentration), diluted with DPBS and filtered through a 0,22 pm membrane. The cell lysate was then concentrated by tangential flow filtration (TFF) using a 500 kDa MWCO membrane (Millipore Pellicon 2 mini). The filtrate was discarded and the retentate was diafiltered against DPBS 5 mM EDTA and further concentrated.

The retentate was then ultracentrifuged at 150.000 g for 2h. The supernatant, containing mostly free proteins, was quantified for the concentration of proteins and lipids and then set apart for further characterization. The pellet, containing the dOMV, was washed with DPBS 5 mM EDTA, ultracentrifuged in the same conditions and resuspended in DPBS 5 mM EDTA.

Purified dOMV was quantified based on (i) the amount of proteins, as measured by a modified Lowry assay and (ii) the concentration of lipids, as measured using FM-64 fluorescent dye (Table 1 ). The purified vesicles had a Z-average particle size (radius) of 54,15 ± 0,26 nm and a polydispersity Index (Pdl) of 0.23, as determined by DLS. The hydrodynamic radius was 40.07 ± 3.33 nm, as determined by SEC/QELS. The purified dOMV were over 85% pure, as determined using SEC/QELS peak areaS at 280 nm. Purified dOMV was stored at -20°C until further use.

Table 1. Protein and lipids concentrations and recovery in dOMV purification steps

Concentration Total (mg) Recovery (%)

(mg/ml)

Proteins Lipids Proteins Lipids Proteins Lipids vo^lume

(ml)

Step I (TFF) 8^3 Ϊ40 830 Ϊ00 Ϊ00 Ϊ00

Supernatant 0,8 1 ,0 82 100 59 12 100

Step II (Final) 2,5 24,5 75 735 54 88 30

Generation of Hib polysaccharide fragments

Native Hib capsular polysaccharide (PRP) was weighted and dissolved in NaPi 10mM pH 7.2 at a final concentration of 10 mg/ml; PRP was reacted with sodium metaperiodate for 30 min under the dark using different molar ratios Hib repeating unit to periodate in order to obtain Hib oligosaccharide (OS) populations of different lengths. Molar ratios Hib:periodate of 1 :0.035, 1 :0.08 and 1 :0.2 were used to generate activated polysaccharides of an apparent molecular size of 62.9, 28.2 and 9.3 kDa, respectively. After the prescribed incubation time, the polysaccharides were purified using a Sephadex G-15 column equilibrated with 0.2 M NaCI and desalted using the same column with H₂0 as running buffer. Hib OS were stored at -20°C.

The concentration of the resulting purified Hib OS was determined by orcinol assay using ribose as a reference; the aldehyde content of Hib OS was determined by BCA assay using glucose as a reference. The average molecular weight of the Hib polysaccharide fragments was determined by SEC-HPLC using a calibration curve of pullulan standards and a profiling of the three products was performed using HPAEC-PAD analysis. The different average distribution among the three OS populations was confirmed by HPAEC-PAD profiling using DP 2, 3 and 4 as standards. The structural identity of the generated OS was ascertained by 1 H

NMR and 31 P NMR and the level of derivatization of Hib OS in terms of content of aldehyde groups was estimated by micro BCA and expressed as degree of activation (DOA; average number of saccharide repeating units per aldehyde).

As shown in Table 2, DOAs of 46.9, 23.2 and 10.3 were obtained for the long, medium and short OS, respectively. This data was corroborated by assessing in the 31 P NMR spectrum the intensity ratios of the signals related to the internal phosphodiesters compared to that of the oxidized residue.

Table 2. Characteristics of the different lengths of Hib OS

Saccharide Aldehydes DOA

Saccharide MW kDa

Sample (mg/ml) (nmol/ml)

(Degree of

(nmol/ml) (SEC-HPLC)

(orcinol assay) (micro-BCA) activation)

Hib LONG 2,425 7030,4 149,9 46,9 62,9

Hib MEDIUM 2,930 8491 ,7 365,6 23,2 28,2

Hib SHORT 3,608 10458,5 965,0 10,8 9,3

Conjugation of purified B. pertussis dOMV to Hib polysaccharide fragments

Aldehyde-containing Hib OS was lyophilized and dissolved in the purified dOMV solution, together with sodium cyanoborohydride (Hib:dOMV:NaBH3CN 5:1 :2 (w/w/w)). The reaction mixture was incubated at 37°C for 72 h. Conjugated OS were purified by tangential flow filtration using a 300 kDa MWCO membrane for the longest Hib OS conjugates and a 100kDa MWCO membrane for the small and medium length. The Hib-dOMV conjugates were concentrated using a 30 kDa MWCO Amicon filter, recovered in PBS 1x and stored at 2-8°C. Purified conjugates were analyzed by Lowry assay for protein content and HPAEC-PAD assay for total and free saccharide content. Characteristics of the final conjugates are described in Table 3. The conjugates were further characterized by SDS-Page, Western Blot, SEC-HPLC and DLS analysis (data not shown).

The saccharide/dOMV was in the range of 0.21-0.33 (w/w) for all the conjugates, with a content of unbound saccharide always inferior to 6%. The DLS profiles for the three conjugates were comparable, with a higher radius and polidispersity found for the three conjugated lengths compared to the naked dOMV (data not shown).

Table 3. Characteristics of the Hib PRP-dOMV conjugates.

Hib free

Conjugate Hib saccharide saccharide dOMV Pertussis Saccharide/dOMV

LAL (EU/_Mg) (Mg/mL) (»/„) ( g/mL) (w/w)

OMV - - 2700,0 - 1 1779

Hib LONG-dOMV 73,9 < 4,4 265,0 0,28 16995

Hib MEDIUM-dOMV 69,4 < 4,5 260,0 0,27 22717

Hib SHORT-dOMV 66,1 < 5,8 314,6 0,21 13517

Flocculation assays

Hib polysaccharides of different length were conjugated to dOMV of Bordetella pertussis.

500 pL of Infanrix Penta was mixed with a corresponding volume of 10 pg of Hib: 41.6; 86.2 and 162.6pL for long, medium and short Hib-dOMV conjugates lots, respectively; 11 pL of Hib- TT was added to 500pL of Infanrix Penta as a reference for comparison. Infanrix Penta is a commercially available vaccine which contains DT, TT, three purified antigens of Bordetella pertussis [pertussis toxoid (PT), pertussis filamentous haemagglutinin (FHA) and pertactin (PRN)j and the purified major surface antigen (HBsAg) of the hepatitis B virus (HBV), adsorbed on aluminium salts. It also contains three types of IPV viruses (type 1 : Mahoney strain; type 2: MEF-1 strain; type 3: Saukett strain).

The mixed samples were observed within 15 minutes with an Olympus 1X51 optical microscope.

A representative set of results obtained with the Hib-short dOMV conjugate is shown in Figure 1. The images obtained with the Infanrix Penta reference show a smooth suspension with small particles of alum, while those obtained with the Hib-TT conjugate show large aggregates of Hib-TT. The incompatibility of the Hib-TT conjugate with Infanrix Penta has been previously observed as significant flocculation when the conjugate is put in contact with the aluminium hydroxide, due to the formation of multiple salt bridges between the Hib polysaccharide and the alum particles. This interaction results in a decrease of the immune response generated by the saccharide, known as“interference”. Surprisingly, and in contrast to the results obtained with Hib-TT, no aggregates were seen when using Hib-dOMV conjugate, suggesting a protective role of the dOMV against the alum particles. Similar results were obtained with the Hib-medium dOMV and the Hib-long dOMV conjugates (data not shown). The experimental results presented in the present application prove that the use of the conjugate or immunogenic composition of the invention decreases the levels of flocculation observed in an equivalent vaccine composition in which the Hib antigen is coupled to a carrier protein, for example TT.

Adsorption assays

The impact of the Hib-dOMV conjugates on the Infanrix Penta antigen adsorption was also evaluated by SDS-PAGE, using the same mixtures described in the flocculation assays. No protein bands were detected in the supernatant of the Infanrix Penta/Hib-dOMV samples, indicating that the antigens remained adsorbed to the alum of the formulation when the Hib- dOMV were added. The different Hib-dOMV conjugates were also tested on their own, showing standard OMV protein patterns (Figure 2).

Evaluation of the immunogenicity of Hib-dOMV conjugates

Infant rats (lco:OFA-SD, n = 8/group, mixed female and male) were immunized intramuscularly on days 0, 14 and 28 with (i) 50 pi of short, medium or long Hib-dOMV (0.5 pg, 1.0 pg or 2 pg in 10 mM PBS NaN3 0.06% (w/w)) and (ii) 50 pi Infanrix Penta (1/10^th human dose) in separate site administrations. Unconjugated, long Hib polysaccharide and 50 pi of Hiberix (a commercially available vaccine containing purified polyribosyl-ribitol-phosphate capsular polysaccharide (PRP) of Haemophilus influenzae type b covalently bound to TT) were used as controls.

Serum from all rats was individually collected seven days after the second and third immunization and tested for the presence of Haemophilus influenzae type b polyribosyl- ribitol- phosphate (PRP) specific IgG antibodies. For logistic reasons, males and females were bled at 6 and 7 days post dose II, respectively. 96-well plates were coated with tyraminated PRP (1 pg/ml) in a carbonate-bicarbonate buffer (50mM) and incubated overnight at 4°C. Rat serum samples were diluted in PBS- Tween 0.05% and a goat anti-Rat IgG (H+L) polyclonal antibody coupled to a peroxidase was added. The colorimetric reaction was observed after the addition of the peroxidase substrate (OPDA) and stopped with HCL (1 M) before reading by spectrophotometry (wavelengths 490-620 nm). For each serum tested and standard added on each plate, a 4-parameter logistic curve was fit to the relationship between the OD and the dilution (Softmaxpro).

Hib-dOMV candidates were immunogenic at all tested concentrations (Figure 3). GMT values induced by HIBERIX were within the range of historical data with 100% positivity whereas infant rats immunized with the unconjugated polysaccharide did not show any antibody response, supporting the validity of the results.

Bactericidal dilution titers against B. pertussis were measured by a serum bactericidal test. Higher SBA titers against B. pertussis were observed in all groups that received Hib-dOMV conjugates co-administered with Infanrix Penta as compared to Hiberix or unconjugated Hib co-administered with Infanrix Penta (Figure 4).

Evaluation of immunogenicity of Hib-dOMV conjugates in adult rat model

Six groups of 10 and one group of 20 six-week old adult rats (OFA) were immunized intramuscularly with each vaccine formulation on days 0, 14 and 28 as indicated below:

A partial bleed was performed on day 21 (7PII) and final bleed on day 35 (7PIII) and anti-Hib (anti-PRR’P) IgG was measured. The immunogenicity of the Hib-dOMV conjugates was confirmed in the adult rat model (Figure 5).

Higher SBA titers against B. pertussis were observed in all groups that received Hib-dOMV conjugates in co-adminstration with Infanrix Penta as compared to Hiberix co-administered with Infanrix Penta (Figure 6). REFERENCES

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Claims

1. An immunogenic conjugate comprising an outer membrane vesicle (OMV) conjugated to a capsular polysaccharide of Haemophilus influenza type b (Hib) and wherein the OMV is obtained from Bordetella.

2. The conjugate according to Claim 1 , wherein the OMV is obtained from Bordetella pertussis.

3. The conjugate according to Claim 1 or 2, wherein the OMV is a native OMV (nOMV).

4. The conjugate according to Claim 1 or 2, wherein the OMV is a non-native OMV.

5. The conjugate according to any one of the preceding claims, wherein the capsular polysaccharide has a molecular weight between from about 5 to about 100 KDa.

6. An immunogenic composition comprising the conjugate according to any one of Claims 1 to 5 and at least one pharmaceutically acceptable carrier or excipient.

7. The immunogenic composition according to claim 6 further comprising an antigen selected from the group consisting of Diphtheria Toxoid (DT), Tetanus Toxoid (TT), an acellular pertussis antigen (aP), Hepatitis B (HepB) antigen and Inactivated Poliovirus (IPV).

8. The immunogenic composition of claim 7 wherein the DT is wild type DT or CRM197; wherein the aP antigen comprises chemically or genetically detoxified pertussis toxin, filamentous haemagglutinin (FHA) and/or 69 kDa outer-membrane protein (also known as pertactin; the HepB antigen is HBsAg; and the IPV component comprises antigens from poliovirus strains Mahoney (type 1 ), MEF-1 (type 2) and Saukett (type 3).

9. The immunogenic composition of any one of Claims 6 to 8 which is a fully liquid stable composition.

10. The immunogenic composition of Claim 9 which is a vaccine.

11. A vaccine according to Claim 9, which comprises: DT (between from about 20 IU to about 100 IU); TT (between from about 40 to about 90 IU); PT (between from about 1 to about 50 micrograms), FHA (between from about 1 to about 50 , Pertactin (between from about 1 to 10 micrograms); HbSAg (between from about 1 to about 50 micrograms); and IPV type 1 (Mahoney strain) (between from about 10 to about 70 D- antigen unit); type 2 (MEF-1 strain) (between from about 10 to about 15 D-antigen unit) and type 3 (Saukett strain) (between from about 5 to about 60 D-antigen unit).

12. A vaccine according to Claim 10, which comprises: DT (not less than 30 IU); TT (not less than 40 IU); PT (25 micrograms), FHA (25 micrograms), Pertactin (8 micrograms); HbSAg (10 micrograms); and IPV type 1 (Mahoney strain) (40 D-antigen unit); type 2 (MEF-1 strain) (8 D-antigen unit) and type 3 (Saukett strain) (32 D-antigen unit).

13. A vaccine according to any one of claims 10 to 12, wherein administration of the capsular polysaccharide of Haemophilus influenza type b generates a Hib antibody response, preferably resulting in an anti-PRP antibody concentration of > 0.15 pg/ml.

14. The conjugate of any one of Claims 1 to 5, the immunogenic composition of any one of Claims 6 to 9 or the vaccine of Claims 10 to 13 for use in inducing an immune response in a vertebrate, preferably a mammal.

15. The conjugate of any one of Claims 1 to 5, the immunogenic composition of any one of Claims 6 to 9 or the vaccine of Claims 10 to 13 for use in prophylaxis.