WO2007026247A1

WO2007026247A1 - Vaccines containing corynebacterium diphtheriae pili

Info

Publication number: WO2007026247A1
Application number: PCT/IB2006/002778
Authority: WO
Inventors: John Telford
Original assignee: Novartis Vaccines And Diagnostics Srl
Priority date: 2005-08-31
Filing date: 2006-08-31
Publication date: 2007-03-08
Also published as: GB0517719D0

Abstract

Diphtheria toxin may not be the sole antigenic component that gives rise to protective immunity in diphtheria vaccines. C.diphtheriae pilus proteins have been found within diphtheria vaccines. These pilus proteins may contribute towards the ability of a vaccine to protect against C.diphtheriae infection, and so it may be desirable to enrich their presence in the vaccines. Conversely, their presence means that diphtheria vaccines are not homogenous, and so it may be desirable for quality control purposes to remove them from vaccines, enriching the toxin as the main immunogenic component while retaining protective efficacy.

Description

VACCINES CONTAINING CORYNEBACTERIUM DIPHTHERIAE PILI

All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

This invention is in the field of vaccines, and in particular vaccines for protecting against Corynebacterium diphtheriae infection for preventing diphtheria.

BACKGROUND ART

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 ('Dt') that is no longer toxic but that remains antigenic and is able to stimulate the production of specific anti-toxin antibodies after injection. Diphtheria toxoids are disclosed in more detail in chapter 13 of reference 1, and they have formed the basis of diphtheria vaccines for many years, as they have been widely accepted as being protective antigens.

SUMMARY OF THE INVENTION

The invention is based on the surprising finding that Dt may not be the sole antigenic component that gives rise to protective immunity in diphtheria vaccines. The inventors have found that C. diphtheriae pilus proteins are present within diphtheria vaccines. As explained below, these pilus proteins may contribute towards the vaccines' ability to protect against C. diphtheriae infection, and so it may be desirable to enrich their presence in the vaccines. Conversely, their presence means that diphtheria vaccines are not homogenous, and so it may be desirable for quality control purposes to remove them from vaccines, enriching Dt as the main immunogenic component while retaining protective efficacy.

Pilin enrichment

In one aspect, therefore, the invention provides a process for preparing a proteinaceous composition, comprising the steps of: (a) preparing a first component comprising a diphtheria toxin or a diphtheria toxoid; (b) preparing a second component comprising a diphtheria pilin; and (c) mixing the first and second components to give the proteinaceous composition vaccine. The proteinaceous composition is suitable for using in the manufacture of a vaccine that protects against C.diphtheriae infection.

The invention also provides a process for preparing a composition, comprising a step in which a diphtheria pilin is added. The composition will typically further include a diphtheria toxin and/or a diphtheria toxoid.

The invention also provides a process for preparing a composition that includes a diphtheria toxin or a diphtheria toxoid, comprising a step in which a diphtheria pilin substantially is not removed.

The invention also provides a composition comprising: (a) a diphtheria toxin or a diphtheria toxoid; and (b) a diphtheria pilin. In one preferred composition, the weight ratio of (total toxin or toxoid) to (total pilin) is at least l:10^A, wherein the value of A is selected from 0, -1, -2, -3, -4, -5 or less. In another preferred composition, the pilin concentration is at least 0.01 ng/ml (e.g. >0.1, >1, >10, >100, >1000, >10000 ng/ml, etc.). In another preferred composition, the ratio of (total toxin or toxoid) to (total pilin) is at least 0.01 ng (e.g. >0.1, >1, >10, >100, >1000, >10000 ng, etc.) for every 100Lf of toxin/toxoid. Components (a) and (b) are preferably the only detectable proteins from C.diphtheriae.

The invention also provides the use of a C.diphtheriae pilin in the manufacture of a medicament for preventing C.diphtheriae infection.

The invention also provides, in a process for preparing a proteinaceous composition for using in the manufacture of a vaccine that protects against C.diphtheriae infection, the improvement consisting of increasing the pilin concentration.

In all cases, one or more C.diphtheriae pilins can be used (e.g. one or more of SpaA, SpaB and SpaC), and references to amounts of pilin refer to the total combined amount of all such pilins. hi general, at least SpaA will be used.

Pilin removal

In another aspect, the invention provides a process for preparing a composition that includes a diphtheria toxin or a diphtheria toxoid, comprising a step in which a diphtheria pilin is specifically removed.

The invention also provides a process for preparing a composition that includes a diphtheria toxin or a diphtheria toxoid, comprising a step in which a diphtheria pilin is removed substantially completely.

The invention also provides a composition comprising: (a) a diphtheria toxin or a diphtheria toxoid; and (b) a diphtheria pilin. In one preferred composition, the weight ratio of (total toxin or toxoid) to (total pilin) is at least 10^A:l, where the value of A is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more i.e. the composition includes less than lOμg of pilin for every lOOμg of toxin/toxoid. In another preferred composition, the pilin concentration is less than lμg/ml e.g. <100ng/ml, <10ng/ml, <lng/ml, <0.1ng/ml, <0.01ng/ml, <0.001ng/ml, etc.. In another preferred composition, the ratio of (total toxin or toxoid) to (total pilin) is at least 10^B Lf of toxin/toxoid for every lμg of pilin, where the value of B is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more. More preferably, component (a) is the only detectable protein from C.diphtheriae.

The invention also provides a composition comprising a diphtheria toxin or a diphtheria toxoid, wherein the composition is essentially free from C.diphtheriae pilin. The invention also provides the use of this composition in the manufacture of a medicament for preventing C.diphtheriae infection.

The invention also provides, in a process for preparing a proteinaceous composition for using in the manufacture of a vaccine that protects against C.diphtheriae infection, the improvement consisting of decreasing pilin concentration. A preferred improvement is substantially complete removal of pilin.

In all cases, one or more C.diphtheriae pilins can be removed (e.g. one or more of SpaA, SpaB and SpaC), and references to amounts of pilin refer to the total combined amount of all such pilins. hi general, at least SpaA will be removed. Pϊlin measurement

The invention also provides a method for analysing a vaccine that protects against C.diphtheriae infection (or for analysing a component used in the manufacture of such a vaccine), comprising the step of detecting the presence or absence of a C.diphtheriae pilin. The detection step may also provide quantitative information about the amount and/or concentration of the pilin in the vaccine or component.

Detection can be achieved by methods such as western blot, etc. Antibodies that specifically recognise the pilin can be obtained by immunising animals with purified pilin protein.

Pilins

The existence of pili in C.diphtheriae has been known for many years {e.g. reference 2 dates from 1976, and reference 3 dates from 1987). More recently, the pili have been studied in detail [4-6], and have been found to be composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Assembled pili can be released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope. All three pilin subunit proteins are synthesized as precursors carrying N-terminal signal peptides and C-terminal sorting signals. Some, but not all, of the six sortase genes encoded in the C.diphtheriae genome are required for precursor processing, pilus assembly or cell wall envelope attachment. Pilus assembly has been proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits.

The DNA and amino acid sequences of SpaA, SpaB and SpaC are known, and can be found in the sequence databases using the following Genlnfo Identifier ("gi") numbers:

Vaccines

Where the invention gives a composition comprising a diphtheria toxin, the toxin is preferably treated to give a toxoid prior to vaccine manufacture. The toxoiding treatment can be performed before, during or after purification of the toxin.

Diphtheria toxoids are preferably prepared by treating the toxin with formalin and/or formaldehyde. Toxoids may be further purified after inactivation e.g. by sterile filtration and/or dialysis.

The Dt in vaccines of the invention is preferably adsorbed onto an aluminium salt, and preferably onto an aluminium hydroxide adjuvant.

The amount of diphtheria toxoid in vaccines of the invention is typically at least 30 IU/dose. Preferably, the diphtheria toxoid component is substantially free from any mercurial preservatives.

Diphtheria toxoids can be used as the sole antigenic component in a monovalent vaccine against C.diphtheriae infection, or can be used as an antigenic component ('D') in a multivalent vaccine for protecting against infection by multiple pathogens. In addition to the 'D' component, vaccines of the invention may comprise one or more of:

• a tetanus toxoid ('T')

• a pertussis antigen ('P'), which may be either cellular ('Pw') or acellular ('Pa')

• a surface antigen from hepatitis B virus (HBV), known as 'HBsAg'

• a hepatitis A virus (HAV) antigen

• a conjugated Haemophilus influenzae type b capsular saccharide ('Hib')

• inactivated polio virus (IP V)

• a conjugated Neisseria meningitidis serogroup C capsular saccharide ('MenC')

• a conjugated Neisseria meningitidis serogroup A capsular saccharide ('MenA')

• a conjugated Neisseria meningitidis serogroup W135 capsular saccharide ('MenW135')

• a conjugated Neisseria meningitidis serogroup Y capsular saccharide ('MenY')

• a conjugated Streptococcus pneumoniae capsular saccharide

• a measles virus antigen

• a mumps virus antigen

• a rubella virus antigen

• a varicella zoster virus antigen

• an influenza virus antigen

More than one of these other antigens can be used. The following combinations of antigens are particularly preferred, where P is Pa or Pw:

• Bivalent vaccines: D-T.

• Trivalent vaccines: D-T-P ; D-T-HBV.

• Tetravalent vaccines: D-T-P-HBV; D-T-P-Hib ; D-T-P-IPV.

• Pentavalent vaccines: D-T-P-Hib-HBV; D-T-P-IPV-HBV ; D-T-P-IPV-Hib.

• Hexavalent vaccines: D-T-P-HBV-Hib-IPV.

• Heptavalent vaccines: D-T-P-HB V-Hib-MenA-MenC; D-T-P-HB V-Hib-IPV-MenC.

• Octavalent vaccines : D-T-P-HB V-Hib-IPV-MenC-Men Y.

In combinations including IPV, the P component is preferably a Pa component.

In addition to these antigens, further antigens may also be present e.g. to given an 8-valent, 9-valent, 10-valent, etc. vaccine.

The invention also provides a multivalent vaccine comprising the components described above, and including a diphtheria pilin. Where a tetanus toxoid is included, it may be adsorbed onto an aluminium hydroxide adjuvant, but this is not necessary (e.g. adsorption of between 0-10% of the total tetanus toxoid can be used). The amount of tetanus toxoid in vaccines of the invention is typically at least 60 IU/dose.

Where a cellular pertussis antigen ('Pw') is included, it may be adsorbed onto or mixed with an aluminium phosphate adjuvant. The amount of wP antigen in vaccines of the invention is typically at least 4 IU/dose.

Where an acellular pertussis antigen ('Pa') is included, it is preferred to use at least two of, and preferably all three of, pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (also known as the '69 kiloDalton outer membrane protein'). Fimbriae (e.g. agglutinogens 2 and 3) may also be used. FHA and pertactin may be treated with formaldehyde prior to use according to the invention. PT is preferably detoxified by treatment with formaldehyde and/or glutaraldehyde. As an alternative to this chemical detoxification procedure the PT may be a mutant PT in which enzymatic activity has been reduced by mutagenesis [7], but detoxification by chemical treatment is preferred. Quantities of acellular pertussis antigens are typically expressed in micrograms.

Where a HBsAg is included, it is preferably prepared by expression in a yeast, such as a Saccharomyces (such as S.cerevisiae) or a Hanensula (such as H.polymorpha). Yeast-expressed HBsAg will generally be non-glycosylated. The HBsAg will generally be in the form of substantially-spherical particles (average diameter of about 20nm), including a lipid matrix comprising phospholipids, including phosphatidylinositol. The HBsAg is preferably 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 [8]. Quantities of HBsAg are typically expressed in micrograms. A typical amount of HBsAg per vaccine dose is lOμg.

Where a conjugated Haemophilus influenzae type b capsular saccharide ('Hib') is included, the conjugate can have excess saccharide or excess protein (by weight), or can have equal weights of both. The carrier protein is preferably a tetanus toxoid or CRMl 97. The saccharide moiety of the conjugate may comprise full-length polyribosylribitol phosphate (PRP) as prepared from Hib bacteria, and/or it may comprise fragments of full-length PRP. Amounts of Hib conjugates are generally given in terms of mass of saccharide (i.e. the dose of the conjugate (carrier + saccharide) as a whole is higher than the stated dose) in order to avoid variation due to choice of carrier. A typical amount of Hib saccharide per vaccine dose is lOμg.

Where inactivated polio virus (IPV) is included, it is preferred to use viruses grown on a Vero cell line. Vero cells can conveniently be cultured microcarriers. Prior to administration to patients, polioviruses must be inactivated, and this can be achieved by treatment with formaldehyde. Poliomyelitis can be caused by one of three types of poliovirus. The three types are similar and cause identical symptoms, but they are antigenically very different and infection by one type does not protect against infection by others. It is therefore preferred to use three poliovirus antigens in the invention: poliovirus Type 1 (e.g. Mahoney strain), poliovirus Type 2 (e.g. MEF-I strain), and poliovirus Type 3 {e.g. Saukett strain). The viruses are preferably grown, purified and inactivated individually, and are then combined to give a bulk trivalent mixture for use with the invention. Quantities of IPV are typically expressed in the 'DU' unit (the "D-antigen unit" [9]).

Where a conjugated Neisseria meningitidis capsular saccharide is included, it can be from one or more of serogroups A, C, Wl 35 and Y. Common nomenclature refers to these four serogroups as 'MenA', 'MenC, 'MenW135' and 'MenY'. Preferred vaccines contain meningococcal conjugates for at least serogroup C, and preferably for both serogroup A and C. MenA-C, MenA-C-W135, MenA-C-Y, MenC-W135-Y and MenA-C-W135-Y combinations are preferred. Meningococcal saccharides are preferably conjugated to a H.influenzae protein D carrier, but they may also be conjugated to a tetanus toxoid carrier, a diphtheria toxoid carrier or a CRMl 97 carrier. A MenC saccharide can be prepared form an OAc+ or OAc- strain, with OAc+ strains being preferred {e.g. CI l). A MenA saccharide preferably has at least 50% of its mannosamine residues O-acetylated at the C-3 position. The saccharide moiety of the conjugate may comprise full-length saccharides as prepared from meningococci, and/or it may comprise fragments of full-length saccharides. Meningococcal conjugates with a saccharide :protein ratio (w/w) of between 1:10 {i.e. excess protein) and 10:1 {i.e. excess saccharide) may be used e.g. ratios between 1:5 and 5:1, between 1:2.5 and 2.5:1, or between 1:1.25 and 1.25:1. Concentrations of meningococcal conjugates are generally given in terms of mass of saccharide {i.e. the dose of the conjugate (carrier + saccharide) as a whole is higher than the stated dose) in order to avoid variation due to choice of carrier. A typical amount of each meningococcal saccharide per vaccine dose is about 5μg or about lOμg.

Where a conjugated Streptococcus pneumoniae capsular saccharide is included, it is preferred to include saccharides from more than one serotype of S. pneumoniae. Conjugate vaccines with polysaccharides from between 5 and 11 different serotypes [10] are known. For example, PrevNar™ [11] contains antigens from seven serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) with each saccharide individually conjugated to CRM₁₉₇ by reductive animation, with 2μg of each saccharide per 0.5ml dose (4μg of serotype 6B), and with conjugates adsorbed on an aluminium phosphate adjuvant. Compositions of the invention preferably include at least serotypes 6B, 14, 19F and 23F. Further serotypes are preferably selected from: 1, 3, 4, 5, 7F, 9V and 18C. 7-valent (as in PrevNar™), 9-valent, 10-valent and 11-valent coverage of pneumococcal serotypes are particularly useful.

Where both diphtheria toxoid and tetanus toxoid are included, various ratios may be used. For instance, some vaccines {e.g. pediatric vaccines) may have a Dt:Tt ratio (measured in Lf units) of between 1.5:1 and 4:1 e.g. between 2:1 and 3:1, such as about 2.5:1 or about 3:1. Other vaccines {e.g. for adolescents) may have a ratio of between 1:1.5 and 1:3 e.g. about 1:2 or about 1:2.5.

Vaccines of the invention can be prepared in liquid format {i.e. where all antigens are in aqueous solution or suspension) during manufacture, or they can be prepared extemporaneously at the time of use by mixing together two components: (a) a first component comprising a diphtheria component; and (b) a second component comprising further antigen(s). The two components are preferably in separate containers {e.g. vials and/or syringes), and the invention provides a kit comprising components (a) and (b). The contents of the first container are preferably aqueous, and the contents of the second container are preferably lyophilised, such that vaccines of the invention can be prepared by reconstituting the lyophilised component with the aqueous component. Typically, the second (lyophilised) component may contain conjugated bacterial saccharide e.g. in a vaccine containing one or more conjugated bacterial saccharides, these saccharides will be lyophilised in the second container whereas all other antigenic components will be in the first container.

Adjuvants

In addition to antigenic components, vaccines of the invention will typically include at least one aluminium salt adjuvant. As mentioned above, the vaccines 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, as neither is a precise description of the actual chemical compound which is present [e.g. see chapter 9 of reference 12]. 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 AlO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OETb, by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070cm^"1 and a strong shoulder at 3090-3100Cm^"1 [chapter 9 of ref. 12].

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 PO₄/AI molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AlPO₄ by the presence of hydroxyl groups. For example, an IR spectrum band at 3164cm^"1 (e.g. when heated to 200⁰C) indicates the presence of structural hydroxyls [ch. 9 of ref. 12].

The adjuvants can take any suitable form (e.g. gel, crystalline, amorphous, etc.).

The PO₄/A1³⁺ 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 Al^3-VmI. 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 0.5-20μm (e.g. about 5-10μm) after any antigen adsorption.

The 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 5.0 and 7.0, more preferably between 5.5 and 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 0 and 10 mg/ml. A preferred concentration is between 2 and 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 1.0 and 2OmM, preferably between 5 and 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 NaCl facilitates the correct measurement of pH prior to adsorption of antigens.

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.

Further components of the vaccine

As well as containing antigens and adjuvant(s), etc., 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 (NaCl) is preferred, which may be present in the final vaccine product at between 1 and 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 preferably contains a preservative. This is particularly important when the vaccine is packaged in multidose containers. Preferred preservatives for inclusion are 2-phenoxyethanol and thimerosal. It is preferred, however, not to use mercurial preservatives (e.g. thimerosal) during the process of the invention. However, the presence of trace amounts 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.

Where aluminium salts are present within the final vaccine, the total amount of aluminium, expressed in terms OfAl³⁺, 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).

During the process of the invention, 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 [13].

A vaccine is preferably sterile. A vaccine is preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. A vaccine is preferably gluten free.

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

Administration of the vaccine

The final vaccines of the invention are suitable for administration to humans, and in particular to children. A typical dosage schedule for the vaccine, or 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.

The vaccine may also be used to complete the primaiy immunization schedule of a different vaccine.

The invention provides a method for raising an immune response in a patient, comprising administering a composition of the invention to the patient.

The invention also provides a composition of the invention, for use in medicine. Various features of medicaments are given above. Preferred medicaments are vaccines.

Medicaments will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. intravenously, subcutaneously, intraperitoneally, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration. In general, however, they are administered by intramuscular injection. Preferred sites for injection are the anterolateral thigh or the deltoid muscle of the upper arm. The uses, methods and medicaments of the invention are preferably for immunisation against the pathogens stated above. The patient is preferably a human, and may be a child (e.g. a toddler or infant), a teenager or an adult, but will generally be a child. Preferred patients are aged between 0-36 months e.g. between 0-24 months, between 0-12 months, or between 0-6 months.

Methods for checking the efficacy of the separate antigens are known in the art.

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.

General

The term "comprising" can mean "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

The term "about" in relation to a numerical value x means, for example, x+10%.

Where bovine materials are used in the culture of C.diphtheriae, or other organisms used with the invention, they should be obtained from sources that are free from bovine spongiform encephalopathy (BSE) or from other transmissible spongiform encephalopathies (TSEs).

Quantities of diphtheria toxoid can be expressed in international units (IU). For example, the NIBSC supplies the 'Diphtheria Toxoid Adsorbed Third International Standard 1999' [14,15], 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 [16]. For example, the NIBSC supplies 'Diphtheria Toxoid, Plain' [17], which contains 300 LF per ampoule, and also supplies 'The 1st International Reference Reagent For Diphtheria Toxoid For Flocculation Test' [18] which contains 900 LF per ampoule.

Quantities of tetanus toxoid can be expressed in international units (IU). For example, the NIBSC supplies the 'Tetanus Toxoid Adsorbed Third International Standard 2000' [19,20], which contains 469 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 [16]. For example, the NIBSC supplies 'The 1st International Reference Reagent for Tetanus Toxoid For Flocculation Test' [21] which contains 1000 LF per ampoule.

Quantities of wP antigens can be expressed in international units (IU). For example, the NIBSC supplies the 'Third International Standard For Pertussis Vaccine' [22], which contains 46 IU per ampoule. Each ampoule contains the freeze-dried residue of 2.0 ml aliquots of an aqueous solution which contained 10 litres of bacterial suspension (equivalent to 180 opacity units in terms of the U.S. Opacity Standard) diluted with eight litres of M/ 15 Sorensen's buffer pH 7.0. As an alternative to the IU system, the 'OU' unit ("opacity units") is also used {e.g. 4 OU may be about 1 IU).

MODES FOR CARRYING OUT THE INVENTION

Patients were immunised with a vaccine containing a diphtheria toxoid. Sera from the patients were collected and used in a western blot against the vaccine. Unsurprisingly, the sera bound to diphtheria toxoid in the vaccine. Surprisingly, a second reactive band was seen, with high molecular weight. We conclude that the pilus of C. diphtheriae is present in the vaccine and is immunogenic in humans.

To confirm this finding, the SpaA, SpaB and SpaC genes from C. diphtheriae are cloned and expressed in a recombinant host. The pilin subunits are then purified and are used to immunise mice to produce polyclonal antibodies. These antibodies are used to probe diphtheria vaccines, with a binding interaction confirming the presence of pilin protein in the vaccines. The recombinant proteins are also incubated with hyperimmune serum from vaccinated patients in order to confirm an immunogenic response.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES (the contents of which are hereby incorporated by reference)

[I] Vaccines, (eds. Plotkin & Orenstein). 4th edition, 2004, ISBN: 0-7216-9688-0. [2] Yanagawa & Honda (1976) Infect Immun 13:1293-5.

[3] Ermolayev et al. (1987) JHyg Epidemiol Microbiol Immunol 31 :313-9.

[4] Ton-That & Schneewind (2003) MoI Microbiol. 50:1429-38.

[5] Ton-That & Schneewind (2004) Trends Microbiol. 12:228-34.

[6] Ton-That et al. (2004) MoI Microbiol. 53:251-61.

[7] Rappuoli et al. (1991) TIBTECH 9:232-238.

[8] US patent 6,013,264.

[9] Module 6 of WHO' s The immunological basis for immunization series (Robertson)

[10] Zielen et al. (2000) Infect. Immun. 68:1435-1440.

II 1] Darkes & Plosker (2002) Paediatr Drugs 4:609-630.

[12] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).

[13] WO2004/110480.

[14] Sesardic et al (2001) Biologicals 29:107-22.

[15] NIBSC code: 98/560.

[16] Module 1 of WHO's The immunological basis for immunization series (Galazka).

[17] NIBSC code: 69/017.

[18] NIBSC code: DIFT.

[19] Sesardic et al. (2002) Biologicals 30:49-68.

[20] NIBSC code: 98/552.

[21] NIBSC code: TEFT.

[22] NIBSC code: 66/303.

Claims

1. A process for preparing a proteinaceous composition, comprising the steps of: (a) preparing a first component comprising a diphtheria toxin or a diphtheria toxoid; (b) preparing a second component comprising a diphtheria pilin; and (c) mixing the first and second components to give the proteinaceous composition.

2. A process for preparing a composition that includes a diphtheria toxin and/or a diphtheria toxoid, comprising a step in which a diphtheria pilin is added.

3. A composition comprising: (a) a diphtheria toxin or a diphtheria toxoid; and (b) a diphtheria pilin, wherein: (i) the weight ratio of (total toxin or toxoid) to (total pilin) is at least 1:1; (ii) the pilin concentration is at least 0.01 ng/ml; and/or (iii) the ratio of (total toxin or toxoid) to (total pilin) is at least 0.01 ng for every 100Lf of toxin/toxoid.

4. The use of a C.diphtheriae pilin in the manufacture of a medicament for preventing C.diphtheriae infection.

5. A process for preparing a composition that includes a diphtheria toxin or a diphtheria toxoid, comprising a step in which a diphtheria pilin is specifically removed.

6. A composition comprising: (a) a diphtheria toxin or a diphtheria toxoid; and (b) a diphtheria pilin, wherein (i) the weight ratio of (total toxin or toxoid) to (total pilin) is at least 10:1; (ii) the pilin concentration is less than 1 μg/ml; and/or (iii) the ratio of (total toxin or toxoid) to (total pilin) is at least 10Lf of toxin/toxoid for every lμg of pilin.

7. A composition comprising a diphtheria toxin or a diphtheria toxoid, wherein the composition is essentially free from C.diphtheriae pilin.

8. A method for analysing a vaccine that protects against C.diphtheriae infection, comprising the step of detecting the presence or absence of a C.diphtheriae pilin.

9. An immunogenic composition comprising: (a) a C.diphtheriae pilin; and (b) one or more of the following antigens: a diphtheria toxoid ('D'); a tetanus toxoid ('T'); a pertussis antigen (T'), which may be either cellular ('Pw') or acellular ('Pa'); a surface antigen from hepatitis B virus, known as 'HBsAg'; a hepatitis A virus antigen; a conjugated Haemophilus influenzae type b capsular saccharide ('HuV); inactivated polio virus (IPV); a conjugated Neisseria meningitidis serogroup C capsular saccharide ('MenC'); a conjugated Neisseria meningitidis serogroup A capsular saccharide ('MenA'); a conjugated Neisseria meningitidis serogroup W135 capsular saccharide ('MenW135'); a conjugated Neisseria meningitidis serogroup Y capsular saccharide ('MenY'); and a conjugated Streptococcus pneumoniae capsular saccharide.

10. The composition of claim 9, wherein component (b) has five antigenic components: D, T, Pw, Hib, HBsAg.

11. The composition of claim 9, wherein component (b) has seven antigenic components: D, T, Pw₅ HBsAg, Hib, MenA, MenC.

12. The composition of claim 9, wherein component (b) has seven antigenic components: D₃ T, Pa, HBsAg, Hib, IPV, MenC.

13. The composition of claim 9, wherein component (b) has eight antigenic components: D₅ T, Pa, HBsAg, Hib, IPV, MenC, MenY.

14. The composition of any one of claims 9 to 13, wherein the diphtheria toxoid is adsorbed onto an aluminium hydroxide adjuvant.