EA045827B1 - IMMUNOGENIC COMPOSITIONS - Google Patents

Description

Field of invention

The invention is an invention in the field of combination vaccines, that is, vaccines containing a mixture of immunogens from more than one pathogen, such that administration of the vaccine allows simultaneous immunization of a subject against more than one pathogen. More specifically, the invention relates to booster vaccines for diphtheria, tetanus and pertussis.

Prior Art

Vaccines that contain antigens from more than one pathogen in a single dose are known as multivalent or combination vaccines. Combination vaccines allow patients to receive fewer injections, which may result in a clinical benefit in the form of greater compliance (eg, see Chapter 29 of Reference [1]). Various combination vaccines are approved for use in humans in the EU and US, including trivalent vaccines to protect against diphtheria, tetanus and pertussis. Such vaccines may be called DTaP and TdaP. Although DTaP and TdaP are both diphtheria-tetanus-pertussis combination vaccines, DTaP vaccines are used for primary immunization, while TdaP vaccines are used for subsequent booster vaccinations. Primary and booster vaccine compositions differ in dose. More specifically, in relation to booster vaccines, these vaccines usually contain lower doses of some antigenic components, for example, BOOSTRIX contains 10 times less diphtheria toxoid than INFANRIX. This is indicated by the lowercase d used to indicate a smaller amount of diphtheria toxoid.

It is also possible to change the ratio of antigenic components. For example, the ratio of diphtheria to tetanus toxoids is 2.5:1 in INFANRIX, but 1:2 in BOOSTRIX. Thus, the dose of diphtheria toxoid in these booster vaccines is significantly reduced, both in absolute quantities and relative to the content of tetanus toxoid.

However, in recent years there has been an increase in the number of cases of disease caused by Bordetella pertussis, even in countries with high vaccination coverage. Although the exact reasons for this increase are unclear, they may include weakened immunity and epidemiological changes in circulating strains.

It is therefore an object of the present invention to provide additional and improved combination vaccines for protection against Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis. It is also an object of the present invention to provide additional and improved TdaP vaccines suitable for use in humans as booster vaccines for adults, adolescents and children four years of age and older who have previously received immunizations intended for children.

Summary of the invention

The invention is based on studies of combination vaccines containing Bordetella pertussis outer membrane vesicles (OMVs). The inventors have found that these combination vaccines result in titers of specific antibodies to the respective antigens with little or no immunological interference between the different antigens. The presence of OMV Bordetella pertussis provides an improved humoral immune response against Bordetella and, surprisingly, also improves the humoral immune response against other antigens of the composition.

Thus, the first aspect provides an immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from Bordetella pertussis. In particular, OMVs are derived from a strain of Bordetella pertussis expressing genetically detoxified pertussis toxoid. More specifically, OMVs are derived from a Bordetella pertussis strain expressing the genetically detoxified pertussis toxoid PT-9K/129G.

Thus, according to the invention, there is provided an immunogenic composition containing (a) outer membrane vesicles (OMVs) of Bordetella pertussis, (b) acellular pertussis antigen, (c) tetanus toxoid and (d) diphtheria toxoid, where OMVs are derived from a strain of Bordetella pertussis, expressing genetically detoxified pertussis toxoid, in particular genetically detoxified pertussis toxoid PT-9K/129G.

The invention also provides an immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs) containing genetically detoxified pertussis toxoid, in particular PT-9K/129G, (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid.

The invention also provides an immunogenic composition containing (a) Bordetella pertussis outer membrane vesicles (OMVs) containing genetically detoxified pertussis toxoid, in particular PT-9K/129G, where lipid A in OMV has a modified structure without glucosamine (GlcN) substitutions at the distal phosphate groups of the central structure, (b) acellular pertussis antigen, (c) tetanus toxoid and (d) diphtheria toxoid.

Genetically detoxified pertussis toxoid PT-9K/129G contains two amino acid substitutions in the S1 subunit, specifically R9K and E129G (see, for example, EP096964). Thus,

- 1 045827 even more specifically, OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified to include the R9K and E129G mutations and expressing the genetically detoxified pertussis toxoid PT-9K/129G. More specifically, 100% of the pertussis toxoid in the outer membrane vesicles is genetically detoxified PT, specifically PT-9K/129G.

In some embodiments, the Bordetella pertussis OMVs used in the invention have a modified lipid A structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure. In some embodiments, the Bordetella pertussis strain from which the OMVs are derived contains an ArnT knockout, specifically a deletion of the gene encoding ArnT (AarnT). Thus, OMVs used in the invention and derived from such strains may have a modified lipid A structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure.

In particular, the OMVs used in the invention are not treated with formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide, or a combination thereof. More specifically, the OMVs used in the invention are not chemically detoxified by treatment with formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide, or a combination thereof.

Suitable acellular pertussis antigens include detoxified pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), fimbrial protein 2 (FIM2), fimbrial protein 3 (FIM3), and combinations thereof. In certain embodiments, the acellular pertussis antigen contains at least two, such as at least three, antigens selected from the group consisting of detoxified pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), fimbrial protein 2 (FIM2) , fimbrial protein 3 (FIM3). Specific combinations of acellular pertussis antigens for use in the invention include: (1) PT, FHA and PRN; (2) PT, FHA, PRN, FIM2 and FIM3; (3) RT and FHA; and (4) PT, FHA, FIM2 and FIM3.

In particular, the immunogenic composition is a vaccine. More specifically, the vaccine is intended for administration to humans. The vaccine may be intended for primary immunization. More specifically, the vaccine is intended for use as a booster vaccine, for example, in a secondary immunization. Even more specifically, diphtheria toxoid is present at a concentration of from about 4 Lf(flocculation units)/ml to about 8 Lf/ml. More specifically, diphtheria toxoid is present at a concentration of approximately 2 Lf per 0.5 mL dose, approximately 2.5 Lf per 0.5 mL dose, approximately 3 Lf per 0.5 mL dose, approximately 3.5 Lf per 0.5 mL dose ml or approximately 4 Lf per 0.5 ml dose. Tetanus toxoid may be present at a concentration of approximately 5 Lf per 0.5 ml dose.

Specifically, tetanus toxoid and diphtheria toxoid are present in a tetanus toxoid:diphtheria toxoid ratio of 1.5:1 to 2.5:1 (when measured in Lf units), such as approximately 2:1 (when measured in Lf units) .

The immunogenic composition may contain an adjuvant, in particular an aluminum salt-based adjuvant.

A second aspect of the invention provides an immunogenic composition for use in a method of inducing an immune response in a patient, comprising the step of administering to the patient an immunogenic composition of the present invention.

A third aspect of the invention provides a method for preparing an immunogenic composition of the present invention, comprising mixing a first component containing outer membrane vesicles (OMVs) and a second component containing acellular pertussis antigen, tetanus toxoid and diphtheria toxoid. In certain embodiments of the third aspect of the invention, the OMVs of the first component are lyophilized and the second component contains the antigens in aqueous form. Thus, the method may further include the step of reconstituting the lyophilized OMVs of the first component with aqueous antigens of the second component.

In a fourth aspect of the invention, there is provided a kit for preparing an immunogenic composition of the present invention, comprising a first component containing OMV and a second component containing acellular pertussis antigen, tetanus toxoid and diphtheria toxoid, where these two components are in separate containers. In certain embodiments of the fourth aspect of the invention, the OMVs of the first component are lyophilized and the second component contains the antigens in aqueous form.

Brief description of graphic materials

Fig. 1: Reactogenicity of W28 9K1129G AarnT vaccine strains compared to W28 9K1129G in vitro. Fig. 1(a) was obtained from a luciferase reporter gene assay for hTLR4 activation by W28 9K/129G (Bp WT) and W28 9K/129G arnTKO (Bp AarnT). Various concentrations of bacteria were used to stimulate HEK293 cells, and the fold induction is shown in comparison with PBS. Fig. 1(b): ELISA for the detection of IL-6 in supernatants of human peripheral blood mononuclear cells (PBMCs) after stimulation with various concentrations of bacteria.

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Fig. 2: OMVs derived from vaccine strains contain FHA, 69K and acellular pertussis antigens PT. Western blots of titrated standards of purified FHA, 69K and subunits of acellular pertussis antigen PT, added in the indicated quantities together with 1 μg of OMV from strains W28 RT 9K/129G (WT) and W28 RT 9K/129G arnTKO (AarnT), obtained by immunostaining with antisera vs. FHA, vs. 69K, and vs. PT.

Fig. 3: Immunization with OMV results in low levels of antibodies against aP antigen in mice. Mice were immunized intraperitoneally with 2.5 μg of OMVs from W28 RT 9K/129G (BP-OMV (WT)) and W28 RT 9K/129G arnTKO (BP-OMV (AArnt)) 3 times with an interval of 3 weeks. Although only anti-69K antibodies were detectable after the 1st immunization, low levels of all 3 antigens (FHA, 69K, and PT) were detectable after the second and third doses using the Luminex assay. Fig. 3(a): Anti-FHA IgG titers. Fig. 3(b): IgG titers against 69K. Fig. 3(c): IgG titers against PT.

Fig. 4: Immunization with TdaP in combination with OMV results in significantly higher levels of antibodies against most vaccine antigens compared to TdaP alone.

Mice were immunized intramuscularly with TdaP (1/5 the human dose) with or without combination with 2.5 mg OMV from the W28 RT 9K/129G arnTKO strain, and after two immunizations, sera were collected and analyzed to measure antibody levels to each from TdaP antigens using the Luminex assay. ***p less than 0.001, **p less than 0.01, *p less than 0.05.

Fig. 5: OMVs lead to the production of antibodies that inhibit B. pertussis adhesion to epithelial cells in vitro, similar to the wP vaccine. Sera obtained from 10 mice of each group immunized with OMV (from W28 9K/129G arnTKO), whole bacteria (W28 9K/129G arnTKO) TdaP vaccine or aluminum hydroxide as a control (Nil) were pooled and serially diluted in infection medium and incubated with wild-type labeled B. pertussis BP536 for 1 h. A549 cells were then infected with bacteria/sera mixtures for 1 h and, after extensive washes to remove unbound bacteria, cell-associated bacteria were quantitated by fluorescence readings at Ex/Em 485/535 nm. Results are presented as the mean plus/minus SD of one representative of three independent experiments, each performed in triplicate.

Fig. 6: OMVs result in a strong protective response, similar to the wP vaccine standard, to intracranial challenge with B. pertussis in the Kendrick test. Mice were immunized once intraperitoneally with whole-cell vaccine or OMV compositions and, 2 weeks after immunization, they were challenged with a suspension of B. pertussis strain 18323 administered intracranially. Survival of mice 2 weeks after infection is shown according to the Kendrick intracranial challenge efficacy test. Vaccine compositions include whole cell pertussis vaccine wP (standard) at a dose of 1/10, 1/50 and 1/250 of the human dose, and OMV from the W28 9K/129G strain (OMV) at the indicated doses.

Fig. 7: FHA-specific total IgG (Fig. 7(a)) and IgG2c (Fig. 7(b)) serum titers are proportional to OMV vaccine dose. FHA-specific antibodies present in the serum of immunized mice on the day of infection were analyzed by ELISA. ***p less than 0.001, **p less than 0.01, *p less than 0.05, ns - not significant. Fig. 7(c): Single immunization with OMV stimulates B. pertussis-specific Th1/Th17 responses. Spleen cells (2x10 ⁶ /ml) from vaccinated C57BL6 mice were cultured in the presence of sonicated B. pertussis (SBP, 5 μg/ml). After 72 hours, the concentrations of IFNy (Th1 indicator), IL-13 (Th2 indicator) and IL-17 (Th17 indicator) in the supernatants were analyzed by ELISA.***p less than 0.001.

Fig. 8: The rate of clearance of B. pertussis from the lungs is directly proportional to the dose of OMV vaccine. C57BL6 mice were immunized with PBS, wP, OMV 0.4, OMV 2, or OMV 10 3 weeks before infection. Mice were then infected with a virulent strain of B. pertussis (Bp338), and the number of bacteria in the lungs was assessed by counting colony-forming units (CFU) in serially diluted lung homogenates at the indicated time points. The broken line shows the detection limit. ***p less than 0.001, wP vs. OMV 0.4; ### p less than 0.001, # p less than 0.05, wP vs. OMV 10; ♦♦♦ p less than 0.θ01, ♦ p less than 0.05, wP versus OMV 2.

Fig. 9: aP formulated with OMV stimulates FHA-specific IgG2c in serum. FHA-specific antibodies present in the serum of immunized mice on the day of infection were analyzed by ELISA. ***p less than 0.001, *p less than 0.05.

Fig. 10: Immunization with wP, aP, aP with OMV and OMV alone provides protection against B. pertussis infection. C57BL6 mice were immunized with PBS, wP, aP, aP with OMV, and OMV alone 3 weeks before challenge. Mice were then infected with a virulent strain of B. pertussis (Bp338), and the number of bacteria in the lungs was assessed by counting CFU in serially diluted lung homogenates at the indicated time points. ***p less than 0.001, **p less than 0.01, *p less than 0.05.

Fig. 11: Addition of OMV to aP vaccine promotes favorable Th1 response. Spleen cells

- 3,045,827 ki (2x10 ⁶ /ml) from vaccinated C57BL6 mice were cultured in the presence of sonicated B. pertussis (SBP, 5 μg/ml). After 72 hours, the concentration of IFNy in the supernatants was analyzed by ELISA. **p less than 0.01, ***p less than 0.001.

Fig. 12: aP boost plus OMV promotes antigen-specific IFNy production in aP primed mice.

Spleen cells (2 x 10 ⁶ /ml) from vaccinated C57BL6 mice were cultured in the presence of FHA (2 μg/ml), PRN (2 μg/ml), sonicated B. pertussis (SBP, 5 μg/ml) or medium itself. After 72 hours, the concentrations of IFNy, IL-17 and IL-13 in the supernatants were analyzed by ELISA. *p less than 0.05, **p less than 0.01, ***p less than 0.001.

Detailed Description of the Invention

The invention is based on studies of compositions containing outer membrane vesicles (OMVs) of Bordetella pertussis. The inventors have discovered that immunogenic compositions containing both OMVs and antigens, such as acellular pertussis antigens, are capable of inducing a stronger immune response than that observed after immunization with OMVs themselves or acellular pertussis antigens themselves. In addition, OMVs also enhance the immune response to other non-Bordetella antigens, such as tetanus toxoid and diphtheria toxoid. The use of the term originating from refers to the source of the OMVs as being derived from Bordetella pertussis, that is, the bacterial strain from which the OMVs are derived or originated. Essentially, the present invention provides an immunogenic composition comprising (a) Bordetella pertussis OMV, (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid.

In a broad sense, the term immunogenic composition refers to any composition that can be administered to induce an immune response, such as a humoral or cellular immune response, against an antigen present in the composition. Thus, the compositions of the invention are immunogenic. When immunogenic compositions prevent, ameliorate, ameliorate or eliminate a disease in a subject, such compositions may be referred to as vaccines. Preferably, the vaccines of the invention are prophylactic (ie, to prevent infection). Preventative vaccines do not provide complete protection against the disease because even if the patient develops antibodies, there may be a delay or delay before the immune system is able to fight the infection. Thus, and for the avoidance of doubt, the term prophylactic vaccine may also refer to vaccines that reduce the severity of the effects of a future infection, for example, by reducing the severity or duration of such infection. The terms protection against infection and/or provision of protective immunity mean that the subject's immune system has been primed (eg, through vaccination) to initiate an immune response and suppress infection. In particular, the initiated immune response is capable of suppressing infection caused by a number of pathogens, such as various strains of bacteria. Thus, a vaccinated subject may be infected but has a better ability to suppress the infection than a control subject.

OMV

OMVs are well known in the art and are spontaneously released by bacteria into the culture medium. OMVs contain components, such as protein and lipid components, of the outer bacterial membrane of cultured bacteria. All OMVs, both native OMVs (nOMVs [2]) and detergent-extracted OMVs (dOMVs), are part of the invention and are collectively referred to herein as OMVs. The term generalized module for membrane antigens can also be used to refer to OMVs obtained from mutant bacteria. In some embodiments of the invention, the OMVs are native OMVs.

OMVs can be obtained from Bordetella pertussis culture. OMV is obtained from the outer membrane of cultured bacteria. Vesicles can be produced by rupture or natural blistering of the outer membrane of a bacterium to form bubbles.

They can be obtained from bacteria grown in liquid or solid culture, for example by separating the bacterial cells from the culture medium (e.g. by filtration or low-speed centrifugation to pellet the cells), lysing the cells (without detergents), and separating the outer membrane fraction from the cytoplasmic molecules (e.g., filtration, differential precipitation or aggregation of outer membranes and/or OMVs, affinity separation methods using ligands that specifically recognize outer membrane molecules, or high-speed centrifugation resulting in sedimentation of outer membranes and/or OMVs).

OMVs can also be obtained from Bordetella pertussis artificially, for example, using detergent treatment (eg deoxycholate or sarkosyl) or non-detergent methods (eg, see reference [3]). Techniques for the artificial production of OMVs involve treating bacteria with a bile salt detergent (e.g., lithocholic acid salts, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid).

- 4 045827 acid, ursocholic acid, etc., with sodium deoxycholate [4 and 5]) at a pH high enough not to precipitate the detergent [6]. Other methods can be carried out virtually without detergents [3] using techniques such as sonication, homogenization, microfluidization, cavitation, osmotic shock, grinding, French press, mixing and so on.

A useful method for purifying OMVs is described in reference [7] and involves ultrafiltration of crude OMVs instead of high-speed centrifugation. The method may include an ultracentrifugation step upon completion of the ultrafiltration.

The OMV preparations used in the present invention will usually be substantially free of whole bacteria, both living and dead. The size of the vesicles allows them to be easily separated from whole bacteria by filtration, such as that typically used for filtration sterilization.

OMVs are capable of inducing an immune response to Bordetella pertussis when administered to a mammal. The immune response may be a cellular or humoral immune response. In particular, the immune response is a humoral immune response. Even more specifically, the immune response is a T cell immune response that can neutralize Bordetella pertussis infection and/or virulence. The immune response elicited by OMVs may be directed toward or against one or more than one of the B. pertussis protein antigens present in OMVs.

Although pertussis toxin is a secreted toxin, it is present in OMVs, such as pertussis toxin, which is of periplasmic origin. As a result, OMVs used in the art are treated with chemical agents such as formaldehyde to chemically detoxify any PT. In addition to the problems of removing residual formalin, chemical processing can negatively affect the immunogenicity of OMVs, for example through protein cross-linking.

Thus, the use of OMVs derived from a Bordetella pertussis strain expressing a genetically detoxified pertussis toxoid provides advantages and has not previously been proposed in the art. In particular, OMVs derived from the Bordetella pertussis strain expressing the genetically detoxified pertussis toxoid PT-9K/129G have advantages. Specifically, for use in the present invention, the inventors isolated OMV from a Bordetella pertussis strain containing the S1 gene modified to include the R9K and E129G mutations and expressing the genetically detoxified pertussis toxoid PT-9K/129G. Due to this, there is no need for chemical detoxification of OMVs through treatment with chemicals such as formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide and combinations thereof. In particular, the OMVs of the invention are not chemically detoxified, more specifically, the OMVs of the invention are not chemically detoxified and/or treated with formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide, and combinations thereof.

The use of Bordetella pertussis strains with knockout or deletion of the ArnT gene also provides benefits. OMVs derived from such strains contain lipid A having a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure. As a result, these OMVs exhibit reduced levels of TLR4 activation compared to OMVs derived from strains with a functional ArnT gene.

The immunogenic compositions of the invention contain both an OMV component (a) and an acellular pertussis antigen component (b). The outer membrane vesicle component (a) contains many proteins associated with the membrane or present within OMVs, including, for example, small amounts of PT, FHA, or pertactin. However, these OMV-associated components should not be considered the acellular pertussis antigen component (b) described below, which will typically be present separately from the OMV in purified form, for example, as isolated recombinant protein(s). (s) antigen(s).

In some embodiments of the invention, compositions containing OMVs originating from Bordetella parapetussis are excluded from the invention.

Diphtheria toxoid

Diphtheria is caused by Corynebacterium diphtheriae, a Gram-positive, non-spore-forming bacterium. This microorganism expresses a phage-encoded ADP-ribosylating exotoxin (diphtheria toxin), which can be treated (eg using formaldehyde) to produce a toxoid that is no longer toxic but remains antigenic and is capable of stimulating the production of specific antibodies against the toxin after injection. Diphtheria toxoids are discussed in more detail in Chapter 13 of the information source [8]. Diphtheria toxoids obtained by treatment with formaldehyde are preferred. Diphtheria toxoid can be prepared by growing C. diphtheriae in a growth medium (eg Fenton's medium or Linggoud & Fenton medium), which may be supplemented with bovine extract, followed by formaldehyde treatment, ultrafiltration and precipitation. The toxoid material can then be processed using techniques including sterile filtration and/or

- 5 045827 dialysis.

The amount of diphtheria toxoid can be expressed in international units (IU). For example, NIBSC [9] supplies Diphtheria Toxoid Adsorbed Third International Standard 1999 [10, 11], containing 160 IU in each ampoule. As an alternative to the ME system, Lf units (flocculating units, lowest flocculating dose or flocculating limit) are defined as the amount of toxoid that, when mixed with one international unit of antitoxin, forms an optimally flocculating mixture [12]. For example, NIBSC supplies Diphtheria Toxoid, Plain [13], containing 300 Lf in each ampoule, and The 1st International Reference Reagent For Diphtheria Toxoid For Flocculation Test ] [14], containing 900 Lf in each ampoule. Conversion between the ME and Lf systems depends on the specific toxoid preparation.

When substances of bovine origin are used in the cultivation of C. diphtheriae, they must be obtained from sources free of bovine spongiform encephalopathy (BSE) or other transmissible spongiform encephalopathies (TSE).

Diphtheria toxoid is typically present in the immunogenic compositions of the invention in an amount capable of eliciting an immune response when administered. Ideally, diphtheria toxoid would induce a protective immune response. The amount of diphtheria toxoid in the immunogenic compositions of the invention is typically 1-50 Lf per dose. Booster vaccines for adolescents and adults typically contain diphtheria toxoid at levels ranging from 4 Lf/ml to 8 Lf/ml, eg 2.5 Lf, preferably 4 Lf, per 0.5 ml dose. Vaccines for children usually contain diphtheria toxoid in amounts of 20 to 50 Lf/ml, for example 10 Lf or 25 Lf per 0.5 ml dose.

In combination vaccines for children, the diphtheria toxoid:tetanus toxoid ratio is usually greater than 1 (that is, childhood vaccines usually have an excess of diphtheria toxoid) and is usually between 2:1 and 3:1 (when measured in Lf units), e.g. 2.5 :1. In contrast, for a booster vaccine administered to adolescents or adults (who have typically already received at least one combination childhood vaccine containing diphtheria toxoid and tetanus toxoid), the tetanus toxoid:diphtheria toxoid ratio is typically greater than 1 (i.e., booster vaccines typically have excess tetanus toxoid) and is usually between 1.5:1 and 2.5:1, e.g. 2:1. Diphtheria toxoid is usually not conjugated.

The total amount of diphtheria toxoid may be equivalent to 1-50 Lf per dose, for example in booster vaccines at a concentration of 4 Lf/ml to 8 Lf/ml, for example 2.5 Lf per 0.5 ml dose or 4 Lf per 0 dose .5 ml, in vaccines for children at a concentration of 20 to 50 Lf/ml, for example, 10 Lf per 0.5 ml dose or 25 Lf per 0.5 ml dose. In certain embodiments, where there is no chemically detoxified diphtheria toxoid, the amount of genetically detoxified diphtheria toxoid present, particularly CRM197, may be equivalent to 1-50 Lf per dose, at a concentration, for example, in booster vaccines, from 4 Lf/ml to 8 Lf/ ml, for example 2.5 Lf per 0.5 ml dose or 4 Lf per 0.5 ml dose, and in childhood vaccines at a concentration of 20 to 50 Lf/ml, for example 10 Lf per 0.5 ml dose or 25 Lf per 0.5 ml dose.

Diphtheria toxoid can be adsorbed to an aluminum hydroxide adjuvant.

Typically, the immunogenic composition containing the diphtheria toxoid antigen is substantially free of any mercury-containing preservatives.

Tetanus toxoid

Tetanus is caused by Clostridium tetani, a gram-positive spore-forming bacillus. This microorganism expresses endopeptidase (tetanus toxin) which can be processed to produce a toxoid that is no longer toxic but remains antigenic and is capable of stimulating the production of specific antibodies against the toxin after injection. Tetanus toxoids are covered in more detail in Chapter 27 of the reference source [1]. Tetanus toxoids obtained by treatment with formaldehyde are preferred. Tetanus toxoid can be produced by growing C. tetani in a growth medium (eg Latham's medium derived from bovine casein) followed by formaldehyde treatment, ultrafiltration and precipitation. The resulting material can then be processed using techniques including sterile filtration and/or dialysis.

The amount of tetanus toxoid can be expressed in international units (IU). For example, NIBSC [15] supplies Tetanus Toxoid Adsorbed Third International Standard 2000 [16, 17], containing 469 IU per ampoule. As an alternative to the ME system, the “Lf” unit is defined as the amount of toxoid that, when mixed with one international unit of antitoxin, forms an optimally flocculating mixture [18]. For example, NIBSC supplies The 1st International Reference Reagent for Tetanus Toxoid For Flocculation Test [19], containing 1000 Lf in each ampoule. Conversion between the ME and Lf systems depends on the specific toxoid preparation.

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If substances of bovine origin are used in the cultivation of C. tetani, they must be obtained from sources free of bovine spongiform encephalopathy (BSE) or other transmissible spongiform encephalopathies (TSE).

Tetanus toxoid is typically present in the immunogenic compositions of the invention in an amount capable of eliciting an immune response when administered. Ideally, tetanus toxoid can induce a protective immune response. The amount of tetanus toxoid in the immunogenic compositions of the invention is typically 1-20 Lf per dose. Booster vaccines for adolescents and adults typically contain 5 Lf of tetanus toxoid per 0.5 ml dose. Vaccines for children typically contain 5 to 10 Lf of tetanus toxoid per 0.5 ml dose.

It will be apparent to one skilled in the art that in some embodiments, tetanus toxoid may be present in both free (unconjugated) and conjugated form, or predominantly in conjugated form, i.e. 80, 85, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99% of tetanus toxoid is present in conjugated form. Thus, the amount of tetanus toxoid in the immunogenic compositions of the invention may refer to unconjugated tetanus toxoid only, conjugated tetanus toxoid only, or the sum of unconjugated and conjugated tetanus toxoid. However, the tetanus toxoid is preferably free, unconjugated. In preferred embodiments, the amount of tetanus toxoid in the immunogenic compositions of the invention refers only to unconjugated tetanus toxoid.

Tetanus toxoid can be adsorbed onto an aluminum hydroxide adjuvant, but this is not necessary (eg, 0-10% adsorption of total tetanus toxoid can be used).

Typically, the immunogenic composition containing tetanus toxoid is substantially free of any mercury-containing preservatives.

Acellular pertussis antigen(s)

Bordetella pertussis is a Gram-negative, non-spore-forming, aerobic bacterium and the causative agent of whooping cough. As described in more detail in Chapter 21 of the reference [1], vaccines against B. pertussis have been available for many years and are divided into two categories: cellular (wP) and acellular (aP). Cellular vaccines contain whole B. pertussis cells that have been killed or inactivated (for example, by treatment with formaldehyde and/or heat), while acellular vaccines contain purified specific B. pertussis antigens, either isolated from native bacteria or isolated after expression in recombinant host.

In this invention, more than one acellular pertussis (aP) antigen may be used in a single vaccine, for example, at least two or at least three of the following well known and well characterized B. pertussis antigens: (1) detoxified pertussis toxin (pertussis toxoid or PT); (2) filamentous hemagglutinin (FHA); (3) pertactin (PRN, also known as 69 kDa or 69K outer membrane protein). Most preferably, all three of these antigens should be used. These three antigens are preferably obtained by isolation from a culture of B. pertussis, for example grown in liquid Stainer-Scholte medium. PT and FHA can be isolated from the liquid fermentation medium (eg by adsorption on a hydroxyapatite gel), while pertactin can be isolated from cells by heating and flocculation (eg using barium chloride). Antigens can be purified in subsequent chromatographic and/or precipitation steps. PT and FHA can be purified by hydrophobic interaction chromatography, affinity chromatography and size exclusion chromatography. Pertactin can be purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. Methods for purifying PT, FHA and pertactin are known in the art.

FHA and pertactin can be treated with formaldehyde before use according to this invention. RT can be detoxified by treatment with formaldehyde and/or glutaraldehyde. As an alternative to this chemical detoxification technique, in preferred embodiments the PT may be a mutant PT whose enzymatic activity has been reduced by mutagenesis [22] (eg, the 9K/129G double mutant). When genetically detoxified RT is included, a smaller amount can be used. For example, the concentration of genetically detoxified pertussis toxoid in the composition may be less than or equal to 5 μg/ml, for example less than 4, less than 3, less than 2.5, less than 2, less than 1 μg/ml, and so on. Thus, in a typical unit dose volume of 0.5 ml, the amount of genetically detoxified pertussis toxoid is less than 2.5 μg, e.g., less than 2, less than 1.5, less than 1, less than 0.5 μg, e.g., from about 0.5 μg to approximately 2.5 mcg.

Other acellular pertussis antigens that may be used include fimbriae (eg, agglutinogens 2 and 3, also called FIM2 and FIM3).

The aP Antigen(s) may be used in an unadsorbed state, but are preferably adsorbed to one or more aluminum salt adjuvants prior to use. Preferably, the aP antigens are adsorbed to an aluminum hydroxide adjuvant.

- 7 045827

Typically, immunogenic compositions containing aP antigens are substantially free of mercury-containing preservatives (eg thimerosal).

Acellular pertussis antigen is typically present in the immunogenic compositions of the invention in an amount capable of eliciting an immune response when administered. Ideally, acellular pertussis antigen can induce a protective immune response. The amount of acellular pertussis antigens is usually expressed in micrograms. The concentration of RT in the vaccine is usually from 5 to 50 μg/ml. Typical RT concentrations are 5 μg/ml, 16 μg/ml, 20 μg/ml, or 50 μg/ml. The concentration of FHA in the vaccine is typically between 10 and 50 mcg/mL. Typical FHA concentrations are 10 μg/ml, 16 μg/ml, or 50 μg/ml. The concentration of pertactin in the vaccine is usually from 5 to 16 μg/ml. Typical pertactin concentrations are 5 μg/ml, 6 μg/ml, or 16 μg/ml. For example, a booster vaccine for adolescents and adults typically contains 2.5 to 8 mcg PT, 4 to 8 mcg FHA (e.g., 4 to 8 mcg FHA), and 2.5 to 8 mcg pertactin (e.g., 2. 5 to 8 mcg pertactin) per 0.5 ml dose. Typically the booster vaccine contains 4 μg PT, 4 μg FHA and 8 μg pertactin, more preferably 5 μg PT, 2.5 μg FHA and 2.5 μg pertactin per 0.5 ml dose. The childhood vaccine typically contains 7 mcg PT, 10 mcg FHA, and 10 mcg pertactin per 0.5 mL dose.

When the aqueous component contains PT, FHA, and pertactin, their weight ratios may vary, but may be, for example, about 16:16:5, about 5:10:6, about 20:20:3, about 25:25:8, or approximately 10:5:3 (PT:FHA:PRN).

Specific immunogenic compositions of the invention

Specifically provided immunogenic compositions according to the invention contain:

(1) OMV B. pertussis, (2) diphtheria toxoid, (3) tetanus toxoid, (4) acellular pertussis antigen;

(1) OMV B. pertussis, (2) diphtheria toxoid, (3) tetanus toxoid, (4) detoxified pertussis toxin, (5) filamentous hemagglutinin, and (6) pertactin;

(1) OMV B. pertussis, (2) diphtheria toxoid, (3) tetanus toxoid, (4) genetically detoxified pertussis toxin, (5) filamentous hemagglutinin, and (6) pertactin;

(1) OMV B. pertussis, (2) diphtheria toxoid, (3) tetanus toxoid, (4) detoxified pertussis toxin, (5) filamentous hemagglutinin, (6) pertactin, (7) polio virus strain type 1 antigen, (8) an antigen of a type 2 polio virus strain; and (9) an antigen of a type 3 polio virus strain.

The immunogenic compositions of the present invention induce an enhanced Th1 and enhanced Th2 response, that is, they increase both IgG1 and IgG2a production. More specifically, the compositions induce an enhanced Th1 and/or enhanced Th2 immune response compared to immunization with OMVs alone or TdaP alone.

Pharmaceutical Methods and Applications

The immunogenic compositions of the invention may further comprise a pharmaceutically acceptable carrier. A typical pharmaceutically acceptable carrier includes any carrier that does not, by itself, induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, lactic acid polymers, glycolic acid polymers, amino acid polymers, amino acid copolymers, sucrose [21], trehalose [22], lactose and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those skilled in the art. Vaccines may also contain diluents such as water, saline, glycerin, and so on. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffers and the like may be present. A typical carrier is sterile pyrogen-free phosphate buffered saline. A detailed discussion of pharmaceutically acceptable excipients is provided in Reference [23].

The compositions of the invention may be presented in aqueous form (ie, solutions or suspensions) or dried form (eg, lyophilized form). If a dried vaccine is used, it will be reconstituted in a liquid medium before injection. Lyophilization of vaccines is known in the art, for example, the Menjugate™ vaccine is presented in lyophilized form. When the immunogenic compositions of the invention include a lyophilized component, this component is typically prepared separately, mixed and then lyophilized. To stabilize antigens during lyophilization, inactive components may be added before lyophilization, for example as stabilizers. Preferred stabilizers for inclusion are lactose, sucrose and mannitol, as well as mixtures thereof, for example mixtures of lactose and sucrose, mixtures of sucrose and mannitol, and so on. Thus, the finished vaccine obtained by reconstituting lyophilized substances in water may contain lactose and/or sucrose. When preparing lyophilized vaccines, it is preferable to use amorphous excipients and/or amorphous buffers [24].

It may be preferable to include a sugar alcohol (eg mannitol) and/or a disaccharide (eg sucrose or trehalose) in the composition, for example in an amount of 1 mg/ml to 30 mg/ml (eg approximately 25 mg/ml).

- 8 045827

The compositions may be presented in vials, or they may be presented in pre-filled syringes. Syringes may come with or without needles. A syringe will contain a single dose of the composition, while a vial may contain a single dose or multiple doses.

The compositions of the invention are preferably administered to patients in unit doses of 0.5 ml. Reference to 0.5 ml doses is intended to include a standard deviation, for example, 0.5 ml plus/minus 0.05 ml. In the case of multiple doses, the entire quantity corresponding to the multiple doses will be dispensed and packaged in a single container, for example, 5 ml for a 10-dose multi-dose container (or 5.5 ml with a 10% excess).

The aqueous compositions of the invention are also suitable for reconstituting other vaccines from lyophilized form. Where the composition of the invention is to be used for such reconstitution immediately prior to use, the invention provides a kit which may contain two vials or may contain one pre-filled syringe and one vial where the contents of the syringe are used to reactivate the contents of the vial before injection.

Vaccines can also be prepared in a form where the vaccine can be prepared directly at the time of use by mixing two components with each other. Such two-component embodiments include liquid-liquid mixing and liquid-solid mixing, for example, by mixing aqueous substances with lyophilized substances.

Thus, a kit that can be used in the invention contains a first component containing an OMV component, and a second component containing (a) acellular pertussis antigen(s), (b) tetanus toxoid, and (c ) diphtheria toxoid, where the two components are in separate containers (for example, vials or syringes). The OMV component of the first component may be lyophilized. In some embodiments, the first component does not contain an adjuvant. The second component may contain aqueous antigens. In some embodiments, the second component contains an adjuvant, for example an aluminum salt adjuvant.

The invention also provides a method for preparing an immunogenic composition according to the invention, comprising mixing a first component containing OMV and a second component containing (a) acellular pertussis antigen(s), (b) tetanus toxoid and (c) diphtheria toxoid. The OMVs of the first component can be lyophilized. The second component may contain aqueous antigens. The method may include the additional step of reconstituting the lyophilized OMVs in the first component with aqueous antigens of the second component. The first component may not contain an adjuvant. The second component may contain an adjuvant, for example an aluminum salt adjuvant.

The compositions of the invention may be packaged in single-dose form or in multi-dose form. For multi-dose formulations, vials are preferred over prefilled syringes. Effective dose volumes can be determined by conventional methods, but a typical human dose of the composition, for example for intramuscular injection, has a volume of 0.5 ml.

The pH of the composition is preferably between 6 and 8, preferably about 7. A stable pH can be maintained using a buffer. Aqueous compositions administered to a patient may have a pH of 5.0 to 7.5, and more typically 5.0 to 6.0 for optimal stability; where diphtheria toxoid and/or tetanus toxoid are present, the pH is ideally between 6.0 and 7.0.

The immunogenic compositions of the invention typically contain a potassium dihydrogen phosphate buffer. The potassium dihydrogen phosphate buffer may contain approximately 1-10 mM potassium dihydrogen phosphate, for example 1.25 mM, 2.5 mM or 5.0 mM. If the composition contains an aluminum hydroxide salt, it is preferable to use a histidine buffer [25]. The composition may be sterile and/or pyrogen-free. The compositions of the invention may be isotonic in humans.

The compositions of the invention are immunogenic and, more preferably, are vaccine compositions. The vaccines of the invention may be prophylactic (ie, to prevent infection) or therapeutic (ie, to treat infection), but will generally be prophylactic. Preventive vaccines do not provide complete protection against the disease because even if the patient develops antibodies, there may be a delay or delay before the immune system is able to fight the infection. Thus, and for the avoidance of doubt, the term prophylactic vaccine may also refer to vaccines that reduce the severity of the effects of a future infection, for example, by reducing the severity or duration of such infection.

The terms protection against infection and/or provision of protective immunity mean that the subject's immune system has been primed (eg, through vaccination) to initiate an immune response and suppress infection. In particular, the initiated immune response is capable of suppressing infection caused by a number of pathogens, such as various strains of bacteria. Thus, a vaccinated subject may be infected but has a better ability to suppress the infection than a control subject. Immunogenic compositions used

- 9 045827 as vaccines, contain an immunologically effective amount of antigen(s), as well as, if necessary, any other components. By immunologically effective amount is meant that administration of that amount to an individual, in a single dose or in a series of doses, is effective for treatment or prevention. Generally, the desired outcome is the development of an immune response that is specific for an antigen (eg, a pathogen) capable of protecting the subject from the pathogen or promoting such protection. This amount will vary depending on the health and physical condition of the individual being treated, the age, the taxonomic group of the individual being treated (e.g., non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the composition vaccines, assessment of the medical situation by the attending physician and other relevant factors. It is expected that this amount will fall within the relatively wide range that can be determined by standard studies.

The compositions of the invention can be prepared in various forms. For example, the compositions can be formulated for injection as liquid solutions or suspensions. The composition may be formulated for pulmonary administration, for example in the form of an inhaler using a fine powder or spray. The composition can be prepared in the form of a suppository or pessary. The composition may be formulated for nasal, ear or ocular administration, for example in the form of a spray, drops, gel or powder (eg references [26] and [27]). Successful nasal administration of pneumococcal saccharides [28, 29], Hib saccharides [30], MenC saccharides [31], and mixtures of Hib and MenC saccharide conjugates [32] have been reported.

The compositions of the invention may contain an antimicrobial agent, particularly when packaged in multi-dose form.

The compositions of the invention may contain a detergent, for example Tween (polysorbate), such as Tween 80. Detergents are usually present in small amounts, for example less than 0.01%.

The compositions of the invention may contain sodium salts (eg, sodium chloride) to provide tonicity. A typical NaCl concentration is 10 plus/minus 2 mg/mL. In some embodiments, a NaCl concentration of 4-10 mg/ml may be used, for example, 9.0, 7.0, 6.75 or 4.5 mg/ml.

The osmolality of the compositions will typically be from 200 mOsm/kg to 400 mOsm/kg, preferably 240-360 mOsm/kg, and more preferably will be in the range of 280-320 mOsm/kg. Osmolality has previously been reported to have no effect on vaccination-related pain [33], but maintaining osmolality within this range is still preferable.

The compositions of the invention will typically contain a buffer. Phosphate buffer is typical.

The compositions of the invention may be administered in combination with other immunoregulatory agents. In particular, the compositions may contain one or more than one adjuvant. Such adjuvants include, but are not limited to, mineral-containing compositions.

Mineral-containing compositions suitable for use in the invention as adjuvants include mineral salts such as aluminum salts and calcium salts (or mixtures thereof). Calcium salts include calcium phosphate (eg, CAP particles disclosed in reference [34]). Aluminum salts include hydroxides, phosphates, sulfates, etc., in any suitable form (eg, gel, crystalline, amorphous, etc.). Adsorption onto these salts is preferred. Mineral-containing compositions can also be prepared in the form of metal salt particles [35].

Adjuvants known as aluminum hydroxide and aluminum phosphate may be used. These names are common names, but are used only for convenience since none of them is an accurate description of the chemical compound actually present (for example, see Chapter 9 of the reference [36]). Any of the hydroxide or phosphate adjuvants commonly used as adjuvants can be used in the invention. Typical adjuvants, known as aluminum hydroxide, are salts of aluminum metahydroxide, usually at least partially crystalline. Typical adjuvants known as aluminum phosphate are aluminum hydroxyphosphates, often also containing a small amount of sulfate (ie aluminum hydroxyphosphate sulfate). They can be prepared by precipitation, and the reaction conditions and concentrations during precipitation affect the extent to which the hydroxyl salt is replaced by phosphate.

Aluminum hydroxide adjuvants typically have a fibrous structure (for example, as seen in transmission electron microscopy micrographs). The pI value of aluminum hydroxide adjuvants is typically approximately 11, meaning the adjuvant itself has a positive surface charge at physiological pH. The adsorption capacity of aluminum hydroxide adjuvants has been reported to be 1.8–2.6 mg protein per mg Al+++ at pH 7.4.

Aluminum phosphate adjuvants typically have a PO4/Al molar ratio of 0.3 to 1.2, preferably 0.8 to 1.2, and more preferably 0.95 plus or minus 0.1. Aluminum phosphate will

- 10 045827 usually amorphous, especially in the case of hydroxyphosphate salts. A typical adjuvant is amorphous aluminum hydroxyphosphate with a PO4/Al molar ratio of 0.84 to 0.92, included at 0.6 mg Ar/ml. Aluminum phosphate will typically be in the form of particles (eg, plate-like structure, as seen in transmission electron microscopy micrographs). Typical particle diameters after adsorption of any antigen are in the range of 0.5-20 μm (eg, approximately 5-10 μm). The adsorption capacity of aluminum phosphate adjuvants has been reported to be 0.7-1.5 mg protein per mg Al ⁺⁺⁺ at pH 7.4.

The point of zero charge (PZC) of aluminum phosphate is inversely related to the degree of hydroxyl substitution by the phosphate, and this degree of substitution can vary depending on the reaction conditions and the concentration of the reactants used to prepare the salt by precipitation. PZC also changes when the concentration of free phosphate ions in solution is changed (more phosphate, the more acidic PZC) or when a buffer such as histidine buffer is added (makes PZC more basic). Aluminum phosphates used according to the invention will typically have a PZC of 4.0 to 7.0, more preferably 5.0 to 6.5, for example about 5.7.

The aluminum salt suspensions used to make the compositions of the invention may contain a buffer (eg phosphate, histidine or Tris buffer), but this is not always necessary. Suspensions are preferably sterile and pyrogen-free. The suspension may contain free aqueous phosphate ions, for example present at a concentration of 1.0 to 20 mM, preferably 5 to 15 mM, and more preferably about 10 mM. Suspensions may also contain sodium chloride.

A mixture of aluminum hydroxide and aluminum phosphate can be used in the invention. In this case, there may be more aluminum phosphate than hydroxide, for example in a weight ratio of at least 2:1, for example 5:1 or more, 6:1 or more, 7:1 or more, 8:1 or more, 9 :1 or more and so on.

The concentration of Al~ ⁺ in the composition for administration to a patient is preferably less than 10 mg/ml, for example 5 mg/ml or less, 4 mg/ml or less, 3 mg/ml or less, 2 mg/ml or less, 1 mg/ml or less and so on. The preferred range is from 0.3 to 1.0 mg/ml. The preferred maximum amount per dose is 0.85 mg.

A typical aluminum phosphate adjuvant is amorphous aluminum hydroxyphosphate with a PO ₄ /Al molar ratio of 0.84 to 0.92, included at 0.6 mg Al ³⁺ /ml. Adsorption at a low dose of aluminum phosphate, for example 50 to 100 μg Al ³⁺ per dose, can be used.

The use of adjuvants based on aluminum salts is particularly preferred, and the antigens are usually adsorbed on such salts. In the compositions of the invention, some antigens may be adsorbed on aluminum hydroxide, and other antigens may be in association with aluminum phosphate. However, it is usually preferable to use only one salt, such as hydroxide or phosphate, but not both. Not all antigens need to be adsorbed, that is, some or all antigens may be present in free form in the solution.

Treatment methods

The invention also provides immunogenic compositions for use in inducing an immune response in a mammal, comprising administering to said mammal an immunogenic composition of the invention. Preferably, the immunogenic composition is capable of inducing an immune response in a mammal. The immune response is preferably protective and preferably includes antibodies and, more preferably, a vaccine. In some embodiments, the vaccine is intended for use in primary vaccination. The immunogenic composition may induce a booster response. The compositions of the invention are preferably administered to patients in doses of 0.5 ml (as discussed above).

The invention also provides a method of inducing an immune response in a mammal, comprising administering to said mammal an immunogenic composition of the invention. The immune response is preferably protective and preferably includes antibodies. The method can induce a booster response. The compositions of the invention are preferably administered to patients in doses of 0.5 ml (as discussed above).

Preferably, the mammal is a human. Where the vaccine is for prophylactic use, the human is preferably a child (eg a toddler or infant, particularly a newborn) or adolescent. A vaccine intended for children may also be administered to adults, for example, to evaluate safety, dose, immunogenicity, and so on. The preferred group of people for treatment are women of reproductive age (eg, adolescence and older). Another preferred group is pregnant women.

In some embodiments, the patient has already been immunized with diphtheria toxoid or a derivative thereof. In other embodiments, the patient has already been immunized with tetanus toxoid or a derivative thereof. In some embodiments, the patient has already been immunized with both a diphtheria toxoid or a derivative thereof and a tetanus toxoid or a derivative thereof.

- 11 045827

The invention also provides a composition according to the invention for use as a medicine.

The invention also provides the use of a composition according to the invention in the manufacture of a medicament for inducing an immune response in a mammal.

Preferably, these uses and methods are for the prevention and/or treatment of a disease caused by one or more of Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis. C. diphtheriae can cause diphtheria; C. tetani can cause tetanus; B. pertussis can cause whooping cough.

The subject in whom the disease is prevented may not be the same subject who receives the immunogenic composition of the invention. For example, an immunogenic composition can be administered to a female individual (before or during pregnancy) to protect the offspring (so-called maternal immunization [37-39]). Immunization of a pregnant female provides antibody-mediated immunity in the offspring through passive maternal immunity. Passive immunity occurs naturally when maternal antibodies are transferred to the fetus through the placenta. Passive immunity is especially important for infants because they are born without any actively acquired immunity. Administration of the compositions according to the invention to a pregnant female increases the immunity of that female, and antibodies are transferred to the newborn through the placenta, providing passive maternal immunity to the infant. However, passive immunity in infants is only temporary and begins to wane after the first weeks or months of life. Due to the temporary nature of passive immunity, it may be important to administer a composition of the invention to an infant to induce active immunity in the infant before passive immunity wanes. Administration of a second immunogenic composition to an infant after birth induces active immunity in that infant and prolongs the immunity transmitted from the mother during pregnancy.

As used herein, an infant is an individual less than one year of age (e.g., less than one day of age, 1 week of age, 2 weeks of age, 3 weeks of age, 4 weeks of age, 2 months of age, 3 months of age , 4 months, 5 months, 6 months, 7 months, 8 months old, 9 months old, 10 months old, 11 months old, less than 12 months old).

The composition of the invention can be administered to a pregnant female at any time during her pregnancy. For example, the composition may be administered to a pregnant female during the first, second or third trimester of her pregnancy. In some embodiments, the composition is administered to female subjects during the last 6-12 weeks of pregnancy (e.g., 28 weeks of pregnancy, 29 weeks of pregnancy, 30 weeks of pregnancy, 31 weeks of pregnancy, 32 weeks of pregnancy, 33 weeks of pregnancy, 34 weeks of pregnancy, 35 weeks pregnancy, 36 weeks pregnant, 37 weeks pregnant, 38 weeks pregnant, 39 weeks pregnant). In particular, the composition of the invention can be administered to a pregnant female at least four weeks before the birth of the child. In some embodiments, the composition is administered to a pregnant female in a single dose regimen at 32-36 weeks of gestation. In other embodiments, the composition is administered to a pregnant female in a two-dose regimen, with the first dose administered at approximately 32 weeks of pregnancy and the second dose administered at approximately 36 weeks of pregnancy.

The composition can be administered to an infant at any time during the first year of life and, if necessary, later. Typically the composition will be administered to the infant one, two, three, four or more times during the first year of life. For example, the composition of the invention may be administered to an infant at one or more time points selected from birth, 2 weeks of age, 4 weeks of age, 6 weeks of age, 2 months of age, 3 months of age, 4 months of age, 6 months of age, 9 months of age and 12 months of age. In particular, the composition according to the invention is administered to the infant at a point in time before maternal antibody titers fall below protective levels. Subsequent administrations can be carried out according to any desired schedule.

In one embodiment, a method is provided for protecting an infant from a disease caused by one or more of Corynebacterium diphtheriae, Clostridium tetani, and Bordetella pertussis, comprising the steps of (a) administering a composition of the invention to a female subject during pregnancy with said infant, and optionally (b) administering the composition according to the invention to the said infant born as a result of this pregnancy.

Thus, also provided is a method of protecting an infant from one or more of diphtheria, tetanus and whooping cough, comprising the steps of (a) administering a composition of the invention to a female subject during pregnancy with said infant, and optionally (b) administering a composition of the invention to said infant, born as a result of this pregnancy.

Preferred compositions of the invention can provide a patient with an antibody titer that exceeds the criterion for serological protection for each antigenic component in an acceptable percentage of human subjects. Antigens with corresponding antibody titers, the excess of which in

- 12 045827 host considered as seroconversion against antigen are well known, and such titers are published by organizations such as WHO. Preferably, seroconversion occurs in more than 80% of a statistically significant sample of subjects, more preferably in more than 90%, even more preferably in more than 93%, and most preferably in 96-100%.

The compositions of the invention will typically be administered directly to the patient. Direct delivery may be by parenteral injection (eg, subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitial tissue) or rectal, oral, vaginal, topical, transdermal, intranasal, ocular, auricular, pulmonary, or other mucosal administration. Preferably intramuscular injection into the thigh or upper arm. The injection may be injection through a needle (eg, a hypodermic needle), but alternatively, needle-free injection may be used. The typical dose volume for intramuscular administration is 0.5 ml.

The invention can be used to provide systemic immunity and/or mucosal immunity.

The immunogenic compositions of the invention can be administered single or multiple times. In the present invention, single administration is preferred. Alternatively, administering another unit dose after the first unit dose may be effective. Typically, the second (or third, fourth, fifth, etc.) unit dose is identical to the first unit dose. Typically, the immunogenic compositions of the invention are administered in three unit doses. Typically, the immunogenic compositions of the invention will be administered intramuscularly, for example by intramuscular injection into the thigh or upper arm.

Multiple administrations can be used in a primary immunization schedule and/or in a booster immunization schedule. The primary administration regimen may be followed by a booster administration regimen. The appropriate interval between primary administrations (eg, 4-16 weeks) and between primary and booster administrations can be determined routinely.

To ensure full effectiveness, a typical primary immunization schedule (particularly for a child) may involve more than one dose. For example, administration can be carried out: at 0 and 6 months (with the first administration at 0 months); at 0, 1, 2 and 6 months; on days 0 and 21, followed by a third dose at 6-12 months; at 2, 4 and 6 months; at 3, 4 and 5 months; at 6, 10 and 14 weeks; at 2, 3 and 4 months; or at 0, 1, 2, 6 and 12 months. Compositions for children can also be used for booster administration, for example, to children in the second year of life.

The compositions can also be used for booster administration, for example, to children in the second year of life. The booster vaccine compositions of the invention for adolescents are administered once to persons aged 10 years and older. The immunogenic composition of the invention can be administered as a booster vaccine to a patient previously vaccinated against both diphtheria and tetanus, and preferably also against pertussis. These patients differ from the general population in having an immunological memory response to a previously administered vaccine. Patients may have received the last of their previous diphtheria and/or tetanus vaccines at least five years before receiving the vaccine of the invention. The age of patients receiving vaccines can range from 4 to 65 years, for example, 11-64 years, 10-18 years, and so on.

Any suitable route of administration may be used. For example, the composition can be administered intramuscularly, intraperitoneally, subcutaneously, transdermally or intradermally. If desired, the composition can be administered by intramucosal routes such as intraoral, intranasal, intravaginal and intrarectal. Administration to the pregnant female and the infant may be carried out by the same route or by different routes. The compositions of the invention can be administered by intramuscular injection, for example into the shoulder or thigh.

The vaccines made according to the invention can be administered to patients simultaneously with another vaccine, for example, simultaneously with a pneumococcal conjugate vaccine such as PREVNAR™, simultaneously with an influenza vaccine, simultaneously with a rotavirus vaccine, simultaneously with a vaccine against measles, rubella, and mumps (MMR). ) and so on.

Where the compositions of the invention contain an aluminum-based adjuvant, precipitation of the components may occur during storage. Therefore, the composition should be shaken before administration to the patient. After shaking, the composition will be a cloudy white suspension.

Incarnations

Embodiment 1. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from Bordetella pertussis.

Embodiment 2. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid.

Embodiment 3. An immunogenic composition according to Embodiment 1 or Embodiment 2, wherein the acellular pertussis antigen is selected from the group consisting of (1) detoxified pertussis

- 13 045827 toxin (PT), (2) filamentous hemagglutinin (FHA) and (3) pertactin (PRN).

Embodiment 4. The immunogenic composition according to embodiment 3, wherein the acellular pertussis antigen contains PT, FHA and PRN.

Embodiment 5. An immunogenic composition according to embodiment 4, wherein PT, FHA and PRN are present in a ratio of 16:16:5 (measured by weight).

Embodiment 6. An immunogenic composition according to any of the previous embodiments, wherein the diphtheria toxoid is present at a concentration of 4 Lf/ml to 8 Lf/ml, for example 4 Lf per 0.5 ml dose.

Embodiment 7. An immunogenic composition according to any of embodiments 1 to 6, wherein the diphtheria toxoid is present at a concentration of 20 to 50 Lf/ml, for example 25 Lf per 0.5 ml dose.

Embodiment 8. An immunogenic composition according to any of the previous embodiments, wherein the tetanus toxoid is present at a concentration of approximately 5 Lf per 0.5 ml dose.

Embodiment 9. An immunogenic composition according to any of embodiments 1 to 8, wherein the tetanus toxoid is present at a concentration of 5 to 10 Lf per 0.5 ml dose.

Embodiment 10. An immunogenic composition according to any of embodiments 1-9, wherein the diphtheria toxoid and tetanus toxoid are present in a diphtheria toxoid: tetanus toxoid ratio greater than 1, for example, from 2:1 to 3:1 (when measured in Lf units), such as 2.5:1.

Embodiment 11. An immunogenic composition according to any one of embodiments 1 to 9, wherein the diphtheria toxoid and tetanus toxoid are present in a ratio of tetanus toxoid: diphtheria toxoid greater than 1, for example, from 1.5:1 to 2.5:1 (when measured in Lf -units), such as 2:1.

Embodiment 12. An immunogenic composition according to any of the previous embodiments, containing an adjuvant.

Embodiment 13. An immunogenic composition according to any of the previous embodiments, containing an aluminum salt adjuvant.

Embodiment 14. An immunogenic composition according to any of the previous embodiments, which is a liquid solution or suspension for injection.

Embodiment 15. The immunogenic composition according to any of embodiments 1-14, which is a lyophilized composition.

Embodiment 16. Immunogenic compositions according to any of the previous embodiments, wherein the composition is free of preservatives.

Embodiment 17. An immunogenic composition according to any of the previous embodiments, which is a vaccine.

Embodiment 18. An immunogenic composition according to any of the previous embodiments, intended for administration to humans.

Embodiment 19. An immunogenic composition according to any of the previous embodiments, intended for use as a medicine.

Embodiment 20. A method of inducing an immune response in a patient, comprising the step of administering to the patient a composition according to any of the previous embodiments.

Embodiment 21. A method of inducing an immune response in a patient, comprising the step of administering to the patient a composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid.

Embodiment 22. A method for preparing an immunogenic composition according to any one of embodiments 1 to 19, comprising mixing a first component containing outer membrane vesicles (OMVs) and a second component containing acellular pertussis antigen, tetanus toxoid and diphtheria toxoid.

Embodiment 23. The method according to embodiment 22, wherein the OMVs of the first component are lyophilized.

Embodiment 24. The method according to embodiment 22 or embodiment 23, where the second component contains aqueous antigens.

Embodiment 25. The method according to embodiment 24, including the additional step of reconstituting the lyophilized OMVs in the first component with aqueous antigens of the second component.

Embodiment 26. The method according to any of embodiments 22-25, where the first component does not contain an adjuvant.

Embodiment 27. The method according to any of embodiments 22-26, where the second component contains an adjuvant, for example an aluminum salt adjuvant.

Embodiment 28. A kit for preparing an immunogenic composition according to any of embodiments 1 to 19, comprising a first component containing OMV and a second component containing acellular pertussis antigen, tetanus toxoid and diphtheria toxoid, where these two components are presented in separate containers.

Embodiment 29. The kit according to embodiment 28, wherein the OMVs in the first component are lyophilized.

Embodiment 30. The kit according to embodiment 28 or embodiment 29, where the second component contains aqueous antigens.

- 14 045827

Embodiment 31. The kit according to any of embodiments 28-30, where the first component does not contain an adjuvant.

Embodiment 32. A kit according to any of embodiments 28-31, wherein the second component contains an adjuvant, for example an aluminum salt adjuvant.

Embodiment 33. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain expressing genetically detoxified pertussis toxoid , in particular genetically detoxified pertussis toxoid PT-9K/129G.

Embodiment 34. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain expressing genetically detoxified pertussis toxoid PT-9K/129G.

Embodiment 35. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified with the inclusion of mutations R9K and E129G, and expressing genetically detoxified pertussis toxoid PT-9K/129G.

Embodiment 36. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified including the R9K and E129G mutations, and expressing the genetically detoxified pertussis toxoid PT-9K/129G, and where the OMVs are not chemically detoxified.

Embodiment 37. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified including the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/129G, and where the OMVs are not chemically detoxified and/or treated with formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide and combinations or derivatives thereof.

Embodiment 38. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified including the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/129G, and wherein Lipid A in OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure.

Embodiment 39. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified incorporating the R9K and E129G mutations, and expressing the genetically detoxified pertussis toxoid PT-9K/129G, where the lipid A of the OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and where the OMVs are not chemically detoxified and/or processed formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide and combinations or derivatives thereof.

Embodiment 40. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) an acellular pertussis antigen selected from the group consisting of (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA), and ( 3) pertactin (PRN), (c) tetanus toxoid and (d) diphtheria toxoid, where OMVs are derived from the Bordetella pertussis strain containing the S1 gene, modified to include the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/ 129G.

Embodiment 41. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) an acellular pertussis antigen selected from the group consisting of (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA), and ( 3) pertactin (PRN), (c) tetanus toxoid and (d) diphtheria toxoid, where OMVs are derived from the Bordetella pertussis strain containing the S1 gene, modified to include the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/ 129G, where the lipid A of the OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and where the OMVs are not chemically detoxified.

Embodiment 42. An immunogenic composition comprising (a) outer membrane vesicles (OMVs), (b) an acellular pertussis antigen selected from the group consisting of (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA), and ( 3) pertactin (PRN), (c) tetanus toxoid and (d) diphtheria toxoid, where OMVs are derived from the Bordetella strain

- 15 045827 pertussis, containing the S1 gene, modified to include the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/129G, where lipid A in OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and wherein the OMVs are not chemically detoxified and/or treated with formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide and combinations or derivatives thereof.

Embodiment 43. An immunogenic composition comprising (a) outer membrane vesicles (OMV), (b) (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA), and (3) pertactin (PRN), (c) tetanus toxoid and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified to include the R9K and E129G mutations and expressing genetically detoxified pertussis toxoid PT-9K/129G.

Embodiment 44. An immunogenic composition comprising (a) outer membrane vesicles (OMV), (b) (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA), and (3) pertactin (PRN), (c) tetanus toxoid and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified to include the R9K and E129G mutations and expressing genetically detoxified pertussis toxoid PT-9K/129G, wherein lipid A in the OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and where the OMVs are not chemically detoxified.

Embodiment 45. An immunogenic composition comprising (a) outer membrane vesicles (OMV), (b) (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA), and (3) pertactin (PRN), (c) tetanus toxoid and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified to include the R9K and E129G mutations and expressing genetically detoxified pertussis toxoid PT-9K/129G, wherein lipid A in the OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and where the OMVs are not chemically detoxified and/or treated with formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide and combinations or derivatives thereof.

Embodiment 46. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain expressing a genetically detoxified pertussis toxoid, in particular genetically detoxified pertussis toxoid PT-9K/129G.

Embodiment 47. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain expressing a genetically detoxified pertussis toxoid PT-9K/129G.

Embodiment 48. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene , modified to include the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/129G.

Embodiment 49. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene , modified to include the R9K and E129G mutations, and expressing the genetically detoxified pertussis toxoid PT-9K/129G, and where the OMVs are not chemically detoxified.

Embodiment 50. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene , modified to include the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/129G, and wherein the OMVs are not chemically detoxified and/or treated with formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide, and combinations or derivatives thereof.

Embodiment 51. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene , modified to include the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/129G, and wherein Lipid A in OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure.

Embodiment 52. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene , modifi

- 16 045827 citated with the inclusion of mutations R9K and E129G, and expressing genetically detoxified pertussis toxoid PT-9K/129G, with knockout or deletion of the ArnT gene, where lipid A in OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure .

Embodiment 53. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene , modified to include the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/129G, wherein Lipid A in the OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and where the OMVs are not chemically detoxified and/ or treated with formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide and combinations or derivatives thereof.

Embodiment 54. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene , modified to include the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/129G, with knockout or deletion of the ArnT gene, where lipid A in OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and where OMVs are not chemically detoxified and/or treated with formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide and combinations or derivatives thereof.

Embodiment 55. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) an acellular pertussis antigen selected from the group consisting of (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA) and (3) pertactin (PRN), (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified to include the R9K and E129G mutations and expressing genetically detoxified pertussis toxoid PT- 9K/129G.

Embodiment 56. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) an acellular pertussis antigen selected from the group consisting of (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA) and (3) pertactin (PRN), (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified to include the R9K and E129G mutations and expressing genetically detoxified pertussis toxoid PT- 9K/129G, where the lipid A of the OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and where the OMVs are not chemically detoxified.

Embodiment 57. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) an acellular pertussis antigen selected from the group consisting of (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA) and (3) pertactin (PRN), (c) tetanus toxoid, and (d) diphtheria toxoid, wherein the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified to include the R9K and E129G mutations, with knockout or deletion of the ArnT gene, and expressing genetically detoxified pertussis toxoid PT-9K/129G, wherein the lipid A of the OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and where the OMV are not chemically detoxified and/or treated with formaldehyde, formalin, glutaraldehyde, peroxide hydrogen and their combinations or derivatives.

Embodiment 58. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA), and (3) pertactin (PRN), ( c) tetanus toxoid and (d) diphtheria toxoid, where the OMVs are derived from a Bordetella pertussis strain containing the S1 gene modified to include the R9K and E129G mutations and expressing genetically detoxified pertussis toxoid PT-9K/129G.

Embodiment 59. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA), and (3) pertactin (PRN), ( c) tetanus toxoid and (d) diphtheria toxoid, where OMV are derived from a Bordetella pertussis strain containing the S1 gene modified to include the R9K and E129G mutations, and expressing genetically detoxified pertussis toxoid PT-9K/129G, where lipid A in OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and where the OMVs are not chemically detoxified.

Embodiment 60. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs), (b) (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA), and (3) pertactin (PRN), ( c) tetanus toxoid and (d) diphtheria toxoid, where OMV

- 17 045827 are derived from a Bordetella pertussis strain containing the S1 gene, modified to include mutations R9K and E129G, and expressing genetically detoxified pertussis toxoid PT-9K/129G, with a knockout or deletion of the ArnT gene, where lipid A in OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure and where the OMVs are not chemically detoxified and/or treated with formaldehyde, formaldehyde, glutaraldehyde, hydrogen peroxide and combinations or derivatives thereof.

Embodiment 61. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs) containing genetically detoxified pertussis toxoid, (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid.

Embodiment 62. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs) containing genetically detoxified pertussis toxoid, wherein Lipid A in the OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure, (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid.

Embodiment 63. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs) containing genetically detoxified pertussis toxoid PT-9K/129G, (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid.

Embodiment 64. An immunogenic composition comprising (a) Bordetella pertussis outer membrane vesicles (OMVs) containing genetically detoxified pertussis toxoid PT-9K/129G, wherein Lipid A in the OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure , (b) acellular pertussis antigen, (c) tetanus toxoid, and (d) diphtheria toxoid.

Embodiment 65. An immunogenic composition according to embodiments 61-64, wherein 100% of the pertussis toxoid in the outer membrane vesicles is genetically detoxified PT.

Embodiment 66. The immunogenic composition according to embodiments 61-65, wherein 100% of the pertussis toxoid in the outer membrane vesicles is genetically detoxified pertussis toxoid PT-9K/129G.

General concepts

The term containing covers including, for example, a composition containing X may contain something additional, for example, X plus Y. The term essentially is not completely exclusive, for example, a composition substantially free of Y may be completely free of Y. B In some embodiments, the term comprising refers to the inclusion of said active agent, such as said polypeptides, as well as the inclusion of other active agents and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, and the like known in the pharmaceutical industry. In some embodiments, the term consisting essentially of refers to a composition whose sole active ingredient is the specified active ingredient(s), such as antigens; however, other compounds may be included that are intended to stabilize, preserve the composition, etc., but not are directly involved in the therapeutic effect of said active ingredient. The use of the transitional phrase consisting essentially means that the scope of the claim should be interpreted as covering the specific materials or steps specified in the claim and those that do not materially affect the essential and novel feature(s) of the claimed invention. See In re Herz, 537 F.2d 549, 55152, 190 USPQ 461, 463 (CCPA 1976) (emphasis added); see also MPEP § 2111.03. Thus, the term consisting essentially of, when used in a claim, should not be construed as equivalent to the term containing. Unless otherwise expressly stated, the term consisting of and its variations means limited. In some areas, instead of essentially consisting of, the term containing an active ingredient consisting of may be used. The term approximately, in relation to the numerical value of x, means, for example, x±10%, x±5%, x±4%, x±3%, x±2%, x±1%. The term essentially is not completely exclusive, for example, a composition substantially free of Y may be completely free of Y. If necessary, the word essentially may be omitted from the definition of the invention. Where the methods indicate steps of administration, for example, (a), (b), (c) and so on, these steps are intended to be sequential, that is, step (c) follows step (b), which is preceded by step (A). Antibodies will generally be specific for their target, that is, their affinity for the target will be higher than for an unrelated control protein such as bovine serum albumin.

Unless otherwise indicated, the method including the step of mixing two or more components does not require any particular order of mixing. Therefore, the components can be mixed in any order. In the case of three components, you can mix two components together, and then mix the resulting mixture with the third component, and so on.

Antibodies will usually be specific for their target. Thus, their affinity for the target will be higher than for an unrelated control protein such as bovine

- 18 045827 serum albumin.

When describing a component as adsorbed to an adjuvant, it is preferred that at least 50% (by weight) of the antigen is adsorbed, for example, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more. If a component is completely adsorbed, it should not be detected in the supernatant of the composition after centrifugation.

Amounts of conjugates are usually indicated by weight of saccharide (ie, the dose of the total conjugate (carrier plus saccharide) above the stated dose) to avoid variability depending on the choice of carrier.

Where substances of animal (and in particular bovine) origin are used in cell culture, they must be obtained from sources that are free from transmissible spongiform encephalopathy (TSE) and, in particular, free from bovine spongiform encephalopathy (BSE).

Embodiments of the Invention

Materials and methods

TdaP vaccine

Commercially available TdaP vaccines were used. The TdaP vaccine contains aluminum hydroxide as an adjuvant and tetanus toxoid, diphtheria toxoid, and acellular pertussis antigens (PT, FHA, and 69K, also called pertactin or PRN).

Bacterial strains and culture conditions

The following B. pertussis strains were used in this study: W28 PT 9K/129G (Pizza et al., 1989) with genetically detoxified pertussis toxin and its derivative arnT knockout (KO) strain without the arnT gene, obtained as described below. Storage and culture of bacteria were generally performed as described in Gasperini et al, 2018 and summarized below. Bacteria were stored at 80°C and reconstituted by plating on Bordet-Gengou (BG) agar plates supplemented with 15% (v/v) sheep blood for 3 days at 37°C. The bacteria were then inoculated at an initial optical density at 600 nm (OD600) of 0.05-0.1 in Stainer-Scholte medium supplemented with 0.4% (w/v) L-cysteine monohydrochloride, 0. 1% (w/v) FeSO ₄ , 0.2% (w/v) ascorbic acid, 0.04% (w/v) niacin, 1% (w/v) reduced glutathione. Cultures were grown in rotary shakers at 37°C. Recombinant E. coli DH5 strains were stored at 80°C and reconstituted by plating on LB agar plates supplemented with 20 g/ml chloramphenicol for 16 hours at 37°C. For liquid cultures, bacteria were inoculated into LB medium supplemented with 20 g/ml chloramphenicol and grown in rotary shakers at 37°C for 16 h.

Construction of arnTKO

To construct an arnT mutant B. pertussis strain similar to that described in Geurtsen et al., 2009, we amplified the portion of DNA upstream of arnT from B. pertussis strain W28 PT-9K/129G using primers -ATAGAATTCACGCGGTGCGGCGCCAGCGC-3 (SEQ ID NO: 1) and ^5' -ATAGGATCCGCCAGGACCTGGCCTGGCC-3 ¹ (seq id NO: 2), containing EcoRI and BamHI sites (underlined). In addition, a DNA fragment located after arnT was obtained by polymerase chain reaction.

5'- ATAGGATCCGGACGAAGCCTTCAAGGGGC-3' reactions (PCR) (SEQ ID with

NO:

primers

3)

5'- ATAAAGCTTCGTCCAGGCGCGCCAGCGC-3' (seq id _m 4) containing BamHI and HindIII sites (underlined). Both PCR products were cloned into pUC19 along with a kanamycin resistance (KanR) cassette containing BamHI sites at both ends. The resulting plasmids pUC19-arnT _up -KanRarnTdown were digested with EcoRI and HindIII restriction enzymes, and the resulting EcoRI-HindIII fragment was ligated into the suicide vector pSORTP1, treated with EcoRI-HindIII restriction enzymes. The final construct, named pSORTP1-arnT _KO , was used to construct arnT mutant B. pertussis (strain W28 PT-9K/129G) via allelic exchange. Transformants were screened by PCR using different sets of primers.

OMV cleaning

OMV was derived from W28 PT-9K/129G and its arnTKO derivative. Isolation of B. pertussis from liquid cultures was carried out after 3 days of cultivation in flasks with baffles with a volume of 250 ml. The ratio of liquid volume to air volume was important for the production yield of OMV and was maintained at 1:5. The bacteria were then pelleted by centrifugation at 5000 g for 30 minutes. Cell-free supernatants were collected and filtered through 0.22 μm Stericup filters (Millipore). After ultracentrifugation at 175,000 g for 2 hours, the resulting OMV pellet was washed with Dulbecco's phosphate buffered saline (D-PBS), another ultracentrifugation at 175,000 g for 2 hours, and finally resuspended in 100 μl of D-PBS. The amount of OMV was determined by Lowry total protein assay (DC Protein Assay, BioRad) following the manufacturer's instructions.

results

Outer membrane vesicle preparations were prepared from two detoxified vaccine strains of B. pertussis, strain W28 PT-9K/129G, expressing a genetically detoxified

- 19 045827 pertussis toxin, and its derivative strain W28 PT-9K/129G arnTKO with a genetically modified lipid A structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure as a result of deletion of the gene encoding ArnT. The arnTKO mutant W28 PT-9K/129G (Bp-ΔamT) exhibits 10-fold less binding to TLR4 (Fig. 1(a)) and induces significantly less IL-6 secretion from PBMCs than strain W28 PT-9K/129G ( Bp WT) (Fig. 1(b)).

Both OMVs of vaccine strain W28 9K/129G (Bp WT) and OMVs of vaccine strain W28 9K/129G arnTKO (Bp ΔamT) were shown to contain small amounts of FHA, 69K, and pertussis toxin components (Fig. 2). And mice immunized with both strains of OMV produced antibodies against all three acellular pertussis components at detectable levels (Fig. 3).

Immunization of mice to obtain mouse antiserum

BALB/c mice (female, 6 weeks old) (Charles River Laboratories International Inc., Wilmington, Massachusetts) received either three intraperitoneal (i.p.) immunizations three weeks apart or two intramuscular (i.m.) immunizations with at intervals of 4 weeks, using 100 μl of the composition (50 μl in each thigh). Sera were obtained 2 weeks after each immunization. OMV was formulated at the doses indicated for each study with 2 mg/ml Al(OH) ₃ . For combination formulations, OMV was included in the formulations at doses of 0.1, 0.5 and 2.5 μg along with 1/5 of the human dose of TdaP antigens (see below) in a total volume of 100 μl in 2 mg /ml Al(OH) ₃ .________________________________________________________________________________

Dose of TdaP vaccine used in humans (0.5 ml) RT FHA 69K D T A1(OH) ₃ 4 mcg 4 mcg 8 mcg 2 Lf (1.2 mcg) 5 Lf (3.2 mcg) 1 mg

Immunological analysis of mouse antisera using the Luminex method

Total IgG titers against all vaccine antigens were analyzed by the Luminex pentaplex immunoassay as described in Agnolon et al., 2015 and below. Titers are expressed in Relative Luminex Units per ml (RLU/ml), obtained by converting the reported median fluorescence intensity (MFI) relative to the hyperimmune control antiserum. The Luminex pentaplex immunoassay, based on MagPlex microspheres, was performed following the manufacturer's instructions to quantify anti-TdaP antibody titers in mouse sera. IgG titers were determined in individual animals after incubation of antigen-coupled microspheres with diluted sera (1:10,000, after 1; 1:20,000, after 2). Phycoerythrin-conjugated secondary antibody (1:400) was used for detection. For IgG measurements, median fluorescence intensity (MFI) was determined on a Luminex FLEXMAP 3D analyzer (Luminex Corporation, Austin, TX) using Bio-Plex Manager 5.0 software (Bio-Rad, Hercules, CA). Antiserum specific for each antigen was used to convert total IgG MFI values to RLU/mL (relative Luminex units). The limit of quantitation (LOQ) was determined for each antigen in the assay and used as the cutoff value to define positive results. The results are shown in Fig. 4.

Adhesion inhibition assay

For the B. pertussis adhesion inhibition assay, bacteria were grown for 16 h in liquid culture, then pelleted at 8000 g for 5 min and resuspended in d-PBS at OD ₆₀₀ 0.5. To fluorescently label B. pertussis cells, a 445-μL bacterial suspension was then mixed with 50 μL of 1 M NaHCO ₃ and 5 μL of Alexa Fluor 488 succinimidyl carboxylic acid ester (Life Technologies, Waltham, MA) and incubated for 15 min at 37°C. . After centrifugation at 8000 g for 5 min at room temperature, the supernatant was removed and the pellet was washed once with 1 ml d-PBS to remove unbound dye and finally the bacteria were resuspended in F12-K medium at OD ₆₀₀ 0.2. Pooled mouse sera were serially diluted 4-fold in F-12K medium containing 1% (v/v) serum from nonimmunized mice and incubated with labeled B. pertussis for 1 h at 37°C in a 1:1 ratio. . One hundred microliters of bacteria/serum mixtures were transferred in triplicate to seeded A549 cells. Infected cells were incubated for 1 h at 37°C. After extensive washing to remove unbound bacteria, excitation/emission fluorescence was measured at 485/535 nm using a Tecan Infinite F200PRO microplate reader.

Immunization of mice with wP or three different doses of OMV resulted in the induction of antibodies that can inhibit the ability of B. pertussis to adhere to cultured A549 epithelial cells in vitro. Sera from mice immunized with inactivated whole bacteria showed the greatest inhibition of bacterial adhesion, and the inhibition of bacterial adhesion induced by OMV vaccines was proportional to the antigen dose. TdaP vaccine did not produce high levels of fluorescently labeled adhesion-inhibiting antibodies

- 20 045827 bacteria, in mice, and only small dilutions of sera (1/40 and higher) led to a decrease in fluorescence in this analysis. It was found that OMV at a maximum dose of 10 μg after vaccination induced antibodies providing the greatest inhibition of bacterial adhesion, which was comparable to that induced in the whole bacteria control (Fig. 5).

Kendrick efficacy test with intracranial challenge

CD1 mice (6 weeks old) were vaccinated once intraperitoneally (ip) with 500 μl of vaccine compositions and 2 weeks after immunization, a challenge was carried out by intracranial injection of 30 μl of a suspension of B. pertussis strain 18323 and the survival of mice was determined for 2 weeks after challenge according to the Kendrick intracranial challenge potency test (Kendrick et al., 1947) and according to the European Pharmacopoeia guidelines. The NIBSC standard was used as a positive control at three doses (1/10, 1/50 and 1/250 of the human dose) and OMV was included in the compositions at doses of 0.4, 2 and 10 μg in 2 mg/ml Al (OH) ₃ .

The mouse protection test after intracerebral challenge (Kendrick test) is effective in determining the effectiveness of whole-cell pertussis vaccines and is the only test that has demonstrated a correlation with protection in children (Xing et al., 2014 [40]). Immunization of mice with escalating doses of OMV (0.4, 2, and 10 μg) provided increasing levels of protection following intracranial challenge with B. pertussis (Fig. 6), which was comparable to the NIBSC wP standard. The protection induced by the OMV vaccines was proportional to the antigen dose and successfully resulted in a strong protective response comparable to that with wP (Fig. 6).

Aerosol control infection

C57BL/6 mice (female, 10 weeks old) were vaccinated with a single intraperitoneal (IP) injection of 100 μl of vaccine formulations and challenged 3 weeks later as previously described ( Misiak et al., 2017a ) and below. Whole cell pertussis (wP) vaccine was used as a positive control at the indicated doses of 1/5 or 1/10 the human dose. OMV in the indicated doses of 0.4, 2 and 10 μg was included in compositions with 2 mg/ml Al(OH)3. For combination formulations, OMV was formulated at a dose of 2.5 μg along with 1/5 of the human dose of TdaP antigens (see below) in a total volume of 100 μl in 2 mg/ml Al(OH) ₃ .

Dose of TdaP used in humans RT FHA PRN TT D.T. Alum (dose volume 0.5 ml) (8 mcg) (8 mcg) (2.5 mcg) (5 Lf) (2Lf) (1 mg)

Serum and spleen samples from immunized mice (4 mice per group) were obtained 3 weeks after vaccination, one day before challenge. Antigen-specific cytokine production by spleen cells and serum antibodies were analyzed by ELISA. The remaining mice were then challenged with a virulent strain of B. pertussis as described below, and lung CFU counts were assessed at days 1, 3, 7, and 14 postinfection.

Airborne infection of mice was carried out by exposing them to an aerosol of B. pertussis (strain BP338; 1x10 ⁹ CFU/ml) for 10 minutes, followed by 10 minutes of rest, using a PARI TurboBOY SX nebulizer. The course of B. pertussis infection was assessed by performing lung CFU counts in groups of three to four mice at specified intervals after challenge. The lungs were aseptically removed and homogenized in 1 ml of sterile saline with 1% casein. Undiluted and serially diluted homogenates (100 μl) from individual lungs were plated in duplicate on Bordet-Gengou agar plates, and bacterial colonies were counted after 6 days of incubation at 37°C.

Antibody titers were measured by ELISA as described previously ( Misiak et al., 2017b ) and below. The amount of FHA-specific antibodies was analyzed by ELISA using FHA (1 μg/ml) adsorbed onto plates, biotin-conjugated anti-mouse IgG1 or IgG2a antibodies, and peroxidase-conjugated streptavidin (BD Pharmingen, Franklin Lakes, NJ). . Antibody levels are expressed as the mean final titer plus/minus SEM, determined by extrapolating the linear portion of the titration curve to 2 SE above the background value obtained using nonimmune serum.

Immunization of mice with wP and three different doses of OMV resulted in the induced production of antibodies specific for B. pertussis. wP resulted in the highest total anti-FHA IgG titers, which were significantly higher than all three OMV doses (Fig. 7). Vaccination with wP resulted in the highest Th1 responses and, accordingly, the highest IgG2c titers against FHA (Fig. 7). Anti-FHA IgG2c was detected in the sera of 1 of 4 and 3 of 4 mice vaccinated with OMV 2 and OMV 10, respectively. No FHA-specific IgG2c was detected in mice immunized with OMV 0.4. Differences in FHA-specific responses indicate significantly lower FHA content in OMV compared to wP.

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High titers of protective antibodies, together with the induction of a Thl response, are a key factor for protection against B. pertussis. Single immunizations with wP, OMV 0.4, OMV 2, and OMV 10 resulted in varying levels of protection against airborne challenge with B. pertussis (Fig. 8). In mice immunized with the wP vaccine, the level of protection was the highest, and in most of them the infection cleared by 7 days after infection. In addition, mice immunized with wP had significantly lower CFU counts at all time points postinfection than mice receiving OMV vaccine at all three concentrations tested. The protection induced by OMV vaccines was proportional to the antigen dose. However, the protection induced by OMV 10 was not as effective as that induced by vaccination with wP. The areas under the clearance curves confirmed that wP provided mice with the best protection against aerosol challenge of B. pertussis. OMV at a maximum dose of 10 μg was found to provide the highest level of protection in a mouse model of B. pertussis challenge. However, their effectiveness was still significantly lower than that of the wP vaccine. It was also found that FHA-specific IgG2c responses were highest in mice receiving wP vaccine, but serum FHA-specific IgG2c levels increased as the dose of OMV vaccine was increased.

The data presented here demonstrate that the level of protection provided by OMV vaccines is directly proportional to the dose of antigen administered. They also show that even at the highest dose selected, OMV vaccines do not provide the same protection as wP.

To compare the effectiveness of protection against B. pertussis challenge that OMVs can have when formulated in combination with acellular pertussis antigens, an aerosol challenge model was used with alum or alum plus OMV added to the acellular pertussis vaccine and with whole cell vaccine as a positive control. . Immunization of mice with wP, aP, and aP plus OMV resulted in high titers of total anti-FHA IgG (Fig. 9). In addition, high titers of total IgG were detected in the OMV-only group. There were no significant differences between the four vaccine groups. High IgG1 titers against FHA were detected in all vaccinated groups, with the highest IgG1 titers being detected in the sera of mice vaccinated with aP and aP plus OMV (Fig. 9). However, no significant differences were found between the groups. FHA-specific IgG2c was detected in the sera of 2 of 4 mice vaccinated with wP, 4 of 4 mice vaccinated with aP plus OMV, and 1 of 4 mice vaccinated with OMV (Fig. 9c). Anti-FHA IgG2c was not detected in the aP group. The addition of OMV significantly enhanced the IgG2c response to aP.

Single immunization of mice with wP, aP, aP plus OMV, as well as OMV alone was found to provide protection against airborne challenge with B. pertussis (Fig. 10). In mice immunized with the wP vaccine, the level of protection was the highest, and the infection resolved by 7 days after infection. Vaccination with aP and OMV provided similar levels of protection. However, the effectiveness of these vaccines was significantly lower than that of the wP vaccine. In contrast, the combination of aP plus OMV was found to provide a level of protection comparable to that induced by the wP vaccine. There were no significant differences between the two vaccines at any of the time points analyzed. The areas under the clearance curves confirmed that wP and aP plus OMV were the most effective of the vaccines analyzed.

In summary, a single immunization with all vaccine compositions tested at a dose equal to 1/5 the human dose induced protective immunity against B. pertussis in C57BL6 mice. It was found that immunization with OMVs themselves also provided significant protection comparable to that induced by the aP vaccine.

Strong Th1 responses were detected in mice vaccinated with wP, aP plus OMV, and OMV alone, but not in mice vaccinated with aP. Enhanced Th1 responses in mice vaccinated with aP plus OMV compared with mice receiving the aP vaccine correlated with high anti-FHA IgG2c titers that were almost absent in mice immunized with aP. The data presented here demonstrate that the addition of OMV to the aP vaccine significantly increases its protective efficacy and results in a switch to anti-FHA IgG2c production and Th1 responses in both prime and booster vaccinations (Fig. 11).

Booster vaccination

In previous experiments, mice that had not previously been immunized were used for immunization. This corresponds to primary vaccination (i.e. childhood vaccines). However, booster vaccination is also required later in life, and most subjects in developed countries will receive currently commercially available vaccines, which are more likely to induce a Th2 response. This situation has been reproduced using a commercially available vaccine for children

- 22 045827 (Infanrix Hexa) for the first (primary) immunization and various commercially available or investigational booster compositions, including TdaP plus OMV, for secondary immunization.

Addition of OMV to the TdaP vaccine significantly enhanced Th1 responses, while various TdaP vaccines, even in the presence of a Th1-inducing adjuvant (Alum-TLR7), did not change Th balance (Fig. 12).

conclusions

With regard to the immunogenicity of the studied vaccine compositions, the following was established.

In mice immunized with all compositions tested, OMV, diphtheria, tetanus and pertussis vaccines resulted in titers of specific antibodies to the corresponding antigens.

No immunological interactions were observed between OMV and combination tetanus/diphtheria/pertussis vaccines.

IgG and functional humoral immune responses to vaccination with the OMV/diphtheria/tetanus/pertussis antigen combination resulted in significantly higher titers than those observed following vaccination with OMV or TdaP vaccine alone.

Addition of OMV to aP vaccine results in a switch to IgG2c production against FHA, and Th1 responses were detected in mice vaccinated with wP, aP plus OMV, and OMV alone, but not in mice vaccinated with aP.

The addition of OMV to the aP vaccine in a combination vaccine significantly increases its protective efficacy compared with the protective responses to OMV or aP alone. In conclusion, there was no evidence of a strong interaction between any of the vaccines studied. Immunization with OMVs alone was found to provide significant protection in the Kendrick efficacy test and in the aerosol challenge model. Addition of OMV to acellular pertussis vaccines results in greater protection than aP or OMV vaccines alone. Without being limited by theory, this may be due to additional protective antigens present in the vesicles and/or the induction of a favorable Th1 profile in both primary and booster vaccinations.

It should be understood that the invention has been described by way of example only and may be modified without departing from the scope and spirit of the invention.

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Claims (15)

CLAIM 1) in a diphtheria toxoid: tetanus toxoid ratio greater than 1, preferably from 2:1 to 3:1, when measured in Lf units, more preferably 2.5:1, or 1) from 4 Lf (flocculation units)/ml to 8 Lf/ml, preferably 4 Lf per 0.5 ml dose, or 1. An immunogenic composition containing (a) outer membrane vesicles (OMVs), (b) acellular pertussis antigen, (c) tetanus toxoid and (d) diphtheria toxoid in immunologically effective quantities, wherein the OMVs are derived from a strain of Bordetella pertussis expressing genetically detoxified pertussis toxoid PT-9K/129G. 2) in a ratio of tetanus toxoid: diphtheria toxoid greater than 1, preferably from 1.5:1 to 2.5:1, when measured in Lf units, more preferably 2:1. 2) from 20 to 50 Lf/ml, preferably 25 Lf per 0.5 ml dose. 2. The immunogenic composition according to claim 1, where the OMVs originate from the Bordetella pertussis strain containing the S1 gene, modified to include the R9K and E129G mutations, and expressing the genetically detoxified pertussis toxoid PT-9K/129G. 3. An immunogenic composition according to claim 2, where the OMVs originate from a Bordetella pertussis strain with a knockout or deletion of the ArnT gene. 4. An immunogenic composition according to any one of claims 1 to 3, wherein Lipid A in OMV has a modified structure without glucosamine (GlcN) substitutions on the distal phosphate groups of the central structure. 5. The immunogenic composition according to any one of claims 1 to 4, wherein the acellular pertussis antigen is selected from the group consisting of (1) detoxified pertussis toxin (PT), (2) filamentous hemagglutinin (FHA), and (3) pertactin (PRN). 6. The immunogenic composition according to claim 5, where the acellular pertussis antigen contains PT, FHA and PRN. 7. Immunogenic composition according to claim 6, where RT is a genetically detoxified pertussis toxoid. 8. An immunogenic composition according to claim 6 or 7, wherein PT, FHA and PRN are present in a ratio of 16:16:5, determined by weight. 9. Immunogenic composition according to any one of claims 1 to 8, where diphtheria toxoid is present in a concentration 10. Immunogenic composition according to any one of claims 1 to 9, where tetanus toxoid is present in a concentration of 5 to 10 Lf per 0.5 ml dose. 11. Immunogenic composition according to any one of claims 1 to 10, where diphtheria toxoid and tetanus toxoid are present: 12. An immunogenic composition according to any one of claims 1 to 11, containing an adjuvant, possibly an adjuvant based on an aluminum salt. 13. Immunogenic composition according to any one of claims 1 to 12, intended for use as a medicine. 14. An immunogenic composition according to any one of claims 1 to 13, intended to induce an immune response in a patient. 15. An immunogenic composition according to claim 14, intended for inducing an immune response against Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis in a patient. -