Immunogenic composition

Abstract

The present invention relates to bacterial strains, in particular for use in the field of vaccines, and in particular in the field of prevention or treatment of infections caused by bacteria of the genus Bordetella.

Description

[Technical field]

[0001] The present invention relates to the field of vaccines, in particular to the prevention or treatment of infections caused by bacteria of the genus Bordetella, in particular Bordetella pertussis. [Background technology]

[0002] Whooping cough, caused by Bordetella pertussis, is a highly contagious respiratory tract infection. Acute infection can lead to severe illness characterized by respiratory failure, pulmonary hypertension, leukocytosis, and death. Whooping cough is a vaccine-preventable disease, and both acellular pertussis (aP) and whole cell pertussis (wP) vaccines are in use worldwide. However, the disease persists in vaccinated populations, and epidemiological data have reported a worldwide increase in whooping cough incidence in recent years.

[0003] Several hypotheses have been postulated, including strain evolution of B. pertussis species and a reduced duration of protection from the aP vaccine compared with the wP vaccine. One area of ​​research aimed at controlling the recurrence of pertussis is targeted at the development of new vaccines.

[0004] Pathogen-derived vesicles have been used to develop immunogenic compositions, such as vaccines. [1] By using outer membrane vesicles (OMVs) or their derivatives, it is possible to potentially deliver a wide range of antigens in their native form.

[0005] OMVs contain bacterial lipopolysaccharide (LPS), which is composed of a highly variable O-antigen, a less variable core oligosaccharide, and a highly conserved lipid moiety called lipid A. Bacterial lipooligosaccharide (LOS) shares a similar lipid A structure with the same set of functional activities as LPS, but lacks the O-antigen unit. In the art, the term "LPS" is often used to refer to both the LPS and LOS bacterial saccharides.

[0006] However, the presence of LPS / LOS in OMVs may present problems from a clinical and regulatory perspective. For example, if LPS / LOS is degraded in the human body, it may induce the production of excessive proinflammatory cytokines. Through their interaction with human TLR4, cytokines may cause several adverse effects (reactogenicity), e.g., fever, chills, shock, and various other symptoms, depending on the particular organism and patient condition.

[0007] The endotoxic activity of LPS / LOS is determined primarily by the composition of its lipid A moiety, which consists of a glucosamine disaccharide substituted with one or two phosphate groups and a variable number of acyl chains [2].

[0008] WO2018 / 167061[3] describes a method for identifying Pseudomonas aeruginosa (LpxA Pa ) or Neisseria meningitidis (LpxA Nm ) to shorten the C3' acyl chain length of Bordetella pertussis. The exogenous LpxA gene was expressed from a plasmid, while the endogenous LpxA gene in the genome was knocked out. Pa Episomal expression of LpxA had the effect of shortening the length of some acyl chains, and the authors noted that TLR4 stimulation was reduced. However, the strain exhibited a strong growth defect. In addition, LpxA Nm Expression of LpxD was also lethal. A similar strong growth defect was observed when an LpxD variant from P. aeruginosa was expressed in an episomal fashion in B. pertussis. While this result confirms that reactogenicity can be modified by manipulation of lipid A, the growth defect of the strains is a major obstacle to using such strains in vaccine production. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there remains a need for improved immunogenic compositions, suitable for example for the production of vaccines. [Means for solving the problem]

[0010] The applicant has developed a novel recombinant Bordetella pertussis bacterial strain which solves the problems observed in the art and is particularly suitable for the preparation of whole cell antigens and / or outer membrane vesicle components. The components of the invention can be used in immunogenic compositions, e.g. in vaccines.

[0011] In a first aspect, the present invention relates to a recombinant Bordetella pertussis bacterium. In particular, the present invention relates to a recombinant Bordetella pertussis bacterium comprising at least one genomic LpxA gene encoding an LpxA protein comprising a mutation at position 170 and / or a mutation at position 229 relative to SEQ ID NO: 1, and / or at least one genomic insertion of a heterologous LpxD gene.

[0012] In this first aspect, the present invention preferably relates to a recombinant Bordetella pertussis bacterium, comprising at least one genomic LpxA gene encoding an LpxA protein comprising a mutation at position 170 and / or a mutation at position 229 relative to SEQ ID NO:1.

[0013] The wild-type LpxA protein from Bordetella pertussis (LpxA Bpe The amino acid sequence of LpxA protein from Bordetella parapertussis (LpxA) is provided as SEQ ID NO: 1. Bpa ) is provided as SEQ ID NO:2.

[0014] The present invention may relate to a recombinant Bordetella pertussis bacterium comprising at least one genomic LpxA gene encoding an LpxA protein, wherein the LpxA gene is derived from Bordetella parapertussis (LpxA Bpa ).

[0015] In such an embodiment, the invention relates to a recombinant Bordetella pertussis bacterium comprising at least one genomic LpxA gene encoding the LpxA protein having SEQ ID NO:2.

[0016] The term "mutation" as used herein refers to a deletion, addition or substitution of an amino acid residue in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide, particularly a wild-type protein or polypeptide.

[0017] The term "substitution" when referring to an amino acid sequence refers to a change of an amino acid to a different amino acid or amino acid moiety. For example, when referring to LpxA, a substitution refers to an amino acid change relative to SEQ ID NO: 1. Substitutions of amino acids at a particular position in a protein sequence are referred to using the following notation: (amino acid residue in wild type protein) (amino acid position) (amino acid residue in mutant protein)". For example, G229A refers to the substitution of a glycine (G) residue at position 229 of the amino acid sequence of a reference protein (here SEQ ID NO: 1) with an alanine (A) residue (in the mutant).

[0018] In particular, the mutation at position 170 is a substitution relative to SEQ ID NO: 1. Even more particularly, the mutation at position 170 is a substitution of a glycine. Even more particularly, the mutation at position 170 is a substitution of a glycine with a serine (G170S).

[0019] In particular, the mutation at position 229 is a substitution relative to SEQ ID NO: 1. Even more particularly, the mutation at position 229 is a substitution of glycine. Even more particularly, the mutation at position 229 is a substitution of glycine with alanine (G229A).

[0020] In particular, the LpxA protein comprises a mutation at position 170 and a mutation at position 229 relative to SEQ ID NO: 1. Even more particularly, the mutation at position 170 is a substitution of glycine and the mutation at position 229 is a substitution of glycine. Even more particularly, the mutation at position 170 is a substitution of glycine with serine (G170S) and the mutation at position 229 is a substitution of glycine with alanine (G229A). Even more particularly, the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229. In particular, in addition to the mutations at positions 170 and / or 229, the LpxA protein has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity to either SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the LpxA protein has 90% sequence identity to SEQ ID NO: 2. In some embodiments, the LpxA protein has the amino acid sequence of SEQ ID NO: 2 (i.e., has 100% sequence identity).

[0021] The LpxD protein from Comamonas testosteroni (LpxD Ct The amino acid sequence of LpxD protein from Pseudomonas aeruginosa (LpxD Pa ) is provided as SEQ ID NO:4.

[0022] In particular, the heterologous LpxD gene encodes an LpxD protein having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity to either SEQ ID NO:3 or SEQ ID NO:4. More particularly, the heterologous LpxD gene encodes an LpxD protein having at least 95% amino acid sequence identity to either SEQ ID NO:3 or SEQ ID NO:4. Even more particularly, the heterologous LpxD gene encodes an LpxD protein having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the heterologous LpxD gene encodes an LpxD protein having the amino acid sequence of SEQ ID NO:3. In some embodiments, the heterologous LpxD gene encodes an LpxD protein having the amino acid sequence of SEQ ID NO:4.

[0023] The term "heterologous LpxD gene" refers to an LpxD gene or nucleotide sequence derived from a species different from that of the host organism into which it is introduced; in the context of the present invention, the heterologous LpxD gene or nucleotide sequence is not normally found in wild-type strains of B. pertussis. In certain embodiments, the nucleotide sequence of the heterologous LpxD gene can be codon-optimized for expression in B. pertussis.

[0024] The term "genomic" refers to the bacterial cell genome, i.e., chromosomal DNA, while "genomic insertion" is used to refer to stable integration into the bacterial cell genome, thereby excluding transient or episomal expression, such as expression of a gene on a plasmid.

[0025] In a first aspect of the invention, the expression or activity of the endogenous LpxA gene in the recombinant B. pertussis bacteria is modified, reduced, suppressed or inactivated relative to wild-type B. pertussis bacteria. In some embodiments of the invention, the expression or activity of the endogenous LpxD gene in the recombinant B. pertussis bacteria may be modified, reduced, suppressed or inactivated relative to expression or activity found in wild-type B. pertussis bacteria. In some embodiments of the invention, the expression or activity of both the endogenous LpxA gene and the endogenous LpxD gene in the recombinant B. pertussis bacteria is modified, reduced, suppressed or inactivated relative to wild-type B. pertussis bacteria.

[0026] In some embodiments, the expression or activity of the endogenous LpxA gene is reduced, suppressed or inactivated by knocking out the endogenous LpxA gene. In some embodiments, the expression or activity of the endogenous LpxA gene is modified, reduced, suppressed or inactivated by replacing the endogenous LpxA gene, for example, via knock-in. In some embodiments, the expression or activity of the endogenous LpxA gene is modified by mutating the endogenous LpxA gene, in particular by mutation at positions 170 and / or 229. In some embodiments, the expression or activity of the endogenous LpxD gene is reduced, suppressed or inactivated by knocking out the endogenous LpxD gene. In some embodiments, the expression or activity of the endogenous LpxD gene is modified, reduced, suppressed or inactivated by replacing the endogenous LpxD gene, for example, via knock-in. As one non-limiting example, a knockout can be achieved by removing, e.g., deleting, part or all of the endogenous gene or by inactivating the endogenous promoter that activates expression of the respective endogenous gene.

[0027] Surprisingly, the recombinant Bordetella pertussis bacterial strain of the invention does not exhibit the growth defects observed in the art.

[0028] In particular, the growth profile of the population of recombinant B. pertussis bacteria of the invention is comparable to the growth profile of the population of a wild-type parent strain of B. pertussis bacteria. More particularly, the growth curve of the population of recombinant B. pertussis bacteria is comparable to the growth curve of the population of a wild-type parent strain of B. pertussis bacteria. For example, the values ​​of elapsed time and growth rate of the population of recombinant B. pertussis bacteria and the population of wild-type Gram-negative bacteria in a standard fermentation process are comparable. More particularly, the values ​​of elapsed time and growth rate of the population of recombinant B. pertussis bacteria and the population of wild-type B. pertussis bacteria vary by less than 20%, such as less than 15%, less than 10% or less than 5%. Even more particularly, the values ​​of elapsed time and growth rate of the population of recombinant B. pertussis bacteria and the population of wild-type B. pertussis bacteria are substantially similar.

[0029] The recombinant Bordetella pertussis bacterial strains of the invention express lipid A that exhibits reduced endotoxin activity relative to lipid A expressed by wild-type Bordetella pertussis bacteria, in particular as measured using a TLR4 stimulation assay, in particular a human TLR4 stimulation assay, for example through NF-κB induction in a recombinant HEK-TLR4 reporter cell line. Those skilled in the art will be aware of other suitable assays.

[0030] In particular, the recombinant Bordetella pertussis bacterial strains of the invention are derived from the Tohama I parent strain, more particularly from the Tohama I PTg strain, a strain of Tohama I that expresses a genetically detoxified pertussis toxoid. As used herein, the term "parent strain" takes its general meaning in the art, referring to the strain of Bordetella pertussis that is used as the starting microorganism to construct a recombinant strain. The parent strain as a whole is the wild-type or comparison strain against which the characteristics of the recombinant strain of the invention are compared. The recombinant Bordetella pertussis bacteria of the invention are as a whole isogenic to its parent strain, except for the genetic modification(s) of the invention described herein.

[0031] In a second aspect of the invention there is provided an outer membrane vesicle (OMV), particularly a population of outer membrane vesicles, derived from the recombinant Bordetella pertussis bacterium of the first aspect. There is also provided a whole cell antigen derived from the recombinant Bordetella pertussis bacterium of the first aspect.

[0032] In a third aspect of the present invention, there is provided an immunogenic composition comprising at least one OMV or a population of OMVs according to the second aspect and a pharma- ceutically acceptable excipient. In particular, the immunogenic composition may also comprise an adjuvant. In some embodiments, the immunogenic composition of the present invention comprises at least one antigen in addition to those present in the OMV(s). In a preferred embodiment of the present invention, the at least one further antigen is selected from the group consisting of: (1) pertussis toxoid (PT), (2) FHA, (3) pertactin (PRN), (4) FIM2 / FIM3, (5) adenylate cyclase, (6) diphtheria toxoid (DT), (7) tetanus toxoid (TT), (8) inactivated poliovirus (IPV), (9) hepatitis B surface antigen, and (10) Hib PRP.

[0033] In a fourth aspect of the invention there is provided a recombinant Bordetella pertussis bacterium according to the first aspect, at least one OMV or population of OMVs according to the second aspect, or an immunogenic composition according to the third aspect for use in inducing an immune response in a suitable mammal.

[0034] In particular, there is provided a recombinant Bordetella pertussis bacterium according to the first aspect, at least one OMV or population of OMVs according to the second aspect, or an immunogenic composition according to the third aspect for use in therapy and / or prophylaxis, e.g. for use as a vaccine.

[0035] In particular, there is provided a method of inducing an immune response in a suitable mammal comprising administering to a suitable mammal a recombinant Bordetella pertussis bacterium according to the first aspect, at least one OMV or population of OMVs according to the second aspect, or an immunogenic composition according to the third aspect.

[0036] In particular, there is provided a recombinant Bordetella pertussis bacterium according to the first aspect, at least one OMV or population of OMVs according to the second aspect, or an immunogenic composition of the third aspect, for use in the manufacture of a medicament, e.g. for the treatment and / or prophylaxis, e.g. for use as a vaccine. In one embodiment there is provided a recombinant Bordetella pertussis bacterium according to the first aspect, at least one OMV or population of OMVs according to the second aspect, or an immunogenic composition of the third aspect, for use in the manufacture of a medicament for the treatment and / or prophylaxis of a disease caused by Bordetella pertussis.

[0037] In a fifth aspect of the invention, there is provided a method for modulating the reactogenicity of lipid A of Bordetella pertussis bacteria, comprising stably integrating into the genome of the bacterium at least one gene selected from the group consisting of LpxA and LpxD, wherein: (i) the LpxA gene encodes an LpxA protein having at least 80% sequence identity to SEQ ID NO: 1 or 2, wherein said protein comprises a serine at position 170 (S170) and / or an alanine at position 229 (A229) when numbered according to SEQ ID NO: 1 or 2; and / or (ii) the LpxD gene encodes an LpxD protein having at least 90% amino acid sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; A method is provided.

[0038] Preferably, in this fifth aspect of the invention, the method for modulating the reactogenicity of lipid A of Bordetella pertussis bacteria comprises stably integrating an LpxA gene into the genome of the bacterium, wherein the LpxA gene encodes an LpxA protein having at least 80% sequence identity to SEQ ID NO: 1 or 2, wherein said protein comprises a serine at position 170 (S170) and / or an alanine at position 229 (A229) when numbered according to SEQ ID NO: 1 or 2.

[0039] In one embodiment, the method of the present invention is directed to Bordetella parapertussis (LpxA Bpa The method includes stably integrating the LpxA gene derived from LpxA into the bacterial genome. [Brief description of the drawings]

[0040] [Figure 1A] FIG. 1 is a diagram of the structure of lipid A from Bordetella pertussis. [Figure 1B] FIG. 1 is a diagram of the structure of lipid A from Bordetella parapertussis, where the dotted lines indicate naturally occurring variant structures, in particular minor variants with one and two secondary C16 acyl chains that may additionally be present, and loss of the C10 chain. [Figure 2-1] FIG. 1 is an alignment of LpxA amino acid sequences from Bordetella genus. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3-1] Fermentation characteristics of Bordetella pertussis Tomaha I PTg. Curve 1: pH is adjusted to 7.2 by addition of acetic acid (curve 3) to avoid basification of the cell broth which would lead to the stopping of the culture, curve 2: dissolved oxygen concentration, curve 4: Dissolved oxygen concentration (DO) is maintained at 25% by manipulating the agitation speed (DO / speed adjustment) to avoid oxygen limitation, curve 5: temperature is adjusted to 35°C. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 4-1] Fermentation characteristics of recombinant Bordetella pertussis strain of the invention expressing LpxA from Bordetella parapertussis (ΔLpxABpe / LpxABpa). Curve 2: Dissolved oxygen concentration (DO) is maintained at 25% by manipulating the agitation speed to avoid oxygen limitation (DO / speed adjustment). Curve 3: Temperature is regulated at 35° C. Curve 4: pH is adjusted to 7.2 by addition of acetic acid (curve 1) to avoid basification of the cell broth, which would lead to the stopping of the culture. Curve 5: Dissolved oxygen concentration. NB: Due to clogging of the exhaust filter, DO and agitation speed dropped to 0 during the filter exchange process for about 20 minutes. This did not affect the fermentation results. [Figure 4-2] This is a continuation of Figure 4-1. [Diagram 5] FIG. 13 shows the agitation rate fermentation characteristics of ΔLpxABpe / LpxABpa (dashed line) and the parent strain, Tohama I PTg (black). [Figure 6A]FIG. 11. Structural analysis of LOS (OMV from ΔLpxABpe / LpxABpa B. pertussis expressing LpxABpa integrated into the genome) by LC-MS. [Figure 6B] 1 is a diagram of the LOS structure determined by LC-MS. The solid line shows the structure of the LOS. The dotted lines illustrate the difference between the LOS structure and that found in the wild-type Bordetella pertussis strain Tohama I. [Figure 7A] FIG. 1 shows the structural analysis of LOS (OMV derived from Bordetella pertussis, expressing LpxABpa in an episomal manner) by LC-MS. [Figure 7B] FIG. 1. LOS structure of minor variants determined by LC-MS (dotted lines illustrate the difference between this LOS and the LOS structure found in wild-type Bordetella pertussis strain Tohama I). [Figure 8A] FIG. 1 shows the structural analysis of LOS (OMV derived from Bordetella pertussis ΔArnT / ΔLpxABpe / LpxABpa, a double knockout variant of ArnT and LpxABpe expressing LpxABpa integrated into the genome) by LC-MS. [Figure 8B] 1 is a diagram of the LOS structure determined by LC-MS. The solid line shows the structure of the LOS. The dotted lines illustrate the difference between the LOS structure and that found in the wild-type Bordetella pertussis strain Tohama I. [Figure 9A] FIG. 1 shows the structural analysis of LOS (OMV derived from Bordetella pertussis ΔLpxABpe / LpxABpa / ΔDNT / LpxDPa, a double knockout variant of LpxABpe and DNT expressing LpxABpa and LpxDPa integrated into the genome) by LC-MS. [Figure 9B] 1 is a diagram of the LOS structure determined by LC-MS. The solid line shows the structure of the LOS. The dotted lines illustrate the difference between the LOS structure and that found in the wild-type Bordetella pertussis strain Tohama I. [Figure 10A]FIG. 1 shows the structural analysis of LOS (OMV derived from Bordetella pertussis ΔLpxABpe / LpxABpa / ΔDNT / LpxDPa / ΔLpxDBpe, a triple knockout variant of LpxABpe, DNT and LpxDBpe expressing LpxABpa and LpxDPa integrated into the genome) by LC-MS. [Figure 10B] 1 is a diagram of the LOS structure determined by LC-MS. The solid line shows the structure of the LOS. The dotted lines illustrate the difference between the LOS structure and that found in the wild-type Bordetella pertussis strain Tohama I. [Figure 11A] FIG. 11. Structural analysis of OMV derived from Bordetella pertussis LpxDPa expressing LOS(LpxDPa in an episomal manner by LC-MS. [Figure 11B] 1 is a diagram of the LOS structure determined by LC-MS. The solid line shows the structure of the LOS. The dotted lines illustrate the difference between the LOS structure and that found in the wild-type Bordetella pertussis strain Tohama I. [Figure 12A-1] FIG. 1 shows the structural analysis of LOS (OMV derived from Bordetella pertussis ΔArnT / PagLBbr, an ArnT knockout variant expressing PagLBbr) by LC-MS. [Figure 12A-2] This is a continuation of Figure 12A-1. [Figure 12B] 1 is a diagram of the LOS structure determined by LC-MS. The solid line shows the structure of the LOS. The dotted lines illustrate the difference between the LOS structure and that found in the wild-type Bordetella pertussis strain Tohama I. [Figure 13A] FIG. 1 shows the structural analysis of LOS (OMV derived from wild-type Bordetella pertussis Tomaha I PTg) by LC-MS. [Figure 13B] FIG. 1 is a diagram of the LOS structure determined by LC-MS. [Figure 14]Figure 1. In vitro activation of TLR4 signaling in HEK-hTLR4 cells. HEK-TLR4 cells were stimulated with OMVs from recombinant Bordetella pertussis strains; OMVs from Bordetella pertussis Tohama I PTg strain (wild type control) based on protein content, or with BEXSERO, QUINVAXEM, or INFANRIX HEXA vaccines based on human dose equivalence (vaccine control). Unstimulated cells were included as negative controls (data not shown). [Figure 15] Figure 1. In vitro activation of TLR4 signaling in HEK-hTLR4 cells. HEK-TLR4 cells were stimulated based on LOS content with OMVs from recombinant Bordetella pertussis spp. strains; OMVs from Bordetella pertussis Tohama I PTg strain (wild-type control); purified LPS from Bordetella pertussis Tomaha I PTg; or detoxified purified LPS from Neisseria meningitis B □MsbB strain (□MsbB). Unstimulated cells were included as negative controls. [Figure 16-1] Figure 1. In vitro activation of TLR4 signaling in HEK-hTLR4 cells. HEK-TLR4 cells were stimulated with OMVs from recombinant Bordetella pertussis strains; OMVs from Bordetella pertussis Tohama I PTg strain (wild type control); or BEXSERO, QUINVAXEM and INFANRIX HEXA vaccines based on protein content. Non-stimulated cells were included as negative controls. [Figure 16-2] This is a continuation of Figure 16-1. [Figure 17-1] FIG. 1. In vitro activation of TLR4 signaling in HEK-hTLR4 cells. HEK-TLR4 cells were stimulated based on LOS content with OMVs from recombinant Bordetella pertussis spp. strains; OMVs from Bordetella pertussis Tohama I PTg strain (wild type control); purified LPS from Bordetella pertussis; or OMVs from detoxified Neisseria meningitidis B □MsbB strain (□MsbB). Stimulation was normalized by LOS content. Unstimulated cells were included as negative control. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18A] FIG. 1 shows the structural analysis of LOS (OMV derived from Bordetella pertussis, which expresses LpxEFn in an episomal manner) by LC-MS. [Figure 18B] FIG. 2 is a diagram of variants of the LOS structure determined by LC-MS, where the variant structure has the phosphate ester removed (position indicated by black arrow). [Figure 19A] FIG. 1 shows the structural analysis of LOS (OMV derived from Bordetella pertussis, which expresses LpxERc in an episomal manner) by LC-MS. [Figure 19B] FIG. 2 is a diagram of variants of the LOS structure determined by LC-MS, where the variant structure has the phosphate ester removed (position indicated by black arrow). [Figure 20A] FIG. 1 shows the structural analysis of LOS (OMV derived from Bordetella pertussis, which expresses LpxFFn in an episomal manner) by LC-MS. [Figure 20B] FIG. 11 is a diagram of the LOS structure determined by LC-MS, showing no identifiable effect on the LOS structure. [Figure 21] Figure 1. In vitro activation of TLR4 signaling in HEK-hTLR4 cells. HEK-TLR4 cells were stimulated with OMVs from recombinant Bordetella pertussis strains expressing LpxEFn based on LOS content. OMVs from Bordetella pertussis strain Tohama I PTg (WT PTg) were used as a positive control and unstimulated cells were included as a negative control. [Figure 22] FIG. 1. In vitro activation of TLR4 signaling in HEK-hTLR4 cells. HEK-TLR4 cells were stimulated based on protein content with OMVs from recombinant Bordetella pertussis strains expressing LpxEFn. OMVs from Bordetella pertussis strain Tohama I PTg (WT PTg) were used as a positive control and unstimulated cells were included as a negative control. [Diagram 23] FIG. 1: Geometric means of individual antibody titers (anti-PRN IgG) measured by ELISA and 95% confidence intervals from a study performed in a nasopharyngeal colonization model in Balb / c mice. [Figure 24] FIG. 1: Individual values ​​and geometric means of bacterial burden, defined by the mean number of colony forming units (CFU) per nasal cavity, shown by group and day, from a study performed in a nasopharyngeal colonization model in Balb / c mice. [Diagram 25] FIG. 1 shows individual anti-PRN IgG titers and geometric mean titers (with error bars representing associated 95% confidence intervals) measured by ELISA on day 28 from a study performed in a pulmonary clearance model in Balb / c mice. [Figure 26] FIG. 1: Individual colony forming unit (CFU) counts (small dots) and geometric means (connected large dots) by group according to day from a study performed in a pulmonary clearance model in Balb / c mice. [Figure 27A] FIG. 1. Six cytokines known to be involved in reactogenic mechanisms (i.e.: IL1a, IL6, TNFa, IL1b, IL10, MIP1a) measured in culture supernatants for various serial dilutions. [Figure 27B] This is a continuation of Figure 27A. [Figure 27C] This is a continuation of Figure 27B. [Figure 27D] Continuation of Figure 27C. [Figure 27E] Continuation of Figure 27D. [Figure 27F] Continuation of Figure 27E. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention relates to recombinant Bordetella pertussis spp. strains comprising an LPS that exhibits reduced endotoxin toxicity relative to the LPS of wild-type Bordetella pertussis bacteria. References to LPS in this application also encompass LOS. The endotoxin toxicity of both LPS and LOS is determined primarily by the composition of the lipid A moiety.

[0042] Surprisingly, the inventors have discovered that the bacteria of the invention also exhibit a bacterial growth profile that is equivalent to that of wild-type, non-recombinant Bordetella pertussis bacteria.

[0043] The term "wild type" is used in reference to bacteria to refer to the normal strain as it exists in nature. Such wild type strains lack the inventive modifications described herein. Thus, the term "wild type" is used to refer to a microorganism of the same strain as the microorganism of the invention, but lacking the genetic modifications of the invention, e.g., the LpxA and / or LpxD genetic modifications described herein. A wild type strain may also be referred to as a parent strain. As used herein, an "endogenous" gene or protein is used to refer to an unmodified gene (nucleic acid sequence) or protein (amino acid sequence) found in the wild type, parental strain of Bordetella pertussis bacteria from which the recombinant bacterium was originally derived.

[0044] In contrast, the term "exogenous" is used to refer to a gene (nucleic acid sequence) or protein (amino acid sequence) that is not naturally found in the wild-type Bordetella pertussis bacterial strain from which the recombinant bacterium is derived. The term "exogenous" can be used interchangeably with the term "heterologous."

[0045] The present invention relates to a recombinant Bordetella pertussis spp. strain, comprising at least one genomic LpxA gene encoding an LpxA protein comprising a mutation at position 170 and / or a mutation at position 229 relative to SEQ ID NO:1; and / or at least one genomic insertion of a heterologous LpxD gene.

[0046] The bacteria of the present invention The recombinant B. pertussis bacteria of the invention can be derived from various parental strains of B. pertussis, including, by way of non-limiting example: Tohama I, e.g. B. pertussis Moreno-Lopez (ATCC: BAA-589), BP165, BPW28, 10536, 18323, I135, BAA-2706, BAA-2705 or PT / 28G [W28] (ATCC: 53894). Preferably, the recombinant B. pertussis bacteria of the invention are derived from strains expressing a genetically detoxified pertussis toxoid, in particular the genetically detoxified pertussis toxoid designated PT-9K / 129G. The recombinant B. pertussis spp. strain expressing the PT-9K / 129G genetically detoxified pertussis toxoid comprises two amino acid substitutions within the S1 subunit of PT, specifically R9K and E129G (see, for example, EP0396964). In particular, the strain of B. pertussis from which the recombinant bacterium is derived is Tohama I. More particularly, the recombinant B. pertussis bacterium is derived from a strain of Tohama I that expresses the genetically detoxified pertussis toxoid. Even more particularly, the recombinant B. pertussis bacterium is derived from a strain of Tohama I that expresses the genetically detoxified pertussis toxoid PT-9K / 129G. Even more particularly, the recombinant B. pertussis bacterium comprises an S1 gene that has been modified to include the mutations R9K and E129G and expresses the genetically detoxified pertussis toxoid PT-9K / 129G.

[0047] Although pertussis toxin is secreted by Bordetella pertussis, it can still be present in isolated whole cells and in outer membrane vesicles. Therefore, the use of strains expressing genetically detoxified PTs is advantageous. Preferably, the recombinant Bordetella pertussis bacteria of the invention do not express the dermonecrotic toxin (DNT) gene.

[0048] Bacterial lipopolysaccharide and lipid A Bacterial lipopolysaccharide (LPS) is composed of a highly variable O antigen, a less variable core oligosaccharide, and a highly conserved lipid moiety called lipid A. Lipid A consists of a glucosamine disaccharide substituted with one or two phosphate groups and several acyl chains of various lengths (Figure 1). The endotoxic activity of LPS is determined primarily by the composition of the lipid A moiety.

[0049] Figure 1(A) shows the structure of lipid A from wild-type Bordetella pertussis. The present invention is based, in part, on the modification of the C2, C2' and / or C3' acyl chain length of lipid A in the LPS of B. pertussis. In a preferred embodiment, the C3' acyl chain of lipid A in the LPS of B. pertussis is modified. In such an embodiment, the C2 and C2' acyl chain lengths of lipid A in the LPS of B. pertussis may also be modified. To this end, the lipid A biosynthetic pathway of B. pertussis has been engineered.

[0050] LpxA acyltransferase The acyltransferase LpxA (referred to as "LpxA") catalyzes the transfer of ester-linked primary β-hydroxymyristate to monosaccharide precursors (UDP-glucosamine) at the 3 and 3' positions of lipid A and determines the length of the acyl chains incorporated at both positions, which varies among Gram-negative bacteria [4, 5, 6].

[0051] The endotoxic activity of LPS is determined primarily by the composition of the lipid A moiety of LPS. Variation in the number of acyl chains on lipid A can affect signaling through TLR4, but the mechanisms by which changes in these chains differentially affect TLR4 signaling are not fully understood [2].

[0052] Therefore, the expression of the heterologous LpxA gene was 10(C 10 ) or 12(C 12 Pseudomonas aeruginosa (LpxA) catalyzes the transfer of acyl chains with a length of 100 carbon atoms. Pa ) and Neisseria meningitidis (LpxA Nm) may reduce the toxicity of endotoxin from LPS LpxA. However, previous studies, such as those described in WO2018 / 167061, have shown that LpxA Pa or LpxA Nm The gene was expressed episomally in Bordetella pertussis and the endogenous LpxA gene was inactivated. a Episomal expression of 14 From C 10 However, the resulting recombinant strain exhibited severe growth defects, making it unsuitable for large-scale fermentation and use. Nm Episomal expression of was lethal.

[0053] Surprisingly, the present inventors have found that LpxA from Bordetella parapertussis (LpxA Bpa We have discovered that genomic expression of LPS reduces the endotoxicity of LPS while also solving the compatibility / growth problems observed in the art.

[0054] The amino acid sequence of LpxA from Bordetella parapertussis is provided as SEQ ID NO: 2. The amino acid sequence of LpxA from Bordetella pertussis is provided as SEQ ID NO: 1.

[0055] The amino acid sequence of LpxA from Bordetella pertussis differs from that of Bordetella parapertussis by two residues corresponding to the amino acids at positions 170 and 229 of SEQ ID NOs: 1 and 2 (Figure 2). The presence of specific substitutions at amino acid positions 170 and / or 229 alters the specificity of LpxA, resulting in the addition of a C3′ residue at the C3′ position of lipid A. 10It is believed that the recombinant B. pertussis spp. strain of the invention can thus express an LpxA gene encoding an LpxA amino acid sequence in which one or both of the amino acids at positions 170 and 229 (when numbered with reference to SEQ ID NO: 1) are substituted with an amino acid residue selected from the group consisting of alanine (A), serine (S), threonine (T) and cysteine ​​(C). In particular, the recombinant B. pertussis spp. strain can express an LpxA gene encoding an LpxA amino acid sequence in which G170 is substituted with serine (G170S) and / or G229 is substituted with alanine (G229A). In particular, the recombinant Bordetella pertussis spp. strain can express an LpxA gene encoding an LpxA amino acid sequence that comprises a serine residue at position 170 and / or an alanine residue at position 229 (when numbered with reference to SEQ ID NO:1).

[0056] Amino acid substitutions or mutations can be introduced by standard molecular biology techniques, such as random mutagenesis, site-directed mutagenesis, directed evolution, gene replacement, and the like.

[0057] Preferably, in addition to the above mutations, the LpxA gene encodes an amino acid sequence having at least 80% sequence identity to SEQ ID NO:2, such as 81%, at least 82%, at least 83%, at least 84%, at least 8%, at least 86%, at least 87%, at least 88%, at least 89% or 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

[0058] In particular, the sequence identity of two sequences can be determined using the GENEPAST algorithm provided by GenomeQuest, Inc. This algorithm, formerly known as "Kerr", performs an arithmetic determination of percent sequence identity without any biological weighting (Dufresne et al, Nature Biotechnology 20, 1269-1271 (2002), or Andree et al, World Patent Information, 2008, vol. 30, issue 4, pages 300-308). In general, sequence identity is calculated over and along the entire (i.e., complete) length of the longest of the sequences being compared.

[0059] LpxD acyltransferase LpxD acyltransferase (LpxD) catalyzes the transfer of amide-linked β-hydroxymyristate at the 2 and 2' positions of lipid A and determines the length of the acyl chains incorporated at both positions, which again varies among Gram-negative bacteria [6].

[0060] Expression of exogenous LpxD variants may improve the endotoxin profile of bacterial LPS. Thus, the recombinant Bordetella pertussis strain of the present invention may comprise at least one heterologous LpxD gene. In particular, the heterologous LpxD gene is integrated into the bacterial genome.

[0061] LpxD from C. testosteronii (LpxD Ct The relevant amino acid sequence of LpxD from Pseudomonas aeruginosa (LpxD) is provided as SEQ ID NO: 3. Pa ) is provided as SEQ ID NO:4.

[0062] Thus, in some embodiments, at least one heterologous LpxD gene is an LpxD gene from Pseudomonas aeruginosa. In some embodiments, at least one heterologous LpxD gene is an LpxD gene from C. testosteroni. In some embodiments, the heterologous LpxD gene replaces the dermonecrotic toxin locus. In some embodiments, the heterologous LpxD gene replaces the endogenous LpxD gene. In some embodiments, the heterologous LpxD gene is under the control of the BP0840 promoter (outer membrane porin promoter). Even more particularly, the wild-type copy of the LpxD gene (LpxD Bp ) has been knocked out.

[0063] In some embodiments, the amino acid sequence encoded by at least one heterologous LpxD gene has at least 90% identity to SEQ ID NO:3.

[0064] Thus, in some embodiments, the amino acid sequence encoded by at least one LpxD gene has at least 90%, at least 95% or 100% identity to SEQ ID NO: 3. More particularly, the LpxD amino acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 3.

[0065] In some embodiments, the amino acid sequence encoded by at least one heterologous LpxD gene has at least 90% identity to SEQ ID NO:4.

[0066] Thus, in some embodiments, the amino acid sequence encoded by at least one LpxD gene has at least 90%, at least 95% or 100% identity to SEQ ID NO: 4. More particularly, the LpxD amino acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 4.

[0067] In particular, the recombinant Bordetella pertussis bacterium of the invention expresses both a heterologous LpxD gene and an LpxA gene encoding an LpxA amino acid sequence comprising a serine residue at position 170 and an alanine residue at position 229. More particularly, the Bordetella pertussis bacterium expresses the LpxA Bpa and LpxD Pa is expressed.

[0068] In some embodiments, at least one LpxA gene and at least one LpxD gene are integrated into the bacterial genome. In other embodiments, the LpxA gene is integrated into the bacterial genome, but the LpxD gene can be expressed in an episomal manner.

[0069] The endogenous LpxA and / or LpxD genes of the recombinant bacterium can be inactivated by means well known to those skilled in the art, for example, both genes can be knocked out.

[0070] Generally, the endogenous LpxA gene is mutated, but can be inactivated or replaced, for example, with SEQ ID NO: 2. Generally, the endogenous LpxD gene is inactivated. In some embodiments, both the endogenous LpxA and endogenous LpxD genes are inactivated.

[0071] In some embodiments, the recombinant Bordetella pertussis bacteria of the invention comprise further genetic modifications, such as the expression of other exogenous genes and / or the inactivation of other endogenous genes. As non-limiting examples, (1) the endogenous ArnT gene (SEQ ID NO: 18) can be inactivated, e.g., deleted or knocked out, (2) the endogenous dermonecrotic toxin (DNT) gene (SEQ ID NO: 19) is inactivated, e.g., deleted or knocked out, (3) the bacterium can express PagL (SEQ ID NO: 16) from Bordetella bronchiseptica, (4) the bacterium can express LpxE protein (SEQ ID NO: 6 or 7) from a heterologous Gram-negative bacterium, and / or (5) the bacterium can express LpxF protein (SEQ ID NO: 8) from a heterologous Gram-negative bacterium.

[0072] Phosphate phosphatases LpxE and LpxF LpxE phosphate phosphatase (LpxE) and LpxF phosphate phosphatase (LpxF) are hydrolases that can act to remove phosphate groups from lipids that contain phosphate groups. When one phosphate group is removed, lipid A is "monophosphoryl lipid A". With respect to lipid A, the activity of LpxE and LpxF can result in the removal of phosphate groups at C1 and C4' of the core disaccharide, respectively.

[0073] Rhizobium leguminosarum (LpxE Rl ) and Francisella novicida (LpxE Fn The LpxE phosphatase from Francisella novicida has been described in the literature

[65] and

[66] . Fn ) was disclosed in

[67] . The authors identified LpxE in E. coli. Rl , LpxE Fn and LpxF Fn

[68] and

[69] further disclose heterologous expression of LpxE or LpxF in Bordetella pertussis. However, these publications do not disclose the use of LpxE or LpxF in Bordetella pertussis.

[0074] The inventors herein tested the expression of exogenous LpxE and LpxF in B. pertussis and demonstrated that the use of heterologous LpxE and / or LpxF can alter the structure of lipid A as well as the properties of lipid A, e.g., the endotoxin profile of B. pertussis LPS.

[0075] Thus, a recombinant B. pertussis spp. strain of the invention may comprise at least one heterologous LpxE gene and / or at least one heterologous LpxF gene, in particular, at least one heterologous LpxE gene and / or at least one heterologous LpxF gene is integrated into the bacterial genome.

[0076] LpxE is a phosphate phosphatase that specifically dephosphorylates the C1 position of the core disaccharide. Preferably, LpxE is from Rhizobium leguminosarum (LpxE Rl ) and Francisella novicida (LpxE Fn ). The appropriate amino acid sequences of LpxE from Rhizobium leguminosarum and Francisella novicida are provided as SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0077] In some embodiments, the heterologous LpxE gene replaces the ArnT gene locus, which encodes glycosyltransferase (ΔArnT). In some embodiments, the heterologous LpxE gene is under the control of the BP0840 promoter (outer membrane porin promoter). In some embodiments, the amino acid sequence encoded by at least one heterologous LpxE gene has at least 90% identity, at least 95% or 100% identity to SEQ ID NO:6 or SEQ ID NO:7.

[0078] In one particular embodiment, the heterologous LpxE gene is LpxE under the control of the BP0840 promoter (outer membrane porin promoter) inserted into the bacterial genome. Fn , which replaces the ArnT gene locus (ΔArnT).

[0079] In one embodiment, a recombinant Bordetella pertussis bacterium of the invention expresses a heterologous LpxE gene and an LpxA gene encoding an LpxA amino acid sequence comprising a serine residue at position 170 and an alanine residue at position 229. More particularly, the B. pertussis bacterium expresses LpxABpa and LpxE Rl and LpxE Fn and an LpxE gene selected from the group consisting of: In some embodiments, the LpxA and LpxE genes are integrated into the bacterial genome. In other embodiments, the LpxA gene is integrated into the bacterial genome, but the LpxE gene is expressed in an episomal manner.

[0080] LpxF is a phosphate phosphatase that specifically dephosphorylates the C4' position of the core disaccharide. Fn The heterologous LpxF gene from Francisella novicida is provided as SEQ ID NO:8.

[0081] In some embodiments, the at least one heterologous LpxF gene is an LpxF gene from Francisella novicida. In some embodiments, the heterologous LpxF gene replaces the ArnT gene locus encoding glycosyltransferase. In some embodiments, the heterologous LpxF gene is under the control of the BP0840 promoter (outer membrane porin promoter). In some embodiments, the amino acid sequence encoded by the at least one heterologous LpxF gene has at least 90% identity, at least 95% or 100% identity to SEQ ID NO:8.

[0082] Lipid A acyl chain In general, lipid A of gram-negative bacteria is composed of β-(1→6)-linked disaccharides acylated with up to eight fatty acids of various length and complexity, and charged substituents such as phosphate groups, phosphoethanolamine residues, or positively charged sugar residues, with numerous variations within lipid A of different bacterial species. Acylation at the 2- and 2'-positions of the disaccharide (reducing and terminal sugar residues, respectively) results in amide-linked lipids, and also at the 3- and 3'-positions when 3-amino-3-deoxy-d-glucosamine is acylated. The hydroxyl groups at the 3- and 3'-positions as well as the 3-hydroxyl group of the fatty acyl chain are also acylated, resulting in ester-linked substituents. The degree of acylation with 4-8 acyl groups attached to the β-(1→6)-linked disaccharides varies from species to species, and multiple forms of lipid A from a given bacterial species are often present.

[0083] In general, the lengths of the acyl chains at the C2, C2', and C3' positions of lipid A in wild-type Bordetella pertussis are C 14 As used herein, "C" with an integer subscript denotes a molecule based on a carbon chain of the length designated by the integer. Thus, C 14 and C 10 would represent molecules with chains of 14 and 10 carbon atoms, respectively.

[0084] The recombinant bacteria of the present invention produce lipid A in which the acyl chain length at the C2 and / or C2' and / or C3' positions is shortened compared to the corresponding acyl chain length of lipid A produced by wild-type Gram-negative bacteria.

[0085] In preferred embodiments of the recombinant bacteria of the invention, the bacteria produce lipid A in which the acyl chain length at the C3' position is shortened compared to the corresponding acyl chain length of lipid A produced by wild-type Gram-negative bacteria. In some embodiments, the bacteria produce lipid A in which the acyl chain length at the C2 and / or C2' positions is also shortened compared to the corresponding acyl chain length of lipid A produced by wild-type Gram-negative bacteria.

[0086] In a preferred embodiment, the length of the acyl chain at the C3′ position is 14 For example, C 6 ~C 12 , C 8 ~C 12 Or C 10 ~C 12 , especially C 10 Or C 12 , e.g., C 6 , C 8 , C 10 Or C 12 In a particularly preferred embodiment, the length of the acyl chain at the C3′ position is 10 It is.

[0087] In detail, the length of the acyl chain at the C2 position is 14 For example, C 6 ~C 12 , C 8 ~C 12 Or C 10 ~C 12 , especially C 10 Or C 12 , e.g., C 6 , C 8 , C 10 Or C 12 In particular, the length of the acyl chain at the C2' position is 14 For example, C 6 ~C 12 , C 8 ~C 12 Or C 10 ~C 12 , especially C 10 Or C 12 , e.g., C 6 , C 8 , C 10 Or C 12 In particular, the length of the acyl chain at the C3' position is 14 For example, C 6 ~C 12 , C 8 ~C 12 Or C 10 ~C 12 , especially C 10 Or C 12 , e.g., C6 , C 8 , C 10 Or C 12 It is.

[0088] In some embodiments, the length of the acyl chain at both the C2 and C2' positions is 14 For example, C 6 ~C 12 , C 8 ~C 12 Or C 10 ~C 12 , especially C 10 Or C 12 , e.g., C 6 , C 8 , C 10 Or C 12 In some embodiments, the length of the acyl chain at both the C2 and C3' positions is 14 For example, C 6 ~C 12 , C 8 ~C 12 Or C 10 ~C 12 , especially C 10 Or C 12 , e.g., C 6 , C 8 , C 10 Or C 12 In some embodiments, the length of the acyl chain at both the C2' and C3' positions is 14 For example, C 6 ~C 12 , C 8 ~C 12 Or C 10 ~C 12 , especially C 10 Or C 12 , e.g., C 6 , C 8 , C 10 Or C 12 In some embodiments, the length of the acyl chains at the C2, C2' and C3' positions is 14 For example, C 6 ~C 12 , C 8 ~C 12 Or C 10 ~C12 , especially C 10 Or C 12 , e.g., C 6 , C 8 , C 10 Or C 12 It is.

[0089] Lipid A produced by the recombinant bacteria of the invention can exist as a mixture of species having different acyl chain lengths.

[0090] Therefore, considering the total population of lipid A in a cell, at least 10% of the acyl chains at the C2 position are C 10 Or C 12 and / or at least 10% of the acyl chains at the C2′ position are C 10 Or C 12 and / or at least 10% of the acyl chains at the C3′ position are C 10 Or C 12 In detail, at least 10% of 10 Or C 12 and at least 12%, at least 15%, at least 20%, at least 24%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% are C 10 Or C 12 Preferably, at least 10% is C 10 Or C 12 and at least 80%, more preferably at least 90%, is C 10 Or C 12 More specifically, approximately 100% is C 10 Or C 12 is

[0091] Bacterial growth The recombinant strains of the invention are stable but also suitable for large scale propagation, e.g., in production. Surprisingly, the bacterial growth profile of the recombinant Bordetella pertussis strains of the invention is comparable to that of the wild-type parent strain, indicating that neither the exemplified LpxA nor LpxD mutations are detrimental to culture growth.

[0092] The recombinant strains of the invention are therefore particularly suitable for the preparation of, for example, whole cell bacterial antigens and / or OMVs, which can then be used as components of immunogenic compositions, such as vaccines.

[0093] Thus, the growth rate of a culture of a recombinant Gram-negative bacteria of the present invention is comparable to the growth rate of a culture of the wild-type, parent strain, specifically, in a standard fermentation process, for example, in a 10 liter fermentation volume, the elapsed time and growth rate values ​​are substantially similar, e.g., varying by less than 20%, less than 15%, less than 10%, or less than 5%.

[0094] Outer membrane vesicles (OMVs) In a second aspect, the present invention relates to an outer membrane vesicle or a population of outer membrane vesicles derived from a recombinant Bordetella pertussis bacterium of the first aspect.

[0095] OMVs are lipid bilayer nanoscale spherical particles (10-500 nm in diameter) that are spontaneously and constitutively released by Gram-negative bacteria during proliferation. OMVs are generated through "budding" of the bacterial outer membrane, and in line with the bacterial outer membrane, OMVs have a composition similar to that of the bacterial outer membrane, including lipopolysaccharide (LPS), glycerophospholipids, outer membrane proteins, and periplasmic components. Usually, outer membrane vesicles are artificially prepared from bacteria and can be prepared using detergent treatment (e.g., with deoxycholate) or by non-detergent means.

[0096] Techniques for forming OMVs include treating the bacteria with bile salt detergents (e.g., salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic acid, etc.) or with sodium deoxycholate [7 and 8]. Other techniques can be performed in the substantial absence of detergents [9 and 10], using techniques such as, for example, sonication, homogenization, microfluidization, cavitation, osmotic shock, disruption, French press, blending, etc. OMVs for use with the present invention can be prepared using an OMV extraction buffer having about 0.5% or less deoxycholate, for example, having about 0.4%, about 0.3%, about 0.2%, about 0.1%, <0.05%, or even zero.

[0097] OMVs may also be formed naturally during bacterial growth and are released into the culture medium. Thus, OMVs can be obtained by culturing the bacteria of the invention in a culture medium and separating the whole cells from the smaller OMVs in the culture medium. Bacterial vesicles can be conveniently separated from the whole bacteria by filtration, for example through a 0.22 μm filter. The bacterial filtrate can be clarified by centrifugation, for example by high-speed centrifugation (for example 20,000×g for about 2 hours). Another useful method of OMV preparation is described in

[11] and involves ultrafiltration of crude OMVs instead of high-speed centrifugation. The method may involve a step of ultracentrifugation after ultrafiltration. A simple method for purifying bacterial vesicles is described in

[12] and includes: i) a first filtration step, in which vesicles are separated from bacteria based on their different sizes, with the vesicles passing through the filtrate, for example using 0.22 μm microfiltration, and (ii) a second filtration step, in which the vesicles are retained in the retentate, for example using 0.1 μm microfiltration. Both steps use tangential flow filtration.

[0098] The OMV of the present invention will contain LOS (also known as LPS). The pyrogenic effect of LOS in OMVs may be less than that seen with the same amount of purified LOS. Thus, preferably, OMVs derived from the bacteria of the present invention will generally be compared to OMVs derived from parent strains when looking at pyrogenicity. LOS levels are expressed in International Units (IU) of endotoxin and can be tested by LAL assay (limulus amebocyte lysate). LOS may be present at less than 2000 IU per μg of OMV protein.

[0099] In some embodiments of the invention, the OMV is a detergent-extracted OMV (dOMV). The term "dOMV" encompasses a variety of proteoliposome vesicles obtained by disrupting the outer membrane of Gram-negative bacteria, usually by a detergent extraction process, and forming vesicles therefrom. Preferably, the OMV is a detergent-extracted OMV (dOMV). In particular, the OMV of the invention is a dOMV prepared using deoxycholate. More particularly, the OMV is about 0.5% or less deoxycholate, for example, about 0.4%, about 0.3%, about 0.2%, about 0.1%, or about 0.05%.

[0100] The dOMVs of the present invention can have a size distribution between about 20 and about 500 nm as measured by dynamic light scattering (DLS) techniques. In particular, the dOMVs of the present invention can have a size distribution between about 50 and about 500 nm. More particularly, the dOMVs of the present invention can have a size distribution between about 75 and about 250 nm, such as between about 75 and about 150 nm. Even more particularly, the dOMVs of the present invention can have an average diameter of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 nm.

[0101] In some embodiments, the bacteria of the invention can be modified to have expression of upregulated or heterologous antigens (i.e., antigens that are not native to a particular bacterial strain). As a result of such modifications, vesicles prepared from such modified bacteria contain higher levels of the upregulated or heterologous antigen. The increase in expression in vesicles of the upregulated antigen (measured relative to the corresponding wild-type strain) is usefully at least 10%, more usefully at least 20%, 30%, 40%, 50%, 75%, 100% or more, measured by mass of the relevant antigen per unit mass of vesicle.

[0102] Whole cell pertussis antigen The recombinant Bordetella pertussis bacteria of the present invention can be used as whole cell pertussis (wP) antigen in the form of inactivated Bordetella pertussis spp. cells. Preparation of cellular pertussis antigen is well documented in the art and can be obtained by heat inactivation of phase I culture of Bordetella pertussis spp. cells. The amount of wP antigen can be expressed in international units (IU). For example, NIBSC supplies the "International Standard For Pertussis Vaccine" (NIBSC code: 94 / 532), which contains 40 IU per ampoule.

[0103] When used in the immunogenic composition of the present invention, the cellular pertussis antigen is typically present in an amount capable of inducing an immune response when administered.Ideally, the cellular pertussis antigen is capable of inducing a protective immune response.The amount of wP antigen in the immunogenic composition of the present invention is typically at least 4 IU / dose.The wP antigen can be adsorbed onto or mixed with an aluminum adjuvant, such as an aluminum phosphate adjuvant.

[0104] TLR4 signaling cascade The bacteria of the invention produce lipid A with reduced endotoxic activity, particularly when measured using a TLR4 stimulation assay, relative to the endotoxic activity of lipid A from wild-type strains.

[0105] TLR4 stimulation assays are well known to those skilled in the art. Stimulation of the TLR4 signaling cascade can result in activation of NF-κB and / or activator protein 1 (AP-1) transcription factors, which contribute to the expression of several proinflammatory cytokines, such as IL-1, IL-6, IL-8 and TNF-α, and thus play a key role in inflammation. Determination of transcriptional activity in certain lines, such as HEK-Blue™-hTLR4 cells (Invivogen), can be used to assess stimulation of the TLR4 signaling cascade. More specifically, the inducible reporter gene secreting embryonic alkaline phosphatase (SEAP) in HEK-Blue™-hTLR4 cells is under the control of the IL-12p 40 minimal promoter fused to five binding sites for NF-κB and AP-1. Upon stimulation with a TLR4 ligand, activation of NF-κB and AP-1 induces secretion of alkaline phosphatase, which is measured at 655 nm (OD 200 nm). 655 ) can be quantified in the supernatant by measuring the optical density.

[0106] The endotoxin activity of the bacteria of the invention, such as the endotoxin activity of whole cell antigens or OMVs derived from the bacteria of the invention, can be determined using a TLR4 stimulation assay, in particular a human TLR4 stimulation assay, more particularly a human TLR4 stimulation assay by optical density (OD) measured at 655 nm. In particular, the endotoxin activity of whole cells or OMVs of the invention is compared to the endotoxin activity of a wild-type parent strain. The ability to activate TLR4 signaling by OMVs derived from the bacteria of the invention can be compared to the ability of OMVs derived from wild-type strains, as well as the ability of OMVs derived from other bacteria or commercially available immunogenic compositions, such as BEXSERO (Meningitis B vaccine containing OMVs derived from Neisseria spp.) or QUINVAXEM (whole cell (wP) pertussis vaccine).

[0107] More specifically, the endotoxin activity of the OMVs of the invention exhibits a reduced or decreased activation of the TLR4 signaling cascade compared to the endotoxin activity of OMVs derived from wild-type bacteria. Specifically, the OD obtained using a TLR4 stimulation assay when testing mutants of LpxA and / or LpxD exhibits a reduced or decreased activation of the TLR4 signaling cascade compared to the endotoxin activity of OMVs derived from wild-type bacteria. 655 Values ​​are OD obtained using unstimulated cells 655 values ​​and / or OD observed in cells stimulated with acellular pertussis (aP) vaccine 655 The value should be similar, for example, to about ±0.5OD. 655 It is required that:

[0108] In some embodiments, TLR4 stimulation assays show a partial reduction in activation of the TLR4 signaling cascade for OMVs of the invention compared to OMVs derived from wild-type bacteria. Specifically, the OD observed for LpxA and / or LpxD bacteria or OMVs of the invention is 655 Values ​​are the OD observed in unstimulated cells. 655 OD values ​​observed in aP vaccine-stimulated cells 655 higher than the value (≧0.5 OD 655 ), whereas the OD observed in cells stimulated with OMVs derived from wild-type bacteria and / or with wP vaccine and / or OMV-containing MenB vaccine 655 may be lower than the value.

[0109] immunogenic composition In a third aspect, the present invention relates to an immunogenic composition comprising at least one outer membrane vesicle derived from a recombinant Bordetella pertussis bacterium of the invention and a pharma- ceutically acceptable excipient.

[0110] The OMVs of the invention are useful as active ingredients in immunogenic compositions, as are whole cell antigens, including whole cells of the recombinant B. pertussis spp. of the invention that have been killed and inactivated (e.g., by treatment with formalin and / or heat) are also useful as active ingredients in immunogenic compositions.

[0111] The term "immunogenic composition" refers broadly to any composition that can be administered to elicit an immune response, such as an antibody or cellular immune response, to one or more antigens present in the composition. Thus, the compositions of the invention are immunogenic.

[0112] If an immunogenic composition prevents, ameliorates, alleviates or eliminates disease from a subject, such a composition can be referred to as a vaccine. Vaccines according to the invention can be either prophylactic (i.e., to prevent infection) or therapeutic (i.e., to treat infection), but will typically be prophylactic. In certain embodiments, the immunogenic composition is a vaccine. A prophylactic vaccine does not guarantee complete protection from disease, because even if a patient develops antibodies, there may be a lag or delay before the immune system is able to fight off the infection. Thus, and for the avoidance of doubt, the term prophylactic vaccine can also refer to a vaccine that ameliorates the effects of future infections, for example, by reducing the severity or duration of such infections.

[0113] The terms "protection against infection" and / or "providing protective immunity" mean that the immune system of a subject has been primed (e.g., by vaccination) to elicit an immune response and fight off infection. In particular, the elicited immune response can fight off infection against Bordetella pertussis. A vaccinated subject may thus become infected, but is more successful at fighting off said infection than a control subject. An immunogenic composition for use as a vaccine comprises an immunologically effective amount of an antigen, as well as any other components as appropriate. An "immunologically effective amount" means that administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. Usually, the desired result is the generation of an antigen (e.g., pathogen)-specific immune response that can or helps to protect the subject against the pathogen. The immunologically effective 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 primates, primates, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating physician's evaluation of the medical situation, and other relevant factors. It is expected that the immunologically effective amount will fall in a relatively broad range that can be determined through routine testing.

[0114] The term "antigen" refers to a substance that, when administered to a subject, induces an immune response directed against that substance. In the context of the present invention, the OMV of the present invention is an antigen. In particular, when administered to a subject, the immunogenic composition will induce an immune response directed against Bordetella, e.g., Bordetella pertussis. In particular, the immune response directed against Bordetella is protective, i.e., capable of preventing or reducing infection or colonization caused by Bordetella, particularly Bordetella pertussis. Thus, the composition can be pharmaceutically acceptable. In general, an immunogenic composition will include components in addition to the antigen, e.g., typically including one or more pharmaceutical carriers and / or excipients. A thorough discussion of such components is available in the literature

[13] .

[0115] A "pharmacologically acceptable carrier" is a carrier that does not itself induce the production of antibodies. Such carriers are well known to those skilled in the art and include, by way of non-limiting example, polysaccharides, polylactic acids, polyglycolic acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (e.g., oil droplets or liposomes). The immunogenic composition may also contain a diluent, such as water, saline, glycerol, and the like. Sterile pyrogen-free phosphate-buffered saline is a common diluent. Such compositions may also contain auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like.

[0116] The composition is generally administered to a subject, such as a patient, particularly to a suitable mammal, such as a human, in an aqueous form.However, the composition may be in a non-aqueous form before administration.For example, some vaccines are produced in an aqueous form and then packaged and distributed, and are also administered in an aqueous form, while other vaccines are freeze-dried during production and reconstituted into an aqueous form when used.Therefore, the composition of the present invention can be dried, such as a freeze-dried formulation.

[0117] The composition may include a preservative, for example thiomersal or 2-phenoxyethanol. However, it is preferred that the vaccine is substantially free of mercurial material (i.e. less than 5 μg / mL), for example thiomersal-free. More preferred is a vaccine that does not contain any mercury. Thiomersal-free vaccines are especially preferred.

[0118] To adjust the osmolarity, a physiological salt can be included, for example a sodium salt: Sodium chloride (NaCl) can be used, which can be present between 1 and 20 mg / ml, for example at about 10±2 mg / ml NaCl.

[0119] The compositions will generally have an osmolality in the range of between 200 mOsm / kg and 400 mOsm / kg, particularly between 240-360 mOsm / kg, more particularly 290-310 mOsm / kg.

[0120] The compositions may include one or more buffers. Common buffers include: phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer, or citrate buffer. Buffers will typically be present in the range of 5-20 mM.

[0121] The pH of the composition will generally be between 5.0 and 8.1, more typically between 6.0 and 8.0, for example between 6.5 and 7.5 or between 7.0 and 7.8.

[0122] The composition is preferably sterile. In particular, the composition is non-pyrogenic, for example containing <1 EU (endotoxin unit, a standard measurement) per dose, for example <0.1 EU per dose. The composition can be gluten-free.

[0123] The composition may contain material for a single immunization or for multiple immunizations (i.e., a "multi-dose" kit). Inclusion of a preservative is preferred in multi-dose configurations. As an alternative (or in addition) to including a preservative in a multi-dose composition, the composition may be contained in a container having a sterile adaptor for removal of the material.

[0124] Immunogenic compositions, such as human vaccines, are typically administered in a dosage volume of about 0.5 ml, although a half dose (ie, about 0.25 ml) can be administered, for example, to children.

[0125] The immunogenic composition of the present invention may also include one or more immunomodulatory agents. Preferably, the one or more immunomodulatory agents include one or more adjuvants. As used herein, "adjuvant" refers to a compound or substance (or a combination of compounds or substances) that, when administered to a subject in conjunction with an antigen(s), e.g., as part of an immunogenic composition or vaccine, improves or enhances the subject's immune response to the administered antigen(s) (compared to the immune response obtained in the absence of the adjuvant). Adjuvants may include TH1 adjuvants and / or TH2 adjuvants, as further discussed below.

[0126] Adjuvants that can be used in the compositions of the invention include mineral-containing compositions, such as aluminum salts and calcium salts. The compositions of the invention can include mineral salts, such as hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates, etc. [see, e.g., Chapters 8 and 9 of ref. 14], or mixtures of various mineral compounds, where the compounds are in any suitable form (e.g., gel, crystalline, amorphous, etc.), and adsorption is preferred. Mineral-containing compositions can also be formulated as particles of metal salts.

[0127] The adjuvants known as "aluminum hydroxide" are typically aluminum oxyhydroxide salts, which are usually at least partially crystalline. Aluminum oxyhydroxide, which can be represented by the formula AlO(OH), can be identified by infrared (IR) spectroscopy, particularly at 1070 cm -1 Absorption band at 3090-3100cm -1 The presence of a strong shoulder at 1000 nm indicates that it is distinct from other aluminum compounds, e.g. aluminum hydroxide, Al(OH). 3and can be distinguished from non-crystalline [Chapter 9 of ref.14]. The degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width at half maximum (WHH) of the diffraction band, where less crystalline particles show greater line broadening due to smaller crystallite size. As the WHH increases, the surface area increases, and adjuvants with higher WHH values ​​have been found to have greater capacity for antigen adsorption. A fibrous morphology (e.g., as seen in transmission electron micrographs) is typical for aluminium hydroxide adjuvants. The pI of aluminium hydroxide adjuvants is typically around 11, i.e. the adjuvant itself has a positive surface charge at physiological pH. For aluminium hydroxide adjuvants, at pH 7.4, the Al +++ Adsorption capacities of between 1.8 and 2.6 mg of protein per mg have been reported.

[0128] The adjuvants known as "aluminum phosphate" are typically aluminum hydroxyphosphates, which often also contain small amounts of sulfate (i.e., aluminum hydroxyphosphate sulfate). Aluminum hydroxyphosphates can be obtained by precipitation, but the reaction conditions and concentrations of the precipitation process affect the degree to which hydroxyl is replaced by phosphate in the salt. Hydroxyphosphates generally have a PO of between 0.3 and 1.2. 4 / Al molar ratio. Hydroxyphosphates are strictly AlPO 4 For example, 3164 cm -1 An IR spectral band at (e.g., when heated to 200 °C) indicates the presence of structural hydroxyls [Chapter 9 of ref. 14].

[0129] Aluminum phosphate adjuvant PO 4 / Al 3+The molar ratio may be between 0.3 and 1.2, in particular between 0.8 and 1.2, more particularly 0.95±0.1. The aluminum phosphate may be amorphous, in particular in the case of a hydroxyphosphate salt. A typical adjuvant is 0.6 mg Al 3+ / ml, with a PO between 0.84 and 0.92 4 Aluminium phosphate is generally in the form of particles (e.g., plate-like morphology as seen in transmission electron micrographs). The typical diameter of the particles is in the range of 0.5-20 μm (e.g., about 5-10 μm) after any antigen adsorption. For aluminium phosphate adjuvants, the adjuvant is at pH 7.4 and contains 100% Al. +++ Adsorption capacities of between 0.7 and 1.5 mg of protein per mg have been reported.

[0130] The point of zero charge (PZC) of aluminum phosphate is inversely proportional to the degree of substitution of hydroxyl with phosphate, which can vary depending on the reaction conditions and reactant concentrations used to prepare the salt by precipitation.PZC can also be modified by changing the concentration of free phosphate ions in solution (more phosphate = more acidic PZC) or by adding a buffer, such as a histidine buffer (making PZC more basic).The aluminum phosphate used according to the present invention can have a PZC between 4.0 and 7.0, more particularly between 5.0 and 6.5, for example about 5.7.

[0131] The suspension of aluminum salt used to prepare the composition of the present invention can contain a buffer (e.g., phosphate or histidine or Tris buffer), but this is not necessary. The suspension is preferably sterile and pyrogen-free. The suspension can contain free aqueous phosphate ions, for example, present at a concentration of between 1.0 and 20 mM, between 5 and 15 mM, particularly about 10 mM. The suspension can also contain sodium chloride.

[0132] In some embodiments, the adjuvant component comprises a mixture of both aluminum hydroxide and aluminum phosphate. In this case, aluminum phosphate may be present in greater amounts than hydroxide, for example in a weight ratio of at least 2:1, such as ≧5:1, ≧6:1, ≧7:1, ≧8:1, ≧9:1, etc. In some embodiments, the adjuvant component comprises aluminum phosphate. In some embodiments, the adjuvant component comprises aluminum hydroxyphosphate sulfate.

[0133] Al in a composition for administration to a patient +++ The concentration can be between 10 mg / ml and 0.01 mg / ml, for example, less than 5 mg / ml, less than 4 mg / ml, less than 3 mg / ml, less than 2 mg / ml, less than 1 mg / ml, for example, about 5 mg / ml, about 4 mg / ml, about 3 mg / ml, about 2 mg / ml, about 1 mg / ml, about 0.3 mg / ml, about 0.05 mg / ml, or about 0.01 mg / ml. A particular range is between about 0.3 mg / ml and about 1 mg / ml. In some embodiments, a maximum of 0.85 mg / dose, for example, about 0.5 mg / l or about 0.3 mg / ml, is preferred.

[0134] Oil emulsion compositions suitable for use as adjuvants in the present invention include squalene-water emulsions such as MF59 [chapter 10 of ref. 14; see also ref. 15] (5% squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) can also be used.

[0135] Saponin preparations can also be used as adjuvants in the present invention. Saponins are a heterogeneous group of sterol and triterpenoid glycosides found in the bark, leaves, stems, roots and flowers of a wide variety of plant species. Saponins from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponins are also commercially available from Smilax ornata (sarsaparilla), Gypsophilla paniculata (brides veil) and Saponaria officinalis (baby's breath). Saponin adjuvant preparations include purified preparations, such as QS21, as well as lipid preparations, such as ISCOMs. QS21 is sold as Stimulon™.

[0136] AS01 is an adjuvant system containing MPL (3-O-desacyl-4'-monophosphoryl lipid A), QS21 ((Quillaja saponaria Molina, fraction 21) Antigenics, New York, NY, USA) and liposomes. AS01B is an adjuvant system containing MPL, QS21 and liposomes (50 μg MPL and 50 μg QS21). AS01E is an adjuvant system containing MPL, QS21 and liposomes (25 μg MPL and 25 μg QS21). In one embodiment, the immunogenic composition or vaccine comprises AS01. In another embodiment, the immunogenic composition or vaccine comprises AS01B or AS01E. In a particular embodiment, the immunogenic composition or vaccine comprises AS01E. AS02 is an adjuvant system containing MPL and QS21 in an oil / water emulsion. AS02V is an adjuvant system containing MPL and QS21 (50 μg MPL and 50 μg QS21) in an oil / water emulsion. AS03 is an adjuvant system containing α-tocopherol and squalene in an oil / water (o / w) emulsion. AS03A is an adjuvant system containing α-tocopherol (11.86 mg tocopherol) and squalene in an o / w emulsion. AS03B is an adjuvant system containing α-tocopherol (5.93 mg tocopherol) and squalene in an o / w emulsion. AS03C is an adjuvant system containing α-tocopherol (2.97 mg tocopherol) and squalene in an o / w emulsion. In one embodiment, the immunogenic composition or vaccine comprises AS03. AS04 is an adjuvant system containing MPL (50 μg MPL) adsorbed onto an aluminium salt (500 μg Al3+). In one embodiment, the immunogenic composition or vaccine comprises AS04. A system involving the use of QS21 and 3D-MPL is disclosed in WO94 / 00153. A composition in which QS21 is quenched with cholesterol is disclosed in WO96 / 33739. A further adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO95 / 17210.In one embodiment, the immunogenic composition further comprises a saponin, which can be QS21. The formulation can also comprise an oil-in-water emulsion and tocopherol (WO 95 / 17210). Unmethylated CpG-containing oligonucleotides (WO 96 / 02555) and other immune modulatory oligonucleotides (WO0226757 and WO03507822) are also preferential inducers of TH1 responses and are suitable for use in the present invention.

[0137] Further adjuvants include Toll-like receptor agonists (particularly Toll-like receptor 2 agonists, Toll-like receptor 3 agonists, Toll-like receptor 4 agonists, Toll-like receptor 7 agonists, Toll-like receptor 8 agonists and Toll-like receptor 9 agonists). The compositions of the invention can include one or more small molecule immune enhancers. For example, the compositions can include TLR2 agonists (e.g., Pam3CSK4), TLR4 agonists (e.g., aminoalkyl glucosaminide phosphates, e.g., E6020), TLR7 agonists (e.g., imiquimod), TLR8 agonists (e.g., resiquimod (also a TLR7 agonist)) and / or TLR9 agonists (e.g., IC31). Any of these agonists ideally have a molecular weight of <2000 Da.

[0138] The TLR7 agonists used in the present invention ideally contain at least one adsorbing moiety. The inclusion of such a moiety in the TLR agonist allows the TLR agonist to be adsorbed to insoluble aluminum salts (e.g., by ligand exchange or some other suitable mechanism), improving their immunological behavior. Phosphorus-containing adsorbing moieties are particularly useful, and thus the adsorbing moiety may include phosphates, phosphonates, phosphinates, phosphonites, phosphinites, and the like. The TLR agonist may include at least one phosphonate group. In certain embodiments, the compositions of the present invention may include a TLR7 agonist that includes a phosphonate group. This phosphonate group may allow the agonist to be adsorbed to insoluble aluminum salts. In some embodiments, the TLR agonist is 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo[f]-[1,7]naphthyridin-8-yl)propanoic acid, as shown below.

[0139] [ka]

[0140] Preferred TLR agonists are water soluble, thus capable of forming a homogeneous solution when mixed in water and aqueous buffer at pH 7 at 25° C. and 1 atmosphere to give a solution having a concentration of at least 50 μg / ml. Thus, the term "water soluble" excludes substances that are only slightly soluble under such conditions.

[0141] The compositions of the invention can be prepared in various forms. For example, the compositions can be prepared as injections, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in a liquid medium prior to injection can also be prepared (e.g., freeze-dried compositions or spray-freeze-dried compositions). The compositions can be prepared for topical administration, for example as ointments, creams or powders. The compositions can be prepared for pulmonary administration, for example as an inhaler using fine powders or sprays. The compositions can be prepared for nasal, aural or ocular administration, for example as liquid drops. The compositions can be in kit form, designed so that the combination composition is reconstituted immediately prior to administration to a patient. Such kits can include one or more antigens in liquid form and one or more freeze-dried antigens.

[0142] Where the compositions are to be prepared extemporaneously prior to use (e.g. where the components are presented in lyophilized form) and are presented as a kit, the kit may comprise two vials, or the kit may comprise one pre-filled syringe and one vial, in which case the contents of the syringe are used to reactivate the contents of the vial prior to injection.

[0143] Combination vaccine The immunogenic compositions of the invention are generally combination vaccines and may include, in addition to the OMVs of the invention, further protective antigens from Bordetella pertussis and from at least one pathogen other than Bordetella pertussis.

[0144] The further protective antigens may be viral and / or bacterial. In addition to Bordetella pertussis, typical bacterial pathogens include, but are not limited to, Corynebacterium diphtheriae, Clostridium tetani, Haemophilus influenzae type b. Typical viral pathogens include, but are not limited to, poliovirus and hepatitis B virus. For the avoidance of doubt, reference to a further antigen is intended to refer to further antigenic components contained in the immunogenic composition of the invention beyond the components of the OMV. For example, although small amounts of pertussis toxoid may be found as a component of the OMV, reference to pertussis toxoid as a further antigen refers to an amount of pertussis toxoid that is specifically added in addition to the OMV components, for example as an isolated protein antigen.

[0145] Acellular pertussis antigen In addition to the OMVs of the invention, the immunogenic compositions of the invention can further comprise one or more acellular pertussis (aP) antigens, particularly selected from the following well-known and well-characterized Bordetella pertussis antigens: (1) detoxified pertussis toxin (pertussis toxoid, or "PT"); (2) filamentous hemagglutinin ("FHA"); (3) pertactin (also known as "PRN" or "69 kilodalton outer membrane protein"); (4) type 2 fimbria ("FIM2"); (5) type 3 fimbria ("FIM3"). It is most preferred to use both detoxified PT and FHA. In some embodiments, PT, FHA and PRN are used. These antigens are preferably prepared by isolation from Bordetella pertussis cultures grown in modified Stainer-Scholte liquid medium. PT and FHA can be isolated from the fermentation broth (e.g., by adsorption to hydroxyapatite gel), while pertactin can be extracted from the cells by heat treatment and flocculation (e.g., using barium chloride). Antigens can be purified by successive chromatography and / or precipitation steps: PT and FHA can be purified by hydrophobic, affinity and size-exclusion chromatography; pertactin can be purified by ion-exchange, hydrophobic and size-exclusion chromatography.

[0146] FHA and pertactin can be treated with formaldehyde before use according to the present invention. PT can be detoxified by treating with formaldehyde and / or glutaraldehyde. As an alternative to this chemical detoxification procedure, preferably, PT can be a mutant PT whose enzymatic activity has been reduced by mutagenesis, for example, a genetically detoxified PT, preferably PT-9K / 129G.

[0147] In some embodiments, the immunogenic compositions of the invention further comprise the fimbrial antigens FIM2 and FIM3.

[0148] The aP antigens may be used in a non-adsorbed state or may be adsorbed onto one or more aluminium salt adjuvants prior to use. Typically, the aP antigens are substantially free of mercurial-based preservatives, such as thimerosal.

[0149] The acellular pertussis antigen may be present in the immunogenic composition of the invention in an amount capable of eliciting an immune response when administered. Ideally, the acellular pertussis antigen is capable of eliciting a protective immune response. The amount of the acellular pertussis antigen is usually expressed in micrograms. The concentration of PT in the vaccine is usually between 5 and 50 μg / ml. Typical PT concentrations are 5 μg / ml, 16 μg / ml, 20 μg / ml or 50 μg / ml, e.g., about 20 μg per dose or about 25 μg per dose. The concentration of FHA in the vaccine is usually between 10 and 50 μg / ml. Typical FHA concentrations are 10 μg / ml, 16 μg / ml or 50 μg / ml, e.g., about 20 μg per dose or about 25 μg per dose. The concentration of pertactin in the vaccine is usually between 5 and 16 μg / ml. Typical pertactin concentrations are 5 μg / ml, 6 μg / ml or 16 μg / ml, e.g., about 3 μg per dose or about 8 μg per dose. FIM2 and FIM3 can be present at concentrations 5 μg / ml and 16 μg / ml. Typical concentrations of FIM2 and FIM3 are 5 μg / ml, 10 μg / ml or 20 μg / ml, e.g., about 5 μg per dose or about 10 μg per dose. Booster vaccines for adolescents and adults typically contain between 2.5-8 μg of PT, 4-8 μg of FHA, and 2.5-8 μg of pertactin per 0.5 ml dose. Typically, booster vaccines contain 4 μg of PT, 4 μg of FHA, and 8 μg of pertactin, more preferably 5 μg of PT, 2.5 μg of FHA, and 2.5 μg of pertactin per 0.5 ml dose. Paediatric vaccines normally contain 7 μg PT, 10 μg FHA and 10 μg pertactin per 0.5 ml dose.

[0150] When the aqueous component comprises each of PT, FHA and pertactin, the weight ratio thereof can vary, but can be, for example, about 16:16:5, about 5:10:6, about 20:20:3, about 25:25:8 or about 10:5:3 (PT:FHA:PRN).

[0151] diphtheria Corynebacterium diphtheriae causes diphtheria. Diphtheria toxin can be treated (e.g., with formalin or formaldehyde) to remove toxicity while retaining the ability to induce specific antitoxin antibodies after injection. Diphtheria toxoids used in diphtheria vaccines are well known in the art. Preferred diphtheria toxoids are those prepared by formaldehyde treatment. Diphtheria toxoids can be obtained by growing Corynebacterium diphtheriae in a growth medium (e.g., Fenton's medium, or Linggoud & Fenton's medium), possibly supplemented with bovine extract, followed by formaldehyde treatment, ultrafiltration, and precipitation. In particular, the growth medium for the growth of Corynebacterium diphtheriae is free of animal-derived components. The toxoided material can then be processed by processes including sterile filtration and / or dialysis. Alternatively, a genetically detoxified diphtheria toxin (eg CRM197) can be used, but may be treated with formaldehyde to maintain long term stability during storage.

[0152] Diphtheria toxoid can be adsorbed onto an adjuvant, such as an aluminium salt adjuvant.

[0153] The amount of diphtheria toxin and / or toxoid in a composition is generally measured in "Lf" units ("Agglutination Units" or "Limit Agglutination Dose" or "Limit of Agglutination"), which is defined as the amount of toxin / toxoid that, when mixed with one international unit of antitoxin, results in an optimally agglutinating mixture [16,17]. For example, NIBSC supplies "Diphtheria Toxoid, Plain"

[18] , which contains 300 LF per ampoule, and also supplies "The 2nd International Reference Reagent For Diphtheria Toxoid For Flocculation Test" (NIBSC Code: 02 / 176), which contains 900 Lf per ampoule. The concentration of diphtheria toxin or toxoid in a composition can be readily determined using an agglutination assay by comparison with a reference material calibrated against such a reference reagent.

[0154] The immunopotency of the diphtheria toxoid in the composition is commonly expressed in International Units (IU). Potency can be assessed by comparing the protection conferred by the composition in laboratory animals (usually guinea pigs) with a reference vaccine calibrated in IU. The NIBSC supplies the "4th WHO International Standard for Diphtheria Toxoid (Adsorbed)" (NIBSC code: 07 / 216), which contains 213 IU per ampoule and is suitable for calibrating such assays.

[0155] A three-dilution assay can be used to determine the potency of the compositions of the invention. After immunization, the guinea pigs are bled or challenged either by subcutaneous or intradermal route. In an alternative embodiment, mice are used instead of guinea pigs. When guinea pigs or mice are bled, the antitoxin levels of the individual animals are titrated by a toxin neutralization test performed using an in vivo or in vitro serological method validated for the type of vaccine being tested. In one embodiment, diphtheria toxoid produced in a fermentation medium containing animal-derived components is used for validation. The potency of the compositions of the invention is calculated using appropriate statistical methods. For a three-dilution assay, the limits of the 95% confidence interval of the potency estimate are within 50-200% of the estimated potency, provided that the lower limit of the 95% confidence interval of the estimated potency does not exceed 30 IU per single human dose. When a one-dilution test is performed, the potency of the tested vaccine is demonstrated to be significantly greater than 30 IU per human dose.

[0156] In IU measurements, compositions generally contain at least 30 IU / dose. Compositions typically contain between 20 and 80 Lf / ml, typically about 50 Lf / ml, of diphtheria toxoid. Booster vaccines for adolescents and adults typically contain between 4 Lf / ml and 8 Lf / ml, e.g., 2.5 Lf, preferably 4 Lf, of diphtheria toxoid per 0.5 ml dose. Paediatric vaccines typically contain between 20 and 50 Lf / ml, e.g., 10 Lf or 25 Lf, of diphtheria toxoid per 0.5 ml dose.

[0157] The purity of a protein preparation can be expressed by the ratio of specific protein to total protein. The purity of the diphtheria toxoid in a composition is generally expressed in units of Lf of diphtheria toxoid per unit mass of protein (nondialysable) nitrogen. By way of example, a very pure toxin / toxoid may have a purity of over 1700 Lf / mg N, indicating that most or all of the protein in the composition is diphtheria toxin / toxoid

[19] .

[0158] tetanus Clostridium tetani causes tetanus. Tetanus toxin can be processed to provide protective toxoids. Toxoids are used in tetanus vaccines and are well known in the art. Thus, the combination vaccine of the present invention can include tetanus toxoid. A preferred tetanus toxoid is one prepared by formaldehyde treatment. Tetanus toxoid can be obtained by growing Clostridium tetani in a growth medium (e.g., Latham's medium derived from bovine casein) followed by formaldehyde treatment, ultrafiltration and precipitation. The growth medium for growing Clostridium tetani may be free of animal-derived components. The material can then be processed by processes including sterile filtration and / or dialysis. The tetanus toxoid can be adsorbed onto an adjuvant, such as an aluminium salt adjuvant.

[0159] The quantity of tetanus toxoid can be expressed in "Lf" units (see below), which is defined as the amount of toxoid that, when mixed with 1 international unit of antitoxin, produces an optimally flocculating mixture.

[14] The NIBSC supplies "The 2nd International Reference Reagent for Tetanus Toxoid For Flocculation Test" (NIBSC Code: 04 / 150), which contains 690 LF per ampoule, allowing the measurement to be calibrated.

[0160] Adolescent and adult booster vaccines typically contain 5 Lf of tetanus toxoid per 0.5 ml dose. Paediatric vaccines typically contain between 5 and 10 Lf of tetanus toxoid per 0.5 ml dose.

[0161] The immune potency of the tetanus toxoid is measured in International Units (IU) and is assessed by comparing the protection provided by the composition in laboratory animals (usually guinea pigs) with a reference vaccine, for example the "Tetanus Toxoid Adsorbed Third International Standard 2000" from NIBSC, which contains 469 IU per ampoule [20, 21]. The potency of the tetanus toxoid in the composition of the invention is at least 35 IU per dose, for example at least 70 IU / ml. More particularly, the potency of the tetanus toxoid in the composition of the invention is at least 40 IU per dose. However, a booster vaccine for adults and adolescents can tolerate a reduced potency of 20 IU / dose due to the reduced antigen content compared to the pediatric vaccine intended for primary immunization.

[0162] A multiple dilution assay can be used to determine the potency of the compositions of the invention. After immunization, the guinea pigs are bled or exposed either by subcutaneous or intradermal route. Alternatively, mice may be used instead of guinea pigs. When guinea pigs or mice are bled, the antitoxin levels of individual animals are titrated by toxin neutralization tests performed using in vivo or in vitro serological methods validated for the type of vaccine being tested. The potency of the compositions of the invention is calculated using appropriate statistical methods. When using a multiple dilution assay, the lower and upper limits of the 95% confidence interval should be within 50-200% of the estimated potency, respectively. The lower 95% confidence limit of the estimated potency of a tetanus vaccine used for primary immunization of children should not be less than 40 IU per single human dose.

[0163] The purity of the tetanus toxoid in a composition is generally expressed in units of Lf of tetanus toxoid per unit mass of protein (non-dialysable) nitrogen. The tetanus toxoid should have a purity of at least 1000 Lf / mg N.

[0164] Hib Haemophilus influenzae type b ("Hib") causes bacterial meningitis. Hib vaccines are usually based on capsular saccharide antigens whose preparation is well documented in the art. Bacteria of the H. influenzae species can be cultured without animal-derived components. Hib saccharides have been conjugated to carrier proteins to enhance their immunogenicity, especially in children. Common carrier proteins for these conjugates are tetanus toxoid, diphtheria toxoid, the CRM 197 derivative of diphtheria toxin, or the outer membrane protein complex (OMPC) from serogroup B Neisseria meningitidis. Thus, the combination vaccine of the invention may comprise Hib capsular saccharides conjugated to a carrier protein.

[0165] Any suitable activation and / or linker chemistry can be used in the conjugation of Hib saccharides. Saccharides are typically activated or functionalized prior to conjugation. Activation can involve, for example, cyanylating reagents such as CDAP (e.g. 1-cyano-4-dimethylaminopyridinium tetrafluoroborate [30, 31]). Other suitable techniques use carbodiimides, hydrazides, active esters, norbornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, see also the introduction in reference 32.

[0166] Linkage via a linker group can be performed using any known procedure, such as those described in references 33 and 34. One type of linkage involves reductive amination of the polysaccharide, coupling the resulting amino group to one end of an adipic acid linker group, and then coupling the protein to the other end of the adipic acid linker group [35, 36, 37]. Other linkers include B-propionamide

[38] , nitrophenyl-ethylamine

[39] , haloacyl halides

[40] , glycosidic linkages [41, 42, 43], 6-aminocaproic acid

[44] , ADH

[45] , C 4 ~C 12As an alternative to the use of linkers, direct linkage can be used. Direct linkage to a protein can involve, for example, oxidation of the polysaccharide followed by reductive amination with the protein, as described in references 46 and 47.

[0167] Tetanus toxoid can be used in a conjugate commonly referred to as "PRP-T", which can be made by activating Hib capsular polysaccharide using cyanogen bromide, coupling the activated saccharide to an adipic acid linker (e.g. (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), usually the hydrochloride salt), and then reacting the linker glycoentity with a tetanus toxoid carrier protein.

[0168] CRM 197 diphtheria toxoid is another preferred Hib carrier protein [47, 48, 49]. Preferred conjugates contain Hib saccharides covalently linked to CRM197 via adipic acid succinic acid diester [50, 51].

[0169] The saccharide portion of the conjugate may comprise full-length polyribosyl ribitol phosphate (PRP) prepared from Hib bacteria, and / or a fragment of full-length PRP. A saccharide:protein ratio (w / w) between 1:5 (i.e. excess protein) and 5:1 (i.e. excess saccharide); For example, conjugates having ratios between 1:2 and 5:1 and between 1:1.25 and 1:2.5 can be used. However, in preferred vaccines, the weight ratio of saccharide to carrier protein is between 1:2.5 and 1:3.5. In vaccines in which tetanus toxoid is present both as the antigen and as the carrier protein, the weight ratio of saccharide to carrier protein in the conjugate can be between 1:0.3 and 1:2

[52] .

[0170] The amount of Hib antigen is usually expressed in μg of saccharide. The concentration of saccharide in the vaccine is typically between 3 and 30 μg / ml, for example 20 μg / ml. Administration of the Hib conjugate preferably results in an anti-PRP antibody concentration of ≧0.15 μg / ml, more preferably ≧1 μg / ml, which are standard response thresholds. In some embodiments of the invention, the Hib polyribosyl ribitol phosphate is synthetic, for example as described in [53 or 54].

[0171] Hepatitis B virus Hepatitis B virus (HBV) is the cause of viral hepatitis. The HBV virion is composed of an inner core surrounded by an outer protein coat or capsid, which contains the viral DNA genome. The major component of the capsid is a protein known as the HBV surface antigen, or more commonly "HBsAg", which is usually a 226 amino acid polypeptide with a molecular weight of approximately 24 kDa. All current hepatitis B vaccines contain HBsAg, which, when administered to normal patients, stimulates the production of anti-HBsAg antibodies that protect against HBV infection. Thus, the immunogenic composition of the present invention can include HBsAg.

[0172] For vaccine production, HBsAg can be produced in several ways, for example by expressing the protein by recombinant DNA techniques. HBsAg for use in the methods of the invention must be recombinantly expressed, for example in yeast cells. Suitable yeasts include hosts of the genus Saccharomyces (e.g. S. cerevisiae), Hanensula (e.g. H. polymorpha), or Pichia. Yeasts can be cultured without animal-derived components. Yeast-expressed HBsAg is generally non-glycosylated, which is the most preferred form of HBsAg for use in the immunogenic compositions of the invention. Yeast-expressed HBsAg is highly immunogenic and can be prepared without the risk of blood product contamination. Many methods are known in the art for purifying HBsAg from recombinant yeast.

[0173] HBsAg is generally in the form of substantially spherical particles (mean diameter about 20 nm) containing a lipid matrix including phospholipids. Yeast-expressed HBsAg particles may contain phosphatidylinositol, which is not found in native HBV virions. The particles may also contain non-toxic amounts of LPS to stimulate the immune system

[55] . If a non-ionic detergent (e.g., polysorbate 20) is used during the yeast disruption process, the particles may retain it

[56] .

[0174] Preferably the HBsAg is from HBV subtype adw2.

[0175] Preferred methods for HBsAg purification involve cell disruption followed by: ultrafiltration, size exclusion chromatography, anion exchange chromatography, ultracentrifugation, desalting, and sterile filtration. After cell disruption, the lysate may be precipitated (e.g., using polyethylene glycol) leaving the HBsAg in solution and available for ultrafiltration.

[0176] Following purification, HBsAg may be subjected to dialysis (e.g., with cysteine) which can be used to remove any mercury-based preservatives, such as thimerosal, that may have been used during HBsAg preparation

[57] .

[0177] The amount of HBsAg is usually expressed in micrograms. Combination vaccines containing HBsAg usually contain between 5 and 60 μg / ml. The concentration of HBsAg in the composition of the invention is preferably less than 60 μg / ml, e.g., ≦55 μg / ml, ≦50 μg / ml, ≦45 μg / ml, ≦40 μg / ml, etc. A concentration of about 20 μg / ml is typical, e.g., 10 μg per dose. In some embodiments of the invention, the composition comprises a "low dose" of HBsAg. This means that the concentration of HBsAg in the composition is ≦5 μg / ml, e.g., <4, <3, <2.5, <2, <1, etc. Thus, in a typical unit dose volume of 0.5 ml, the amount of HBsAg is less than 2.5 μg, e.g., <2, <1.5, <1, <0.5, etc.

[0178] Poliovirus Poliovirus causes polio. As disclosed in more detail in Chapter 24 of Reference 1, inactivated poliovirus vaccines (IPV) have been known for many years. Thus, the combination vaccine of the present invention can include an inactivated poliovirus antigen.

[0179] Poliovirus can be grown in cell culture, the preferred culture uses the Vero cell line derived from monkey kidney. Vero cells can be conveniently made into cultured microcarriers. After growth, the virions can be purified using techniques such as ultrafiltration, diafiltration and chromatography. If animal (especially bovine) material is used in the cell culture, it must be obtained from a source that is free of transmissible spongiform encephalopathies (TSE), especially free of bovine spongiform encephalopathy (BSE). Preferably, poliovirus is grown in cells that are cultured in a medium that is free of components of animal origin.

[0180] Prior to administration to a patient, the poliovirus must be inactivated, which can be done by treatment with formaldehyde (or preferably a non-aldehyde agent). Polio can be caused by one of three types of poliovirus. The three types are similar and cause the same symptoms, but antigenically are very different and infection with one type does not protect against infection with the others. Thus, the present invention preferably uses three poliovirus antigens: poliovirus type 1 (e.g., Mahoney strain), poliovirus type 2 (e.g., MEF-1 strain) and poliovirus type 3 (e.g., Saukett strain). Other strains of poliovirus types 1, 2 and 3 are known in the art and can also be used. These viruses are preferably grown, purified and inactivated individually and then combined to provide a bulk trivalent mixture for use in the present invention.

[0181] The amount of IPV is usually expressed in "DU" units ("D antigen units"

[58] ). A combination vaccine usually contains between 1 and 100 DU of each poliovirus type per dose, e.g., about 40 DU of type 1 poliovirus, about 8 DU of type 2 poliovirus, and about 32 DU of type 3 poliovirus, although lower doses [59, 60] can be used, e.g., 10-20 DU of type 1, 2-4 DU of type 2, and 8-20 DU of type 3. A combination vaccine of the invention can contain a "low dose" of poliovirus. For type 1 poliovirus, low dose means that the virus concentration in the composition is ≦20 DU / ml, e.g., <18, <16, <14, <12, <10, etc. For type 2 poliovirus, low dose means that the virus concentration in the composition is ≦4 DU / ml, e.g., <3, <2, <1, <0.5, etc. For type 3 poliovirus, low dose means a virus concentration in the composition of ≦16 DU / ml, e.g., <14, <12, <10, <8, <6, etc. When all three poliovirus types 1, 2 and 3 are present, the three antigens can be present in a DU ratio of 5:1:4, respectively, or in any other suitable ratio, e.g., 15:32:45 when using the Sabin strains.

[61] Low dose antigens from the Sabin strains are particularly useful, where type 1 is ≦10 DU, type 2 is ≦20 DU, and type 3 is ≦30 DU (per unit dose, usually 0.5 ml).

[0182] Where IPV components are used and the poliovirus is grown in Vero cells, the vaccine composition preferably contains less than 10 ng / ml Vero cell DNA, preferably < 1 ng / ml, such as < 500 pg / ml or < 50 pg / ml, e.g. less than 10 ng / ml Vero cell DNA that is > 50 base pairs in length.

[0183] Preparation of combination vaccines Antigen components from the above pathogens for use in vaccines are commonly referred to by the following abbreviations: "D" for diphtheria toxoid, "T" for tetanus toxoid, "P" for pertussis antigen, where "aP" is acellular (e.g. comprising at least the OMV, PT and FHA of the invention and optionally pertactin and / or FIM2 / FIM3), HBsAg for Hepatitis B surface antigen, "Hib" for conjugated Haemophilus influenzae b capsular saccharide, and "IPV" for trivalent inactivated poliovirus.

[0184] Embodiments of the present invention include, but are not limited to, combination vaccines comprising the following components: - D, T, aP - D, T, aP, IPV - D, T, aP, HBsAg - D, T, aP, Hib - D, T, aP, Hib, IPV - D, T, aP, HBsAg, Hib - D, T, aP, HBsAg, IPV - D, T, aP, HBsAg, IPV, Hib

[0185] These combination vaccines may consist essentially of the antigens listed above, but may further include antigens derived from additional pathogens. In particular, these combination vaccines contain only the antigens listed above as active ingredients, but may further include excipients, such as adjuvants, buffers, etc. In some embodiments, the aP component consists of the OMV, PT (preferably genetically detoxified PT) and FHA of the present invention. In some embodiments, the aP component consists of the OMV, PT (preferably genetically detoxified PT), FHA and PRN of the present invention. In some embodiments, the aP component consists of the OMV, PT (preferably genetically detoxified PT), FHA and FIM2 / FIM3 of the present invention. In some embodiments, the aP component consists of the OMV, PT (preferably genetically detoxified PT), FHA, PRN and FIM2 / FIM3 of the present invention.

[0186] For combination pediatric vaccines, the D:T ratio is typically greater than 1 (i.e., pediatric vaccines usually have an excess of D in Lf units), and is generally between 2:1 and 3:1 (measured in Lf units), e.g., 2.5:1. In contrast, for booster vaccines administered to adolescents or adults (who have usually received at least one combination pediatric vaccine containing D and T), the T:D ratio is typically greater than 1 (i.e., booster vaccines usually have an excess of T in Lf units), and is generally between 1.5:1 and 2.5:1, e.g., 2:1.

[0187] One useful vaccine comprises an OMV of the invention and, per unit dose, 2Lf D, 5Lf T, 4μg PT-9K / 129G, 4μg FHA, and 8μg pertactin. Another useful vaccine comprises an OMV of the invention and, per unit dose, 25Lf D, 10Lf T, 25μg PT-9K / 129G, 25μg FHA, and 8μg pertactin.

[0188] When antigen components are combined to prepare a multivalent composition, the antigens may be added individually or may be premixed. If the combined vaccine comprises D and T antigens and a further antigen, a premixed DT component may be used in preparing the combined vaccine. This bivalent component may be combined with the further antigen. When a DT mixture is used to prepare a combined vaccine, the ratio of diphtheria toxoid to tetanus toxoid in the mixture may be between 2:1 and 3:1 (measured in Lf units), preferably between 2.4:1 and 2.6:1, for example preferably 2.5:1.

[0189] Treatment Method In a fourth aspect of the invention, the OMV or immunogenic composition may be used to induce an immune response in a mammal, particularly a suitable mammal, preferably a human, and the immune response is preferably protective and preferably involves antibodies.

[0190] The immunogenic and pharmaceutical compositions of the present invention are for in vivo use to elicit an immune response against an immunogen of interest. As disclosed herein, a method of raising an immune response in a mammal is provided, comprising administering an effective amount of an immunogenic or pharmaceutical composition of the present invention. The immune response is preferably protective and preferably involves antibodies and / or cell-mediated immunity. The method can raise a booster response.

[0191] The invention also provides an immunogenic or pharmaceutical composition of the invention for use in a method of raising an immune response in a mammal.

[0192] The invention also provides the use of an OMV or population of OMVs of the invention in the manufacture of a medicament for raising an immune response in a mammal.

[0193] The uses and methods of the present invention can raise an immune response in a mammal, thereby protecting the mammal against a variety of diseases and / or infections, such as the bacterial and / or viral diseases discussed above. The OMVs and compositions are immunogenic, and more preferably are vaccine compositions. Vaccines according to the present invention can be either prophylactic (i.e., to prevent infection) or therapeutic (i.e., to treat infection), but will typically be prophylactic.

[0194] Mammals, particularly suitable mammals, can be humans or large veterinary mammals (e.g. horses, cows, deer, goats and pigs). If the vaccine is intended for prophylactic use, the human is preferably a child (e.g. a toddler or infant) or a teenager. If the vaccine is intended for therapeutic use, the human is preferably a teenager or an adult. Vaccines intended for children can also be administered to adults to assess, for example, safety, dosage, immunogenicity, etc.

[0195] Vaccines prepared according to the present invention can be used to treat both children and adults. Thus, the human patient can be less than 1 year old, less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. In some embodiments, the child is an individual less than 1 year old, e.g., less than 1 day old, about 1 week old, about 2 weeks old, about 3 weeks old, about 4 weeks old, about 2 months old, about 3 months old, about 4 months old, about 5 months old, about 6 months old, about 7 months old, about 8 months old, about 9 months old, about 10 months old, about 11 months old, about 12 months old.

[0196] Particular patient groups that may receive the vaccine include the elderly (e.g., 20-50 years old, up to 60 years old, preferably up to 65 years old), younger people (e.g., up to 5 years old or infants under about 12 months of age), hospitalized patients, health care workers, military personnel and soldiers, pregnant women, and chronically ill or immunocompromised patients. However, the vaccine is not only suitable for these groups, but may be used more generally in the population.

[0197] The compositions of the present invention will generally be administered directly to the patient. Direct delivery can be by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal, or into the interstitial space of tissue). Alternative delivery routes include rectal, oral (e.g., tablets, sprays), buccal, sublingual, vaginal, topical, transdermal or transdermal, intranasal, ocular, otic, pulmonary or other mucosal administration. Intradermal and intramuscular administration are two preferred routes. Injection can be via a needle (e.g., a hypodermic needle), but needle-free injection can be used instead. A typical intramuscular dose is 0.5 mL.

[0198] The present invention can be used to induce systemic and / or mucosal immunity, preferably to induce enhanced systemic and / or mucosal immunity.

[0199] The uses and methods of the invention are preferably for the prevention and / or treatment of diseases caused by Bordetella pertussis and optionally one or more of Corynebacterium diphtheriae, Clostridium tetani, Haemophilus influenzae serotype b, poliovirus and hepatitis B. In a preferred embodiment, the uses and methods of the invention are for the prevention of diseases caused by Bordetella pertussis.

[0200] The subject whose disease is prevented may not be the same as the subject who receives the immunogenic composition of the present invention. For example, the immunogenic composition may be administered to a woman (before or during pregnancy) to protect her offspring (so-called "maternal immunization"). The immunization of a pregnant woman provides antibody-mediated immunity to the infant through passive immunity due to transferred maternal antibodies ("passive maternal immunity"). Passive immunity occurs naturally when maternal antibodies are transferred to the fetus through the placenta. Passive immunity is particularly important for infants, since infants are born without any active acquired immunity. Administration of the composition of the present invention to a pregnant woman boosts the immunity of the woman and transfers antibodies through the placenta to the newborn, thus conferring passive maternal immunity to the infant. However, the passive immunity of the infant is only temporary and begins to decline several weeks or months after birth. Because passive immunity is only temporary, it may be important for the infant to receive the composition of the present invention to induce active immunity in the infant before the passive immunity fades. Administration of the second immunogenic composition to the infant after birth induces active immunity in the infant, extending the immunity inherited from the mother during pregnancy.

[0201] Dosing may be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or a booster immunization schedule. In a multiple dose schedule, various doses may be given by the same route or by different routes, e.g., a parenteral prime and a mucosal boost, a mucosal prime and a parenteral boost, etc. Multiple doses will typically be administered at least one week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In one embodiment, multiple doses may be administered at approximately 6 weeks, 10 weeks and 14 weeks after birth, e.g., at 6 weeks, 10 weeks and 14 weeks of age, as is often used in the World Health Organization's Expanded Program on Immunisation ("EPI"). In an alternative embodiment, two primary doses are administered about two months apart, e.g., about 7, 8, or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the second primary dose, e.g., about 6, 8, 10, or 12 months after the second primary dose. In a further embodiment, three primary doses are administered about two months apart, e.g., about 7, 8, or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the third primary dose, e.g., about 6, 8, 10, or 12 months after the third primary dose.

[0202] Methods for modulating lipid A reactogenicity In a fifth aspect, the present invention provides a method for modulating the reactogenicity of lipid A of Bordetella pertussis bacteria, comprising stably integrating at least one gene selected from the group consisting of LpxA and LpxD into the genome of the bacterium, wherein: (i) the LpxA gene encodes an LpxA protein having at least 80% sequence identity to SEQ ID NO: 1 or 2, wherein said protein comprises a serine at position 170 (S170) and / or an alanine at position 229 (A229) when numbered according to SEQ ID NO: 1 or 2; and / or (ii) the LpxD gene encodes an LpxD protein having at least 90% amino acid sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; Methods As mentioned above, the reactogenicity of bacterial lipid A in Gram-negative bacteria, particularly Bordetella pertussis, can be modulated by expression of exogenous enzymes from the lipid A biosynthetic pathway, such as the acetyltransferases LpxA and LpxD.

[0203] Genomic integration of the LpxA and LpxD genes allows stable expression of exogenous DNA without the need for antibiotic selection. In addition, the inventors surprisingly discovered that such strains do not suffer from the growth defects seen in the art.

[0204] definition As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise, i.e., "a" means "one or more" unless expressly stated otherwise.

[0205] The term "and / or" as used in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A," and "B." Similarly, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0206] The term "comprising" includes "including", e.g., a composition "comprising" X can include more, e.g., X+Y. The term "substantially" does not exclude "completely", e.g., a composition "substantially free" of Y can be completely free of Y. In some implementations, the term "comprising" refers to including the indicated active agent, such as a recited polypeptide, as well as other active agents, and pharma- ceutically acceptable carriers, excipients, emollients, stabilizers, and the like, known in the pharmaceutical industry. In some implementations, the term "consisting essentially of" refers to a composition in which the only active ingredient of the composition is the specified active ingredient(s), e.g., an antigen, but which may include other compounds for purposes such as stabilizing the formulation, preserving it, and the like, but not directly involved in the therapeutic effect of the specified active ingredient. Using the transitional phrase "consisting essentially of" means that the scope of the claim is to be construed to include the particular materials or steps recited in the claim and which do not materially affect the basic and novel characteristics of the claimed invention. See In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03. Thus, the term "consisting essentially of," when used in the claims of the present invention, is not intended to be construed as equivalent to "comprising." The term "consisting of" and variations thereof mean "limited to," unless expressly specified otherwise. In certain areas, the term "comprising an active ingredient consisting of" may be used in place of "consisting essentially of."

[0207] The term "about" in reference to a numerical value x means within an acceptable contextual error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system or the degree of precision required for a particular purpose, e.g., x±10%, x±5%, x±4%, x±3%, x±2%, x±1%.

[0208] The term "substantially" does not exclude "completely", for example, a composition "substantially free" of Y may be completely free of Y, but in all important respects it functions as a free condition, but the numerical value may include a condition indicating the presence of some impurity or substance. If necessary, the term "substantially" may be omitted from the definition of the present invention. When a particular value is used in the present specification and claims, unless otherwise specified, the term "substantially" means that there is an acceptable error range for the particular value.

[0209] When a method refers to steps of administration, e.g., (a), (b), (c), etc., it is intended that these are sequential, i.e., step (a) precedes step (b), which is followed by step (c). An antibody will generally be specific for its target, i.e., have a higher affinity for the target than for an unrelated control protein, e.g., bovine serum albumin.

[0210] All publications, patents, or patent applications cited within this patent specification are hereby incorporated by reference.

[0211] Specific embodiments of the present invention Embodiment 1. A recombinant Bordetella pertussis bacterium, comprising: (i) at least one genomic LpxA gene encoding an LpxA protein comprising a mutation at position 170 and / or a mutation at position 229 relative to SEQ ID NO:1; and / or (ii) at least one genomic insertion of a heterologous LpxD gene; A recombinant Bordetella pertussis bacterium comprising:

[0212] Embodiment 2. A recombinant Bordetella pertussis bacterium of embodiment 1, comprising at least one genomic LpxA gene encoding an LpxA protein comprising a substitution at position 170 and / or a substitution at position 229 relative to SEQ ID NO:1.

[0213] Embodiment 3. The recombinant Bordetella pertussis bacterium of embodiment 1 or 2, wherein the LpxA protein comprises a serine residue at position 170 and / or an alanine residue at position 229 relative to SEQ ID NO:1.

[0214] Embodiment 4. The recombinant Bordetella pertussis bacterium of embodiment 3, wherein the LpxA protein comprises (i) a serine residue at position 170 and (ii) an alanine residue at position 229, when numbered according to SEQ ID NO:1.

[0215] Embodiment 5. The recombinant Bordetella pertussis bacterium of embodiment 4, comprising an LpxA gene encoding an LpxA protein having at least 80% amino acid sequence identity to SEQ ID NO: 1 or 2.

[0216] Embodiment 6. The LpxA gene from Bordetella parapertussis, which encodes the LpxA protein (LpxA Bpa ), a recombinant Bordetella pertussis bacterium of any one of the preceding embodiments.

[0217] Embodiment 7. The recombinant Bordetella pertussis bacterium of any one of the preceding embodiments, comprising an LpxA gene encoding an LpxA protein having SEQ ID NO:2.

[0218] Embodiment 8. The recombinant Bordetella pertussis bacterium of any one of the preceding embodiments, wherein the heterologous LpxD gene encodes an LpxD protein having at least 90% amino acid sequence identity to SEQ ID NO:3 or SEQ ID NO:4.

[0219] Embodiment 9. The recombinant Bordetella pertussis bacterium of embodiment 8, wherein the heterologous LpxD gene encodes an LpxD protein having at least 95% amino acid sequence identity to SEQ ID NO:3 or SEQ ID NO:4.

[0220] Embodiment 10. The recombinant Bordetella pertussis bacterium of embodiment 9, wherein the heterologous LpxD gene encodes an LpxD protein having an amino acid sequence selected from the group consisting of SEQ ID NO:3 or SEQ ID NO:4.

[0221] Embodiment 11. The recombinant Bordetella pertussis bacterium of any one of the preceding embodiments, wherein the endogenous LpxA gene is inactivated and / or the endogenous LpxD gene is inactivated.

[0222] (i) at least 30% of the C3' acyl chains are from about C10 to about C12 in length; and / or (ii) at least 30% of the C2' acyl chains are from about C10 to about C12 in length; and / or (iii) at least 30% of the C2 acyl chains are from about C10 to about C12 in length The recombinant Bordetella pertussis bacterium of any one of the preceding embodiments, which produces lipid A.

[0223] Embodiment 13. The recombinant Bordetella pertussis bacterium of any one of the preceding embodiments, wherein (i) the bacterium produces lipid A, wherein at least 30% of the C3' acyl chains are from C10 to about C12 in length.

[0224] Embodiment 14. The recombinant Bordetella pertussis bacterium of any one of the preceding embodiments, wherein (i) the bacterium produces lipid A in which at least 30% of the C3' acyl chains are C10 in length.

[0225] Embodiment 15. The recombinant Bordetella pertussis bacterium of any one of the preceding embodiments, which produces lipid A in which substantially all of the C3' acyl chains of the lipid A have a length of C10.

[0226] Embodiment 16. The recombinant Bordetella pertussis bacterium of any one of the preceding embodiments, which produces lipid A having reduced endotoxin activity compared to the endotoxin activity of lipid A produced by the parent strain, particularly as measured using a TLR4 stimulation assay, particularly a human TLR4 stimulation assay.

[0227] Embodiment 17. The recombinant Bordetella pertussis bacterium of any one of the preceding embodiments, wherein a bacterial population growth curve of said bacterium is comparable to a population growth curve of the parent strain.

[0228] Embodiment 18. The recombinant Bordetella pertussis bacterium of any one of the preceding embodiments, derived from the Tohama I strain and expressing the genetically detoxified pertussis toxoid PT-9K / 129G.

[0229] Embodiment 19. An isolated outer membrane vesicle (OMV) derived from the recombinant Bordetella pertussis bacterium of any one of embodiments 1 to 18, comprising a modified lipid A incorporated into the membrane, wherein: Substantially all of the C3' acyl chains of lipid A have a length of C10; and / or An OMV in which substantially all of the C2 and C2' acyl chains of lipid A are C12 in length.

[0230] Embodiment 20. An isolated outer membrane vesicle according to embodiment 19, comprising a modified lipid A incorporated into the membrane, wherein substantially all of the C3' acyl chains of lipid A have a length of C10.

[0231] Embodiment 21. An isolated outer membrane vesicle according to embodiment 19 or 20, which is substantially free of dermonecrotic toxin and / or contains endogenous genetically detoxified pertussis toxoid.

[0232] Embodiment 22. An immunogenic composition comprising at least one isolated OMV according to embodiments 19-21 and a pharma- ceutically acceptable excipient.

[0233] Embodiment 23. The immunogenic composition of embodiment 22, further comprising at least one additional antigen selected from the group consisting of: (1) pertussis toxoid (PT), (2) FHA, (3) pertactin (PRN), (4) FIM2 / FIM3, (5) adenylate cyclase, (6) diphtheria toxoid (DT), (7) tetanus toxoid (TT), (8) inactivated poliovirus (IPV), (9) hepatitis B surface antigen, and (10) Hib PRP.

[0234] Embodiment 24. The OMV of embodiments 19 to 21 or the immunogenic composition of embodiment 22 or 23 for use in inducing an immune response in a suitable mammal, such as a human.

[0235] Embodiment 25. The OMV of embodiments 19 to 21 or the immunogenic composition of embodiment 22 or 23 for use in prophylaxis or as a vaccine, for example in the prevention of disease caused by Bordetella pertussis.

[0236] Embodiment 26 The OMV of embodiments 19 to 21 or the immunogenic composition of embodiment 22 or 23 for use in the prevention of disease caused by Bordetella pertussis.

[0237] Embodiment 27. A method of modulating the reactogenicity of lipid A of Bordetella pertussis bacteria, comprising stably integrating into the genome of the bacterium at least one gene selected from the group consisting of LpxA and LpxD, wherein: (i) the LpxA gene encodes an LpxA protein having at least 80% sequence identity to SEQ ID NO: 1 or 2, wherein said protein comprises a serine at position 170 (S170) and / or an alanine at position 229 (A229) when numbered according to SEQ ID NO: 1 or 2; and / or (ii) the LpxD gene encodes an LpxD protein having at least 90% amino acid sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; method.

[0238] Embodiment 28. A recombinant Bordetella pertussis bacterium comprising at least one genomic LpxA gene encoding an LpxA protein, wherein the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), and wherein the LpxA gene encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2.

[0239] Embodiment 29. A recombinant Bordetella pertussis bacterium comprising at least one episomal LpxA gene encoding an LpxA protein, wherein the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), and wherein the LpxA gene encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2.

[0240] Embodiment 30. A recombinant Bordetella pertussis bacterium comprising: (i) at least one genomic LpxA gene encoding the LpxA protein, a genomic LpxA gene, wherein the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), and the LpxA gene encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2; (ii) at least one genomic insertion of a heterologous LpxD gene encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3 or SEQ ID NO:4.

[0241] Embodiment 31. A recombinant Bordetella pertussis bacterium comprising: At least one episomal LpxA gene encoding the LpxA protein, an episomal LpxA gene, wherein the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), and the LpxA gene encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2; (ii) at least one genomic insertion of a heterologous LpxD gene encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3 or SEQ ID NO:4.

[0242] Embodiment 32. A recombinant Bordetella pertussis bacterium comprising at least one genomic insertion of a heterologous LpxD gene encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3 or SEQ ID NO:4.

[0243] Embodiment 33. A recombinant Bordetella pertussis bacterium comprising at least one genomic LpxA gene encoding an LpxA protein, the LpxA protein comprising a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), the LpxA gene encoding an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2, the bacterium being derived from the Tohama strain and expressing the genetically detoxified pertussis toxoid PT-9K / 129G.

[0244] Embodiment 34. A recombinant Bordetella pertussis bacterium comprising at least one episomal LpxA gene encoding an LpxA protein, the LpxA protein comprising a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), the LpxA gene encoding an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2, the bacterium being derived from the Tohama I strain and expressing the genetically detoxified pertussis toxoid PT-9K / 129G.

[0245] Embodiment 35. A recombinant Bordetella pertussis bacterium comprising: (i) at least one genomic LpxA gene encoding the LpxA protein, a genomic LpxA gene, wherein the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), and the LpxA gene encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2; (ii) at least one genomic insertion of a heterologous LpxD gene encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3 or SEQ ID NO:4; wherein the bacterium is a recombinant Bordetella pertussis bacterium derived from the Tohama I strain and expressing the genetically detoxified pertussis toxoid PT-9K / 129G.

[0246] Embodiment 36. A recombinant Bordetella pertussis bacterium, comprising: At least one episomal LpxA gene encoding the LpxA protein, an episomal LpxA gene, wherein the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), and the LpxA gene encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2; (ii) at least one genomic insertion of a heterologous LpxD gene encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3 or SEQ ID NO:4; wherein the bacterium is a recombinant Bordetella pertussis bacterium derived from the Tohama I strain and expressing the genetically detoxified pertussis toxoid PT-9K / 129G.

[0247] Embodiment 37. A vector comprising at least one genomic insertion of a heterologous LpxD gene encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3 or SEQ ID NO:4, Recombinant Bordetella pertussis bacteria derived from the Tohama I strain and expressing the genetically detoxified pertussis toxoid PT-9K / 129G.

[0248] Embodiment 38. A recombinant Bordetella pertussis bacterium comprising at least one genomic LpxA gene encoding an LpxA protein, the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), and the LpxA gene encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2; The bacterium is derived from the Tohama I strain, expresses the genetically detoxified pertussis toxoid PT-9K / 129G, and does not express the dermonecrotic toxin (DNT) gene (SEQ ID NO: 19). Recombinant Bordetella pertussis bacteria.

[0249] Embodiment 39. A recombinant Bordetella pertussis bacterium comprising at least one episomal LpxA gene encoding an LpxA protein, the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), and the LpxA gene encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2; The bacterium is derived from the Tohama I strain, expresses the genetically detoxified pertussis toxoid PT-9K / 129G, and does not express the dermonecrotic toxin (DNT) gene (SEQ ID NO: 19). Recombinant Bordetella pertussis bacteria.

[0250] Embodiment 40. A recombinant Bordetella pertussis bacterium comprising: (i) at least one genomic LpxA gene encoding the LpxA protein, a genomic LpxA gene, wherein the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), and the LpxA gene encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2; (ii) at least one genomic insertion of a heterologous LpxD gene encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3 or SEQ ID NO:4; wherein said bacterium is a recombinant Bordetella pertussis bacterium derived from the Tohama I strain, expressing the genetically detoxified pertussis toxoid PT-9K / 129G and not expressing the dermonecrotic toxin (DNT) gene (SEQ ID NO: 19).

[0251] Embodiment 41. A recombinant Bordetella pertussis bacterium comprising: At least one episomal LpxA gene encoding the LpxA protein, an episomal LpxA gene, wherein the LpxA protein comprises a serine residue at position 170 and an alanine residue at position 229 (relative to SEQ ID NO: 1), and the LpxA gene encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 or 2; (ii) at least one genomic insertion of a heterologous LpxD gene encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3 or SEQ ID NO:4; wherein said bacterium is a recombinant Bordetella pertussis bacterium derived from the Tohama I strain, expressing the genetically detoxified pertussis toxoid PT-9K / 129G and not expressing the dermonecrotic toxin (DNT) gene (SEQ ID NO: 19).

[0252] Embodiment 42. A vector comprising at least one genomic insertion of a heterologous LpxD gene encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3 or SEQ ID NO:4, Recombinant Bordetella pertussis bacteria derived from the Tohama I strain, expressing the genetically detoxified pertussis toxoid PT-9K / 129G and not expressing the dermonecrotic toxin (DNT) gene (SEQ ID NO: 19).

[0253] Embodiment 43. An isolated outer membrane vesicle (OMV) derived from the recombinant Bordetella pertussis bacterium of any one of embodiments 28 to 42, comprising a modified lipid A incorporated into its membrane, wherein substantially all of the C3' acyl chains of lipid A have a length of C10, and / or substantially all of the C2 acyl chains and C2' acyl chains of lipid A have a length of C12.

[0254] Embodiment 44. An isolated outer membrane vesicle (OMV) derived from the recombinant Bordetella pertussis bacterium of any one of embodiments 28 to 42, comprising a modified lipid A incorporated into the membrane, wherein substantially all of the C3' acyl chains of the lipid A have a length of C10.

[0255] Embodiment 45. An immunogenic composition, particularly a pharmaceutical composition, such as a vaccine, comprising an immunologically effective amount of an isolated OMV according to embodiment 43 or 44 and a pharma- ceutically acceptable carrier.

[0256] Embodiment 46 The recombinant Bordetella pertussis bacterium of any one of embodiments 1 to 18, comprising a genomic insertion of a heterologous LpxE gene.

[0257] Embodiment 47. The heterologous LpxE gene is from Rhizobium leguminosarum (LpxE Rl 47. A recombinant Bordetella pertussis bacterium according to embodiment 46.

[0258] Embodiment 48. The recombinant Bordetella pertussis bacterium of embodiment 47, wherein the amino acid sequence encoded by the heterologous LpxE gene is SEQ ID NO:6.

[0259] Embodiment 49. The heterologous LpxE gene is from Francisella novicida (LpxE Fn 47. A recombinant Bordetella pertussis bacterium according to embodiment 46.

[0260] Embodiment 50. The recombinant Bordetella pertussis bacterium of embodiment 49, wherein the amino acid sequence encoded by the heterologous LpxE gene is SEQ ID NO:7.

[0261] Embodiment 51 The recombinant Bordetella pertussis bacterium of any one of embodiments 46 to 50, wherein the heterologous LpxE gene is under the control of the BP0840 promoter.

[0262] Embodiment 52 The recombinant Bordetella pertussis bacterium of any one of embodiments 46 to 51, wherein the heterologous LpxE gene is inserted into the bacterial genome.

[0263] Embodiment 53. The recombinant Bordetella pertussis bacterium of embodiment 52, wherein the heterologous LpxE gene replaces the ArnT gene locus (ΔArnT).

[0264] Embodiment 54. The recombinant Bordetella pertussis bacterium of any one of embodiments 1 to 18, comprising a genomic insertion of a heterologous LpxF gene.

[0265] Embodiment 55. The heterologous LpxF gene is from Francisella novicida (LpxF Fn ), a recombinant Bordetella pertussis bacterium of embodiment 54.

[0266] Embodiment 56. The recombinant Bordetella pertussis bacterium of embodiment 55, wherein the amino acid sequence encoded by the heterologous LpxF gene is SEQ ID NO:8.

[0267] Embodiment 57. The recombinant Bordetella pertussis bacterium of any one of embodiments 54 to 56, wherein the heterologous LpxF gene is under the control of the BP0840 promoter.

[0268] Embodiment 58 The recombinant Bordetella pertussis bacterium of any one of embodiments 54 to 57, wherein a heterologous LpxF gene is inserted into the bacterial genome.

[0269] Embodiment 59. The recombinant Bordetella pertussis bacterium of embodiment 58, wherein the heterologous LpxF gene replaces the ArnT gene locus (ΔArnT).

[0270] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Bacterial strains and gene sequences Tables 1-3 list the key bacterial strains, DNA and amino acid sequences used:

[0271] [Table 1]

[0272] [Table 2]

[0273] [Table 3]

[0274] Creation and cultivation of recombinant bacterial strains Heterologous Lpx genes were expressed in B. pertussis either by genomic integration or by episomal (non-genomic) expression.

[0275] Genomic integration Desired LpxA Bpa The synthetic sequence containing the sequence (SEQ ID NO: 9) and the upstream and downstream homology arms (SEQ ID NO: 14 and 15) enabling homologous recombination was cloned into the vector pSORTP1

[62] using the flanking EcoRI and HindIII restriction sites. The vector pSORTP1 contains an origin of conjugal transfer, selection markers for gentamicin and ampicillin, and a copy of the rpsL gene that confers sensitivity to streptomycin.

[0276] The pSORTP1 vector was transformed into E. coli SM10 λpir and recombinant clones were co-cultivated with the nalidixic acid / streptomycin-resistant B. pertussis strain Tohama I to allow conjugative transfer of the vector. Streptomycin resistance in this strain is conferred by a mutation in the rpsL gene. The plasmid cannot replicate in B. pertussis and must be integrated into the bacterial genome by homologous recombination to confer gentamicin resistance. Recombinant B. pertussis was obtained by simultaneous selection with gentamicin (to eliminate wild-type B. pertussis) and nalidixic acid (to eliminate the E. coli SM10 donor strain). Genomic transfer of the pSORTP1 vector containing a functional copy of the rpsL gene sensitizes B. pertussis to streptomycin. Upon selection with streptomycin, a second homologous recombination event forces removal of the vector backbone opposite the transgene, resulting in a markerless mutation. Using this approach, a recombinant Bordetella pertussis Tohama I strain was obtained that expresses the LpxA sequence from Bordetella pertussis. Several further strains were constructed as follows:

[0277] ΔArnT / PagL BbrIn the strain, the ArnT locus in the B. pertussis genome was replaced with a copy of the Bordetella bronchiseptica PagL gene under the control of the promoter region of B. pertussis BP0840 (outer membrane porin). A recombinant sequence was constructed with the upstream and downstream regions of ArnT containing the B. bronchiseptica PagL sequence. The recombinant sequence was cloned into the vector pSORTP1 using the flanking EcoRI and HindIII restriction sites. Following transformation, as described above, the resulting strain combines knockout of the ArnT gene with expression of the PagL gene.

[0278] ΔArnT / ΔLpxA Bpe / LpxA Bpa The strain knocks out the ArnT locus (SEQ ID NO: 20) in the Bordetella pertussis genome.

[0279] ΔLpxA Bpe / LpxA Bpa / ΔDNT / LpxD Pa / ΔLpxD Bpe In strains, LpxD Pa (SEQ ID NO: 21) was integrated into the genome, replacing the dermonecrotic toxin (DNT) gene (SEQ ID NO: 19). Bp was knocked out.

[0280] LpxE Fn / ΔArnT / ΔLpxA Bpe / LpxA Bpa In the strain, the ArnT locus (SEQ ID NO: 20) in the B. pertussis genome was replaced with a copy of the Francisella novicida LpxE gene (SEQ ID NO: 22) under the control of the promoter region of B. pertussis BP0840 (outer membrane porin SEQ ID NO: 17). A recombinant sequence was constructed with the upstream and downstream regions of ArnT containing the Francisella novicida LpxE sequence. The recombinant sequence was cloned into the vector pSORTP1 using the flanking EcoRI and HindIII restriction sites. Following transformation, as described above, the resulting strain combines knockout of the ArnT gene with expression of the LpxE gene.

[0281] Episomal expression The synthetic sequence was cloned into the vector pMMB67EH (ATCC Ref.: 37622) using the flanking Acc65I and HindIII sites. Ct、 LpxD Pa、 LpxE Fn , LpxE Rl and LpxF Fn The coding sequence was codon-optimized for expression in Bordetella pertussis (PriorityGENE service, GENEWIZ).

[0282] The pMMB67EH vectors containing the heterologous LpxD, LpxE or LpxF genes were transformed into E. coli SM10 λpir and recombinant clones were co-cultured with the nalidixic acid / streptomycin resistant B. pertussis strain Tohama I to allow conjugal transfer of the vector. Recombinant B. pertussis containing the pMMB67EH plasmid were then selected with ampicillin and nalidixic acid. Stable transformation of B. pertussis Tohama I with the respective pMMB67EH constructs was confirmed by PCR detection of the heterologous Lpx sequence.

[0283] B. pertussis recombinant strains were grown under standard conditions in commercially available media (see 63), which was supplemented with 1 mM IPTG for episomal expression.

[0284] Recombinant Bordetella pertussis ΔLpxA Bpe / LpxA Bpa Growth assessment of; To assess any potential effects on bacterial growth, the recombinant strain ΔLpxABpe / LpxA Bpa was tested in a 10 L fermentation.

[0285] Briefly, two complete Bordet-Gengou agar plates were used to inoculate two 30 ml, 24 hour precultures. The 30 mL precultures were then used to inoculate two additional 24 hour precultures of 1 L. These two second precultures (approximately 1.5 L) were then used to inoculate the OD 650nm The 10 L fermenter was inoculated with 1.

[0286] A decrease in dissolved oxygen (DO) concentration was observed at approximately 9 hours. This decrease was associated with an increase in oxygen consumption due to the exponential growth rate of the culture. DO was then controlled by the agitation rate.

[0287] The end of the fermentation process after 37.5 h was marked by a decrease in the agitation rate caused by a decrease in oxygen consumption, evidence of the exhaustion of the main carbon source (Na-L-glutamate). 650nm ) was 10.1, and cell survival was 6.3 10 10 (CFU / ml).

[0288] Overall, ΔLpxA Bpe / LpxA Bpa The fermentation characteristics of the Tohama I PTg strain were comparable to those of the Tohama I PTg strain, with similar fermentation times and elapsed time to DO (Table 5, Figures 3-5).

[0289] [Table 4]

[0290] Preparation of OMVs Outer membrane vesicles (OMVs) were generated from bacterial cultures for mass spectrometric analysis of LPS structure and in vitro stimulation of the human TLR4 receptor. OMVs were generated by detergent extraction from bacterial pellets after 24 h of culture.

[0291] Briefly, bacterial pellets were resuspended in 20 mM Tris-HCl, 2 mM EDTA and 50 U / mL benzonase, pH 8.6, and incubated for 30 min at room temperature. For detergent extraction of OMVs, 0.1% DOC was added and the suspension was incubated for 30 min at 40°C. Cell debris was removed by centrifugation at 20,000g for 30 min (4°C) and the supernatant was sterile filtered (0.22 μm). The sterile supernatant was then ultracentrifuged (145,000g, 2 h, 4°C), resuspended in 1x PBS (with 5 mM EDTA), ultracentrifuged a second time (same conditions), resuspended in 1x PBS (without EDTA) and sterile filtered (0.22 μm).

[0292] Characterization of LPS structure by mass spectrometry OMV preparations were analyzed by liquid chromatography-mass spectrometry (LC-MS) to determine the effect of genomic mutations in LpxA and / or LpxD (or LpxE) and the effect of episomal expression of heterologous LpxA and / or LpxD, LpxE or LpxF genes on the structure of LPS. OMV preparations from wild-type strain Tohama I PTg were used as controls.

[0293] Briefly, OMV preparations were precipitated with 95% ethanol, the pellets were dried, dissolved in 50% methanol, and sonicated, the sonicated material was centrifuged, and 5 μL of the supernatant was injected onto an HPLC column for MS analysis of the LOS structure.

[0294] LpxA recombinant strain LpxA Bpa Genomic integration and expression of recombinant B. pertussis ΔLpxA Bpe / LpxA Bpa This has a profound effect on the LPS structure of the derived OMV, where 100% of the acyl chains at the C3′ position are C 14 From C 10 (Figure 6(B) - the dotted line indicates the shortening from C14 to C10). In contrast, the episomal LpxA in Bordetella pertussis Tomaha I BpaExpression of LpxA had a very low, unquantifiable effect (strain LpxA Bpa ). C 14 From C 10 The variant to episomal LpxA Bpa Although it is present only as a minority, Figure 7(B) shows where it may differ from the wild type (C 14 From C 10 For reference, the structure of the LOS derived from wild-type Bordetella pertussis Tomaha I Ptg is shown in FIG.

[0295] LpxA Bpa Genomic integration and expression of has a similar effect on LPS structure when combined with ΔArnT, where 100% of the acyl chains at the C3′ position are C 14 From C 10 The length of the sarcoid was shortened to 10 mm (Figure 8).

[0296] Importantly, LpxA Bpa Genomic integration and expression of ΔLpxA Bpe / LpxA Bpa / ΔDNT / LpxD Pa strains and ΔLpxA Bpe / LpxA Bpa / ΔDNT / LpxD Pa / ΔLpxD Bpe The same effect was seen on the C3' acyl chain length of the strains in which LpxD from Pseudomonas was also expressed (Figures 9 and 10). Expression of the exogenous LpxD gene in combination with inactivation of the respective endogenous LpxD had no detrimental effect on the activity of the exogenous LpxA gene. In addition, no significant growth differences were observed.

[0297] Similarly, in strains in which LpxEFn was also expressed, the effect of genomic integration and expression of LpxABpa on C3′ acyl chain length was not affected, and in this case 100% was C 10 It exhibited a length of .

[0298] LpxD recombinant strain LpxD from Pseudomonas aeruginosa (LpxDPa ) had only a partial effect on the LPS structure of recombinant Bordetella pertussis-derived OMVs (Figure 11), where only 36% of the total LPS displayed shortened length, either in both the C2 and C2' acyl chains (24%) or in only one of the C2 or C2' acyl chains (12%).

[0299] LpxD Pa Genomic expression of LpxD enhanced its effect on LPS, shortening the length of both C2 and C2' acyl chains in 70% of LPS and shortening the length of only C2 or C2' in 30% of LPS (Figure 9). Bpe Removal of the residual activity of ΔLpxA resulted in 100% chain shortening at both positions (ΔLpxA Bpe / LpxA Bpa / ΔDNT / LpxD Pa / ΔLpxD Bpe , Figure 10).

[0300] ArnT / PagL recombinant strain Inactivation of ArnT and genomic integration of PagL resulted in 100% removal of the C10 ester at C3 (Figure 12). For reference, the structure of the OMV derived from wild-type Bordetella pertussis Tomaha I Ptg is shown in Figure 13.

[0301] LpxE recombinant strain LpxE from F. novicida (strain B2982, 9.8); Fn In the case of LpxE, episomal expression resulted in 25% monophosphoryl lipid A. Fn Genomic expression of LpxE from R. leguminosarum (strain B2983, 9.9) increased the percentage of monophosphoryl lipid A to 91%. However, expression of the same construct in an LpxA mutant background surprisingly produced only 24% monophosphoryl lipid A. Similar results were observed with LpxE from R. leguminosarum (strain B2983, 9.9).

[0302] LpxE in a wild-type background Fn and LpxERc Structural data for recombinant strains expressing the α-terminal β ...

[0303] LpxF recombinant strain For LpxF from F. novicida (strain B2981, 9.10), no identifiable effect on the LOS structure was observed in a limited series of experiments (Figure 20).

[0304] In vitro assessment of TLR4 signaling activation in HEK-hTLR4 cells Stimulation of the TLR4 signaling cascade can lead to the activation of NF-κB and / or activator protein 1 (AP-1) transcription factors, which contribute to the expression of several proinflammatory cytokines, such as IL-1, IL-6, IL-8, and TNF-α, and thus play a key role in inflammation.

[0305] To analyze whether signaling activation of the TLR4 pathway was reduced when recombinant Bordetella pertussis-derived purified OMVs were used, TLR4-dependent transcriptional activity was determined in HEK-Blue™-hTLR4 cells (Invivogen).

[0306] Upon stimulation with TLR4 ligand, activation of NF-κB and AP-1 induces secretion of alkaline phosphatase, which can be detected in the supernatant using the QUANTI-Blue™ colorimetric assay. TLR4 receptor response was determined by measuring NF-κB and AP-1 dependent SEAP production in the supernatant measured at 655 nm. For each sample, three different dilutions were tested in triplicate. The percentage of signal due to TLR4 stimulation was visualized through specific blocking of the TLR4 receptor with anti-TLR4 antibodies, using an irrelevant antibody (anti-M72) as a control. The ability of OMVs from different recombinant B. pertussis spp. strains to activate TLR4 signaling was compared to that of OMVs from the Tohama I PTg strain (Figures 14-17).

[0307] For assays normalized by protein content, BEXSERO (MenB vaccine containing MenB OMVs), QUINVAXEM (wP vaccine) and INFANRIX HEXA (aP vaccine) were included as controls. For assays normalized by LPS content, purified LPS from Bordetella pertussis, detoxified (LpxL1 mutant) purified LPS from Neisseria, and a negative control without stimulation were included.

[0308] HEK-hTLR4 data showed that the LpxA standalone mutant ΔLpxA Bpe / LpxA Bpa The activation profile was comparable to that of INFANRIX HEXA (±0.5 OD), i.e., activation of the TLR4 signaling cascade was low.

[0309] Similar results were obtained for LpxA Bpa All recombinant B. pertussis strains carrying the genomic insertion of ΔArnT / ΔLpxA Bpe / LpxA Bpa , ΔLpxA Bpe / LpxA Bpa / ΔDNT / LpxD Pa , and ΔLpxA Bpe / LpxA Bpa / ΔDNT / LpxD Pa / ΔLpxD Bpe was obtained.

[0310] According to their partial effect on LPS structure, such LpxD mutants, in which LpxD is expressed in an episomal manner, induced a more moderate decrease in the activation of the TLR4 signaling cascade. More specifically, the OD observed with the LpxD mutants was 1.0-fold lower than that observed with the LpxD mutants. 655 Values ​​are the OD observed in unstimulated cells. 655 OD values ​​observed in cells stimulated with and / or aP vaccine 655 was higher than the value (≧0.5OD 655) but not the OD observed in cells stimulated with OMVs derived from wild-type Gram-negative bacteria. 655 The reduction was also observed in assays using both normalized proteins and LPS.

[0311] ΔLpxA Bpe / LpxA Bpa The decrease in TLR4 stimulation caused by the mutations had already saturated the readout, and no further decrease could be observed. Bpe / L pxA Bpa The effect of the ΔArnT mutation in the strain cannot be easily deduced. Bbr In the OMVs derived from the strains, an increase in TLR4 stimulation is observed. Given that such an increase is not observed in either ΔArnT mutant strain, this increase is likely due to the PagL Bbr It is hypothesized that this is caused by removal of the C3 acyl chain during activation.

[0312] Interestingly, although the LpxE mutant has an altered structure of lipid A, no significant reduction in TLR4 stimulation was detected in the assays used (FIGS. 21 and 22).

[0313] Efficacy studies conducted in a nasopharyngeal colonization model in Balb / c mice The goal of this study was to evaluate the impact of a dOMV candidate vaccine when co-administered with DTPa in a nasopharyngeal (NP) colonization model, compared with mice vaccinated with DTPa or DTPw vaccines, or with mice previously exposed to the wild-type Bordetella pertussis Tohama I strain (convalescent mice).

[0314] Experimental Design: Twenty-five 6-week-old female BALB / c mice were assigned to one of the groups listed in Tables 6 and 7 below. Table 6 details the first study, which included 25 mice. All of these mice were infected with wild-type Bordetella pertussis Tohama I strain 10 6CF / 10 μl (recovery mice). This study is called "LIMS 20190306." Table 7 details the second study, called "LIMS 20190307."

[0315] Both studies were performed with Bordetella pertussis Tohama I strain 10 5 They were designed to have the same-day challenge with CF / 10 μl, so convalescent mice (in LIMS 20190306) were older than vaccinated mice (in LIMS 20190307).

[0316] The distribution of the mean number of colony forming units (CFU), measured by counting colony growth on charcoal-cephalexin agar plates or by quantification by PCR, is assumed to be log-normal. The statistical method is an analysis of variance (ANOVA) of log10 values ​​with two factors (vaccine and day) and their interaction by method (classical or quantitative).

[0317] [Table 5]

[0318] [Table 6]

[0319] result: The humoral immune response is shown in Figure 23, which presents the individual antibody titers anti-PPRN IgG measured by ELISA on day 28 as well as the geometric mean titers (GMTs) and their 95% confidence intervals derived from the ANOVA model. Note that groups in Study 20190306 were evaluated on days 28 and 56.

[0320] Immunogenicity is observed in all vaccinated groups (Infanrix, Easy Five, OMV LPXA and OMV WT) for anti-PRN IgG measured by ELISA on day 28. However, lower immunogenicity is observed after Easy Five vaccination compared to the other vaccinated groups (GMR>3). Furthermore, convalescent mice show significantly lower immunogenicity compared to the vaccinated groups at the two time points D28 and D56 (GMT of 136 and 346, respectively).

[0321] The assessment of protection relates to the bacterial load defined by the mean number of colony forming units (CFU) per nasal cavity at each collection time (+2 hours and 4, 7, 13, 21 days post-challenge). Descriptive statistics are presented in FIG.

[0322] A significant difference in bacterial load was highlighted between the non-vaccinated group (i.e., Unvaccinated Group 1 in Table 7) and all other groups, suggesting some degree of protective immune response against nasal colonization after vaccination or reinfection.

[0323] A greater than 10 log CFU reduction (GMR=258) was observed in convalescent animals (Table 6) compared to unvaccinated animals, confirming the suitability of this group as a positive control.

[0324] Taken together, this suggests that the model is capable of detecting reduced nasal colonization.

[0325] A greater than 1 log CFU reduction was observed with OMV LpxA-Infanrix (GMR=61) (group 5 in Table 7) and OMV WT-Infanrix (GMR=48) (group 4 in Table 7) compared to unvaccinated animals and compared to Infanrix alone (GMR=17.7 and 13.8, respectively), but not compared to the unvaccinated (convalescent mice) group (GMR=4.2 and GMR=5.4, respectively).

[0326] Vaccination with both OMV vaccines in coadministration with DTPa resulted in a similar reduction in nasal colonization following challenge with Bordetella pertussis Tohama I strain compared to both unvaccinated and Infanrix (DTPa) vaccinated mice, indicating added value of OMVs in preventing nasopharyngeal colonization compared to the DTPa vaccine. It also indicates that detoxification of lipid A did not adversely affect the reduction in nasal colonization.

[0327] Efficacy studies performed in a Balb / c mouse lung clearance model The primary goal of this study was to evaluate the added value of a dOMV vaccine when co-administered with DTPa in a mouse model of protective efficacy against the PRN(-) strain of Bordetella pertussis.

[0328] Protective efficacy was assessed by determining the bacterial burden per lung at each harvest time (+2 hours and days 2, 5 and 8 post-challenge) by the mean number of colony forming units (CFU - log10).

[0329] Experimental design 26-week-old female BALB / c mice were randomly assigned to one of the study groups detailed in Table 8. On day 29, 5 × 10 6 Mice were challenged with CFU / 50 μl.

[0330] [Table 7]

[0331] result: The humoral immune response is shown in Figure 25. Figure 25 presents the individual anti-PRN IgG titers measured by ELISA on day 28 (one day before challenge) as well as the geometric mean titers (GMTs) and their 95% confidence intervals derived from the ANOVA model. Vaccine uptake was confirmed as seen by anti-PRN IgG in all vaccinated groups in Figure 25. All groups induced similar anti-PRN responses, except for the "Easy-Five" group, which had an estimated GMT approximately one-third lower than the other groups. These differences were detected as statistically significant.

[0332] The evaluation of protection against pertussis relates to the bacterial load defined by the mean number of colony forming units (CFU) per lung at each collection time (+2 hours and days 2, 5, 8 after exposure). Descriptive statistics are shown in Figure 26. As shown in Figure 26, significant differences between the no vaccine group and the other groups are highlighted, suggesting a protective immune response after vaccination with a pertussis-containing vaccine. Partial protection was observed in the groups immunized with Infanrix or EasyFive. Significant differences were observed between the groups immunized with Infanrix and those that also received dOMV at the same time, indicating the added value of dOMV in addition to the DTPa vaccine in the pulmonary clearance of PRNneg strains.

[0333] It was expected that different kinetic profiles would be obtained depending on the group (e.g. Infanrix and Easy five should be different from all other groups), which would be represented by a significant interaction between group and day in the statistical analysis.

[0334] A higher CFU reduction is observed after vaccination with OMVs compared to the unvaccinated group (GMR between 130000 and 42000 on day 5 and equal to 127439 on day 8) and compared to the Infanrix or Easy five groups (GMR less than 2000 on days 5 and 8). No differences are observed between the three OMV groups. These results are positive, since they show that OMV LpxA and OMV LpxA / LpxD are as effective as OMV WT in reducing lung clearing from colonization with B. pertussis PRN-negative strains.

[0335] Stimulation of human PBCs Experimental Design: Experiments were performed to assess reactogenic biomarker profile (innate cytokine) secretion by stimulated human peripheral blood mononuclear cells (PBMCs). Frozen PBMCs collected from three healthy blood donors were thawed and individually seeded in 96-well plates at a concentration of 5E6 cells per ml. Cells were then incubated with protein equivalents of the formulations for 24 hours at 37°C. Eight serial dilutions were prepared with 5-fold steps between dilutions as detailed in Table 9. At the end of incubation, cytokines were measured using a multiplex assay in harvested culture supernatants.

[0336] Human standards and controls were prepared for the study. The following cytokines were tested in this study: IL-6, IL-10, TNF-α ("TFNa"), IL-1α ("IL-1a"), IL-Iβ ("IL-1b"), and MIP-1α ("MIP1a").

[0337] [Table 8]

[0338] result: Six cytokines known to be involved in reactogenic mechanisms (i.e., IL-6, IL-10, TNF-α ("TFNa"), IL-1α ("IL-1a"), IL-Iβ ("IL-1b") and MIP-1α ("MIP1a")) were measured in culture supernatants for various serial dilutions. Figure 27 represents the average of three donors by stimulation and by cytokine (excluding dilution #1, which altered cell survival under these experimental conditions).

[0339] FIG. 27 shows that OMV LpxA exhibited reduced innate cytokine secretion by human PBMCs compared to OMV wild type (WT), suggesting reduced reactogenicity of OMVs harboring the LpxA mutation.

[0340] From the Kruskal-Wallis area under the curve (AUC) analysis grouping cytokines and dilutions at all dilutions (but excluding dilution #1) (see FIG. 27 and Table 9), it was observed that dOMV WT adsorbed on Al(OH)3 was the only condition that induced statistically significantly higher innate cytokines than Infanrix Hexa. No statistical differences were observed between the other conditions. Non-adsorbed dOMVs displaying mutations on LpxA or LpxA / LpxD showed a trend towards reduced innate cytokine secretion by human PBMCs compared to OMV WT, suggesting reduced reactogenicity of OMVs harboring LpxA or LpxA / D mutations. The level of innate cytokine secretion induced by OMV LpxA was in the same range as Bexsero and tended to be lower than the whole cell vaccine.

[0341] The invention has been described by way of example only, and it will be understood that modifications are possible whilst remaining within the scope and spirit of the invention as defined in the appended claims.

[0342] References [1] Acevedo R. et al., Bacterial outer membrane vesicles and vaccine applications. Front. Immunol. 2014 Mar 24;5:121. [2] Maeshima N., Fernandez R.C. (2013) Recognition of lipid A variants by the TLR4 / MD-2 receptor complex. Frontiers in Cellular and Infection Microbiology 3:3. [3] WO2018 / 167061 [4] Sweet C.R., Preston A., Toland E., Ramirez S.M., Cotter R.J., Maskell D.J., Raetz C.R. (2002) Relaxed acyl chain specificity of Bordetella UDP-N-acetylglucosamine acyltransferases. Journal of Biological Chemistry 277(21):18281-90. [5] Bartling C.M., Raetz C.R. (2009) Crystal structure and acyl chain selectivity of Escherichia coli LpxD, the N-acyltransferase of lipid A biosynthesis. Biochemistry 48(36):8672-83 [6] Steeghs L, Berns M, ten Hove J, de Jong A, Roholl P, van Alphen L, Tommassen J, van der Ley P. (2002) Expression of foreign LpxA acyltransferases in Neisseria meningitidis results in modified lipid A with reduced toxicity and retained adjuvant activity. Cell Microbiol. 2002 Sep;4(9):599-611. [7] EP0011243 [8] Fredriksen et al. (1991) Production, characterization and control of MenB-vaccine "Folkehelsa": an outer membrane vesicle vaccine against group B meningococcal disease..NIPH Ann. 14(2):67-80 [9] WO2004 / 019977

[10] US6,558,677

[11] WO2005 / 004908

[12] WO2011 / 036562

[13] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.

[14] Vaccine Design (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum.

[15] WO90 / 14837.

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

[17] Lyng (1990) Biologicals 18:11-17

[18] NIBSC code: 69 / 017

[19] Kuhmlann & Rieger (1995) Immunol Infect Dis 5:10-4

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

[21] NIBSC code: 98 / 552

[22] Lindberg (1999) Vaccine 17 Suppl 2:S28-36

[23] Buttery & Moxon (2000) J R Coll Physicians Lond 34:163-168

[24] Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii

[25] Goldblatt (1998) J. Med. Microbiol. 47:563-567

[26] EP-A-0477508

[27] US patent 5,306,492

[28] WO1998 / 42721

[29] Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114

[30] Lees et al. (1996) Vaccine 14:190-198

[31] WO1995 / 08348

[32] WO1998 / 42721

[33] US patent 4,882,317

[34] US patent 4,695,624

[35] EP-B-0477508

[36] Mol. Immunol. 1985, 22, 907-919

[37] EP-A-0208375

[38] WO2000 / 10599

[39] Gever et al. (1979) Med. Microbiol. Immunol. 165:171-288

[40] US patent 4,057,685

[41] US patent 4,673,574

[42] US patent 4,761,283;

[43] US patent 4,808,700

[44] US patent 4,459,286

[45] US patent 4,965,338

[46] US patent 4,663,160

[47] Research Disclosure, 453077 (Jan 2002)

[48] Anderson (1983) Infect Immun 39(1):233-238

[49] Anderson et al. (1985) J Clin Invest 76(1):52-59

[50] Kanra et al. (1999) The Turkish Journal of Paediatrics 42:421-427

[51] Ravenscroft et al. (2000) Dev Biol (Basel) 103: 35-47

[52] WO96 / 40242

[53] Verez-Bencomo V, Fernandez-Santana V, Hardy E, Toledo ME, Rodriguez MC, Heynngnezz L, Rodriguez A, Baly A, Herrera L, Izquierdo M, Villar A, Valdes Y, Cosme K, Deler ML, Montane M, Garcia E, Ramos A, Aguilar A, Medina E, Torano G, Sosa I, Hernandez I, Martinez R, Muzachio A, Carmenates A, Costa L, Cardoso F, Campa C, Diaz M, Roy R. A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b. Science. 2004 Jul 23;305(5683):522-5.

[54] WO2018 / 020046

[55] Vanlandschoot et al. (2005) J Gen Virol 86:323-31

[56] WO2007 / 054820

[57] WO2003 / 066094

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

[59] WO2008 / 028956

[60] WO2008 / 028957

[61] Liao et al. (2012) J Infect Dis. 205:237-43

[62] Stibitz S, Black W, Falkow S. The construction of a cloning vector designed for gene replacement in Bordetella pertussis. Gene. 1986;50(1-3):133-140.

[63] Goffin P, Slock T, Smessaert V, De Rop P, Dehottay P. A versatile, non genetically modified organism (GMO)-based strategy for controlling low-producer mutants in Bordetella pertussis cultures using antigenic modulation. Biotechnol J. 2015 Aug;10(8):1269-80.

[65] Karbarz M.J., Kalb S.R., Cotter R.J., Raetz C.R. (2003) Expression cloning and biochemical characterization of a Rhizobium leguminosarum lipid A 1-phosphatase. Journal of Biological Chemistry 278(41):39269-79.

[66] Wang X., Karbarz M.J., McGrath S.C., Cotter R.J., Raetz C.R. (2004) MsbA transporter-dependent lipid A 1-dephosphorylation on the periplasmic surface of the inner membrane: topography of Francisella novicida LpxE expressed in Escherichia coli. Journal of Biological

[67] Wang X., McGrath S.C., Cotter R.J., Raetz C.R. (2006) Expression cloning and periplasmic orientation of the Francisella novicida lipid A 4’-phosphatase LpxF. Journal of Biological Chemistry 281(14):9321-30.

[68] WO2013 / 130779

[69] WO2015 / 179270

Claims

1. A recombinant Bordetella pertussis bacterium comprising: At least one genomic LpxA gene encoding an LpxA protein comprising an amino acid sequence having at least 90% identity to SEQ ID NO:2; A recombinant Bordetella pertussis bacterium, wherein, in the amino acid sequence, the amino acid residue corresponding to position 170 of SEQ ID NO:1 is a serine residue, and the amino acid residue corresponding to position 229 of SEQ ID NO:1 is an alanine residue.

2. A recombinant Bordetella pertussis bacterium as described in claim 1, wherein the amino acid sequence has at least 99% identity to sequence number 2.

3. A recombinant Bordetella pertussis bacterium as described in claim 1, wherein the amino acid sequence has 100% identity to sequence number 2.

4. A recombinant Bordetella pertussis bacterium as described in claim 1, further comprising at least one genomic insertion of a heterologous LpxD gene encoding an LpxD protein having an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO:

4.

5. 5. The recombinant Bordetella pertussis bacterium of any one of claims 1 to 4, wherein the endogenous LpxA gene is inactivated.

6. A recombinant Bordetella pertussis bacterium as described in claim 4, in which the endogenous LpxD gene is inactivated.

7. (i) At least 30% of the C3' acyl chains are approximately C 10 From about C 12 and / or (ii) At least 30% of the C2' acyl chains are approximately C 10 From about C 12 and / or (iii) At least 30% of the C2 acyl chains are approximately C 10 From about C 12 is A recombinant Bordetella pertussis bacterium according to any one of claims 1 to 6, which produces lipid A.

8. A recombinant Bordetella pertussis bacterium according to any one of claims 1 to 7, which produces lipid A with reduced endotoxin activity compared to the endotoxin activity of lipid A produced by the parent strain, in particular when measured using a TLR4 stimulation assay, in particular a human TLR4 stimulation assay.

9. A recombinant Bordetella pertussis bacterium according to any one of claims 1 to 8, wherein the bacterial population growth curve of said bacterium is equivalent to the population growth curve of the parent strain.

10. An isolated outer membrane vesicle (OMV) derived from a recombinant Bordetella pertussis bacterium according to any one of claims 1 to 9, comprising a modified lipid A incorporated into a membrane, Substantially all of the C3' acyl chain of lipid A is C 10 and / or Substantially all of the C2 and C2' acyl chains of lipid A are C 12 having a length of Isolated outer membrane vesicles (OMVs).

11. 11. The isolated outer membrane vesicle of claim 10, which is substantially free of dermonecrotic toxin and / or contains endogenous genetically detoxified pertussis toxoid.

12. 12. An immunogenic composition comprising at least one isolated OMV of claim 10 or 11 and a pharma- ceutically acceptable excipient.

13. The immunogenic composition of claim 12, further comprising at least one additional antigen selected from the group consisting of: (1) pertussis toxoid (PT), (2) FHA, (3) pertactin (PRN), (4) FIM2 / FIM3, (5) adenylate cyclase, (6) diphtheria toxoid (DT), (7) tetanus toxoid (TT), (8) inactivated poliovirus (IPV), (9) hepatitis B surface antigen, and (10) Hib PRP.

14. 14. An OMV as claimed in claim 10 or 11, or an immunogenic composition as claimed in claim 12 or 13, for use in inducing an immune response in a suitable mammal, such as a human.

15. 14. An OMV as claimed in claim 10 or 11, or an immunogenic composition as claimed in claim 12 or 13, for use in prophylaxis or as a vaccine.

16. 1. A method for modulating the reactogenicity of lipid A of Bordetella pertussis bacteria, comprising: stably integrating into the genome of said bacterium at least one genomic LpxA gene encoding an LpxA protein comprising an amino acid sequence having at least 90% identity to SEQ ID NO:

2. Including, A method wherein, in the amino acid sequence, the amino acid residue corresponding to position 170 of SEQ ID NO:1 is a serine residue, and the amino acid residue corresponding to position 229 of SEQ ID NO:1 is an alanine residue.

17. The method described in claim 16, further comprising a step of stably incorporating at least one heterologous LpxD gene encoding an LpxD protein having an amino acid sequence having at least 90% identity to SEQ ID NO:4 into the genome of the bacterium.

Images