Abstract:
A composition is provided comprising  N. meningitidis  outer membrane vesicles, wherein said outer membrane vesicles are enriched with at least one antigenic component. The composition is suitable for use in vaccines and for treatment of infection, particularly meningococcal infection, demonstrating a broad spectrum of protection. A number of preferred antigenic components are described and include antigenic proteins and proteoglycans derived from  N. meningitidis.

Description:
REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a Continuation-in-Part of U.S. application Ser. No. 12/289,098, filed Oct. 20, 2008, now pending, which is a continuation of application Ser. No. 10/320,800, filed Dec. 17, 2002, now abandoned, which is a Continuation-in-Part of U.S. application Ser. No. 09/830,854 filed on Aug. 29, 2001, now U.S. Pat. No. 6,821,521 B1, filed as application No. PCT/GB99/03626 on Nov. 2, 1999, the contents of each of these applications are fully incorporated herein by reference. 
     
    
     BACKGROUND 
       [0002]    The present invention relates to a multicomponent vaccine and methods for preparing a multicomponent vaccine that confers protective immunity to a broad spectrum of infection by Gram negative pathogens. In particular the present invention relates to a multicomponent vaccine that provides both passive and active protective immunity to meningococcal disease. 
         [0003]    Meningococcal meningitis is a major problem worldwide and in many countries incidence of infection is increasing.  Neisseria meningitidis  is the causative agent of the disease and is also responsible for meningococcal septicaemia, which is associated with rapid onset and high mortality, with around 22% of cases proving fatal. 
         [0004]    At present, vaccines directed at providing protective immunity against meningococcal disease provide only limited protection because of the many different strains of  N. meningitidis . Vaccines based upon the serogroup antigens, the capsular polysaccharides, offer only short lived protection against infection and do not protect against many strains commonly found in North America and Europe. A further drawback of these vaccines is that they provide low levels of protection for children under the age of 2 years, one of the most vulnerable groups that are commonly susceptible to infection. 
         [0005]    The meningococcal transferrin receptor is made up of two types of component protein chain, Transferrin binding protein A (TbpA) and TbpB. The receptor complex is believed to be formed from a dimer of TbpA which associates with a single TbpB (Boulton et al. (1998)). Epitopes present in TbpA are known to be masked within the interior of the protein (Ala&#39;Aldeen (1996)). Vaccines against meningococcal meningitis based on TbpB from one strain alone show some cross reactivity and there is evidence of a cross-reactive immune response in rabbits immunised with TbpB alone (Feirreros et al. (1998)). 
         [0006]    It would be of advantage, nevertheless, to provide a vaccine that gives a broader range of protective immunity to infection from a wider spectrum of strains of  N. meningitidis . It would be of further advantage to provide a vaccine that confers protective immunity to infants as well as adults and whose protection is long term. It would also be of advantage to provide a vaccine that protects against sub-clinical infection, i.e. where symptoms of meningococcal infection are not immediately apparent that the infected individual may act as a carrier of the pathogen. 
         [0007]    It is an object of the present invention to provide compositions containing Tbps, and vaccines based thereon, that meet or at least ameliorate the disadvantages in the art. In particular, it is an object of the invention to provide a vaccine composition that consistently and reliably induces protective immunity to meningococcal infection. 
         [0008]    Accordingly, a first aspect of the present invention provides a composition comprising both transferrin binding proteins A (TbpA) and B (TbpB), suitably in a molar ratio of about 2:1 (TbpA:TbpB). In a preferred embodiment of the present invention the molar ratio of TbpA to TbpB is 2:1. 
         [0009]    The composition may be combined with a pharmaceutically acceptable carrier—for example the adjuvant alum although any carrier suitable for oral, intravenous, subcutaneous, intraperitoneal or any other route of administration is suitable—to produce a pharmaceutical composition for treatment of meningococcal disease. 
         [0010]    The present invention thus provides for a vaccine comprising both TbpA+B proteins, preferably with a molar ratio of between 1.8 and 2.2 molecules of TbpA to one molecule of TbpB, more preferably 2 molecules of TbpA to one of TbpB. This particular combination of components, surprisingly, can provide higher protective immunity to meningococcal infection, compared to vaccination with TbpB alone. In a specific embodiment of the invention, described in more detail below, a 1:1 combination of A:B is more protective against challenge than B alone. This is surprising as TbpA has previously been considered to be non-protective. The present results differ from this established view with some experiments (described in more detail below) showing that, when administered as a vaccine, TbpA is also able to provide protective immunity to meningococcal infection. However, the present results most strikingly demonstrate the consistent performance of vaccines that comprise both Tbps A and B compared to those comprising Tbp A or B alone. It is this lack of variability between compositions and the consistently high level of protection to infection induced in response to vaccination with Tbp A+B, that enables the compositions of the invention to demonstrate significant advantage over the vaccines of the prior art. 
         [0011]    Transferrin binding proteins are known to be located on the outer membranes of a number of Gram negative bacteria such as  N. meningitidis . Formulations of the composition of the present invention with conventional carriers or adjuvants provide a composition for treatment of infection by these bacteria. 
         [0012]    It is an advantage that following administration of a composition according to the present invention antibodies may be raised against epitopes that consist of sequences from TbpA and TbpB in juxtaposition. Thus, the immune response obtainable using such a composition may be improved compared with that from prior art vaccine compositions which comprise only one component of the Tbp complex and in which the full range of potential Tbp epitopes are unavailable. It is a further option in the present invention for one Tbp subunit component of the TbpA+B complex to be from a first strain of  N. meningitidis  and another from a second strain different from the first. For example, the TbpA dimer is taken from the first strain and the TbpB is from the second. The TbpA and TbpB proteins may be selected independently from strains K454, H44/76 and B16B6. In all aspects of the invention the Tbps can be directly isolated from the bacterial source or can be produced by recombinant methods commonly known in the art. Combinations of proteins from other strains are also envisaged, and the combining of components from different strains of bacteria offers the potential for providing an individual with a broader spectrum of protection against meningococcal infection. It is further optional for a composition or vaccine of the invention to contain a mixture of A proteins from different strains or a mixture of B proteins from different strains, broadening further the potential spectrum of protection conferred by the invention. A still further option is for Tbps to be obtained from or derived from other bacteria, including  N. gonorrhoeae, N. lactamica  and  Moraxella catarrhalis.    
         [0013]    In the present invention, the term “transferrin binding protein” or “Tbp” refers to a protein which either alone binds to transferrin or can be part of a complex of proteins that binds transferrin. The term also embraces fragments, variants and derivatives of such a protein provided that antibodies raised against the fragment, variant or derivative bind the protein. Thus, TbpA and TbpB either dissociated or associated into a complex are considered to be Tbp. Moreover, mutants, fusion proteins or fragments of either TbpA or B or other derivatives of the TbpA+B complex with a common antigenic identity are also considered to be represented by the term Tbp in the present invention. 
         [0014]    A second aspect of the invention provides a composition comprising a complex of two TbpAs and one TbpB. The proteins are thus held together in the ratio seen in the native receptor. The individual proteins may be linked, for example, by hydrogen bonds or covalent bonds. In the latter case, each TbpA is covalently linked to the TbpA, either directly or indirectly. In a preferred embodiment, the complex of TbpA and TbpB assumes a native configuration. 
         [0015]    A native TbpA+B complex may be isolated and purified from  N. meningitidis . Alternatively, the invention also provides for synthesis of recombinant Tbp protein followed by assembly of the TbpA+B complex in vitro. The TbpA+B complex may be formed by admixture, or may be crosslinked by physical (e.g. UV radiation) or chemical methods known to the art, resulting in a combination of Tbps that will remain together and cannot dissociate from each other. In a further example, a single chain recombinant protein comprising two TbpA sequences, preferably in the form of the TbpA dimer, is then covalently linked with TbpB protein to form a complete TbpA+B complex in vitro. Another example of the invention in use provides that TbpA and B are mutated so as to introduce cysteine residues that facilitate the formation of disulphide bridges between the TbpA and TbpB subunits, so as to obtain a covalently bound complex. 
         [0016]    In preparation of a recombinant protein TbpA and TbpB genes may also be truncated so that only those domains known to contribute to the antigenicity of the protein are incorporated in the Tbp complex. 
         [0017]    In some compositions of the invention the TbpA+B complex is able to act as a transferrin receptor and binds to human transferrin. In other compositions of the invention the TbpA+B complex is non-functional in the sense that it does not bind transferrin, but it nevertheless provides an antigenic component that elicits an appropriate immune response. 
         [0018]    A third aspect of the invention provides for a composition comprising a Tbp and  N. meningitidis  outer membrane vesicles. An advantage of this composition is that when administered to a vaccinee or patient it presents a different combination of  N. meningitidis  antigens, and particularly antigens that are in a formation substantially as present on the membrane of live infecting organisms. The combination offers the potential for a more effective protection against infection or a broader spectrum of protection than existing vaccines. Known methods of outer membrane vesicle isolation, such as by desoxycholate treatment, are suitable for preparation of compositions of the invention. In various preferred embodiments the Tbp is Tbp A, Tbp B or TbpA+B either in a native complex or in dissociated form. 
         [0019]    The outer membrane vesicles may further be pretreated in vitro with Tbp so as to enrich the vesicle membrane with Tbp. By “enrich” and like terms, we refer to an outer membrane vesicle to which has been added Tbps so as to increase the concentration or density of Tbps in that vesicle. Preferably, the enrichment results in an outer membrane vesicle having an increased number of transferrin receptors located in the membrane, due to an increased concentration of TbpA and TbpB following their addition to the vesicle and their association into receptors or receptor-like structures. A particular advantage of such embodiments of the invention is that the Tbps, regarded as key antigenic components of the vaccine, are presented in a highly antigenic environment that closely mimics the environment in which transferrin receptors are presented on live, infecting bacteria. 
         [0020]    As mentioned previously, the Tbp components of the composition need not be wild-type Tbps. They can be made recombinantly, and in so doing sequence alterations may be introduced. In one typical example of the invention, recombinant TbpB is modified so as to comprise a membrane binding domain. A preferred membrane spanning domain is a hydrophobic alpha helical region added to either the N or C terminus of the Tbp B protein. However, a membrane anchoring region need not only be an alpha helix, addition of a fatty acid or lipid chain would also facilitate membrane anchoring. In fact, wild type TbpB is believed to anchored to the bacterial outer membrane via such a lipid chain anchor. In a further example of the invention in use, the outer membrane vesicles are pretreated in vitro with membrane binding recombinant TbpB so as to enrich the vesicle membrane with TbpB. OMVs enriched with Tbps are additionally generated by inducing high levels of Tbp expression in  N. meningitidis  and then isolating OMVs via one of the methods described previously. This latter method is typically achieved by transforming the  N. meningitidis  host with a suitable expression vector into which has been inserted a gene or genes encoding the Tbps of choice. Suitable expression vectors for use in neisserial species include the plasmid pMGC10 (Nassif et al. (1991)). 
         [0021]    It is preferred that the composition of the present invention comprises outer membrane vesicles and TbpA+B complexes isolated from a range of different strains of  N. meningitidis . Other preferred compositions of the invention comprise other  N. meningitidis  proteins including surface antigens, periplasmic proteins, superoxide dismutase and glycoproteins. 
         [0022]    The composition of the third aspect of the invention may instead of or in addition to outer membrane vesicles comprise one or more liposomes, each liposome including TbpA and/or TbpB preferably including TbpA and TbpB associated into a receptor or into a receptor-like complex. Thus a further means of presenting the transferrin receptor antigens is provided. 
         [0023]    In a further embodiment of the third aspect of the invention, the composition comprises 22 kD antigen (Neisserial surface protein A (NspA)) as well as or instead of outer membrane vesicles. NspA and its preparation are described by Martin et al, 1997. 
         [0024]    A fourth aspect of the invention provides for a vaccine comprising a composition of the invention as described above. A vaccine of the invention may also comprise antibodies to Tbp and thus provide a level of passive immunity to bacterial infection. 
         [0025]    A fifth aspect of the invention provides for a method of manufacturing a composition that comprises combining TbpA, TbpB and  N. meningitidis  outer membrane vesicles with a pharmaceutically acceptable carrier. It is preferred that the molar ratio of TbpA to TbpB is about 2:1. The outer membrane vesicles can be pretreated in vitro with native TbpA+B so as to enrich the vesicle membrane with Tbp complex. However, the outer membrane vesicles may also be pretreated with other protein components so as to enrich them for these antigenic components also. The outer membrane vesicles may also be pretreated with antigenic proteins and proteoglycans from several different strains of  N. meningitidis.    
         [0026]    A further aspect of the invention provides for a composition comprising a Tbp and a Cu,Zn-Superoxide dismutase (Cu,Zn-SOD). 
         [0027]    Cu,Zn-Superoxide Dismutase (Cu,Zn-SOD) is an metalloenzyme found in many prokaryotic and eukaryotic organisms. It catalyses the reduction of the superoxide radical anion, O 2   − , to hydrogen peroxide and molecular oxygen, thus playing an important role in the removal of cytotoxic free radicals from the organism. In bacteria Cu,Zn-SODs have been identified in the periplasm of a number of Gram negative species including  N. meningitidis . The enzyme can exist as a dimer or a monomer, and accordingly in a preferred embodiment the present invention provides for a composition comprising a Tbp and a Cu,Zn-SOD of the dimeric type. 
         [0028]    As mentioned previously with regard to the use of the term “Tbp”, the Cu,Zn-SOD of the present invention is also considered to encompass fragments, variants and derivatives of such a protein provided that antibodies raised against the fragment, variant or derivative bind the wild type Cu,Zn-SOD. 
         [0029]    In examples of the invention in use, compositions are provided that comprise a Cu,Zn-SOD and either a TbpA, a TbpB or a TbpA+B complex. In the latter example it is preferred that the molar ratio of TbpA to Tbp B is between 1.8 and 2.2, with the most suitable compositions having a ratio of 2:1 (TbpA:TbpB). In further compositions of the invention the Tbps are from a different strain of  N. meningitidis  to that of the Cu,Zn-SOD, thus facilitating the formation of a broader spectrum of immune response to meningococcal infection. 
         [0030]    The invention also provides for compositions wherein the Tbps and Cu,Zn-SOD are from different bacterial species, typically different Gram negative species. Such compositions thereby allow an even broader spectrum of immune response to be elicited when administered as a vaccine. Typical compositions comprise a neisserial TbpA+B complex as well as a monomeric Cu,Zn-SOD—from  E. coli  for example—and optionally a further dimeric Cu,Zn-SOD—for example from  Haemophilus parainfluenzae.    
         [0031]    Other aspects of the invention provide for methods of manufacturing compositions that provide protective immunity to Gram negative bacterial infection, comprising combining a covalently linked complex of TbpA and TbpB with either or both of  N. meningitidis  outer membrane vesicles and a Cu,Zn-SOD, plus a pharmaceutically acceptable carrier. 
         [0032]    A further aspect provides for use of Tbps A and B in the manufacture of a medicament for human vaccination. It is preferred that such a medicament is suitable for vaccination against meningococcal infection, although some compositions of the invention provide broad spectrum protection to infection from a wider range of bacterial pathogens. 
         [0033]    We have identified that combining  Neisseria meningitidis  outer membrane vesicles (OMVs) and an antigenic component isolated from  Neisseria meningitidis  increases the cross-protective capability of a meningococcal disease vaccine against a range of meningococcal strains. 
         [0034]    The invention thus provides a composition comprising  Neisseria meningitidis  outer membrane vesicles (OMVs) and at least one antigenic component. 
         [0035]    It is an option for the composition to comprise a plurality of antigenic components—for example, at least 2, 3, 4 or 5 antigenic components. Said plurality of antigenic components may be from different sources, for example, from different strains of  Neisseria meningitidis.    
         [0036]    We have also identified that enriching  Neisseria meningitidis  outer membrane vesicles (OMVs) with an antigenic component isolated from  Neisseria meningitidis  increases the cross-protective capability of a meningococcal disease vaccine against a range of meningococcal strains. 
         [0037]    The invention thus provides  Neisseria meningitidis  outer membrane vesicles (OMVs) that are enriched with at least one antigenic component. 
         [0038]    It is an option for the  Neisseria meningitidis  outer membrane vesicles to be enriched with a plurality of antigenic components—for example, at least 2, 3, 4 or 5 antigenic components. Said plurality of antigenic components may be from different sources, such as different strains of  Neisseria meningitidis.    
         [0039]    By “enrich” (and like terms), we refer to an outer membrane vesicle to which has been added at least one antigenic component, so as to increase the concentration or density of antigenic components in that vesicle. A particular advantage of this aspect of the invention is that the antigenic components are presented in a highly antigenic environment that closely mimics the environment in which these antigenic components are presented on live, infecting bacteria. 
         [0040]    The antigenic component may, in one embodiment, comprise or consist of a  N. meningitidis  antigenic component, such as a  N. meningitidis  antigenic protein or an  N. meningitidis  antigenic proteoglycan. 
         [0041]    In one embodiment, the antigenic component may comprise or consist of a  N. meningitidis  antigenic protein selected from the group consisting of TbpA, TbpB, NspA, PorA, OMP85, FrpB (now know as FetA), PiIQ, Hsf, SodC, MafA. Thus, in one embodiment, the invention thus provides  Neisseria meningitidis  outer membrane vesicles (OMVs) that are enriched with at least one antigenic component selected from the group consisting of  N. meningitidis  TbpA, TbpB, Neisserial Surface Protein A (NspA), PorA, OMP85, FrpB (now known as FetA), PiIQ, Hsf, SodC and MafA. 
         [0042]    In one embodiment, said antigenic component comprises or consists of a peptide having a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 and 72, or a sequence having at least 90 or 95% sequence identity thereto. 
         [0043]    In one embodiment, the antigenic component comprises or consists of an antigenic proteoglycan, such as a  N. meningitidis  antigenic proteoglycan selected from the group consisting of a surface antigen, a periplasmic protein, a superoxide dismutase and a glycoprotein. 
         [0044]    The antigenic component need not be a wild-type antigenic component. In one embodiment, the antigenic component is made recombinantly and, in doing so, it is an option for sequence alterations (eg. insertions, deletions or substitutions) to be introduced. 
         [0045]    In one embodiment, said outer membrane vesicles are from a first strain of  Neisseria meningitidis  and said antigenic component is from a second strain of  Neisseria meningitidis , wherein said second strain is different from said first strain. By way of example, said first and second different strains of  Neisseria meningitidis  may be selected from strain NZ98/254 or strain 44/76. 
         [0046]    The invention also provides a composition comprising  Neisseria meningitidis  outer membrane vesicles (OMVs) that are enriched with at least one antigenic component. 
         [0047]    Said composition may comprise a plurality of said  Neisseria meningitidis  outer membrane vesicles, wherein said plurality of  Neisseria meningitidis  outer membrane vesicles are enriched with different antigenic components. Alternatively, or in addition, said composition may comprise a plurality of said  Neisseria meningitidis  outer membrane vesicles that are from different strains  Neisseria meningitidis.    
         [0048]    The compositions of the invention may comprise a pharmaceutically acceptable carrier (any carrier suitable for oral, intravenous, subcutaneous, intraperitoneal or any other route of administration is suitable). 
         [0049]    The compositions of the invention may, in one embodiment, be vaccine compositions. 
         [0050]    Thus, in one embodiment, the invention provides a vaccine composition comprising  Neisseria meningitidis  outer membrane vesicles (OMVs) and at least one antigenic component. 
         [0051]    In one embodiment, the invention provides a vaccine composition comprising  Neisseria meningitidis  outer membrane vesicles (OMVs) that are enriched with at least one antigenic component. 
         [0052]    A vaccine composition may optionally comprise one or more conventional excipients, adjuvants, immunostimulatory compounds, antibodies, or anti- Neisseria  drugs. 
         [0053]    The invention also provides a method of manufacture of a composition, comprising combining  Neisseria meningitidis  outer membrane vesicles and an antigenic component (such as a  Neisseria meningitidis  antigenic component). 
         [0054]    In one embodiment, said method comprises:
       (a) extracting an antigenic component from an outer membrane of a bacteria, such as  Neisseria meningitidis , and preparing an aqueous solution of said antigenic component;   (b) extracting outer membrane vesicles (OMVs) from a culture of  Neisseria meningitidis , and preparing an aqueous solution of said OMVs; and   (c) admixing the solution prepared in (a) and the solution prepared in (b).       
 
         [0058]    Step (a) of said method optionally comprises the initial step of recombinantly expressing a DNA that encodes the antigenic component in the bacteria (eg.  Neisseria meningitidis ), prior to extracting said antigenic component from the outer membrane of said bacterium, and preparing the aqueous solution of said antigenic component. 
         [0059]    In one embodiment, the method further comprises combining the OMVs and antigenic component with a pharmaceutically acceptable carrier. 
         [0060]    The invention also provides a method of manufacture of a composition comprising  Neisseria meningitidis  outer membrane vesicles that are enriched with at least one antigenic component (such as a  Neisseria meningitidis  antigenic component). In one embodiment, said method comprises:
       (a) recombinantly expressing a DNA that encodes the antigenic component in the  Neisseria meningitidis  bacteria; and   (b) extracting outer membrane vesicles (OMVs) enriched with said antigenic component from a culture of said recombinant  Neisseria meningitidis , and preparing an aqueous solution of said OMVs.       
 
         [0063]    In one embodiment, the method further comprises combining the OMVs with a pharmaceutically acceptable carrier. 
         [0064]    In one embodiment of said methods, the antigenic component is selected from any of the antigenic components (eg.  Neisseria meningitidis  antigenic components) described above. In one embodiment of said methods, the outer membrane vesicles and/or the antigenic components are obtained from different strains of  Neisseria meningitidis.    
         [0065]    Known methods of isolating OMVs, such as by desoxycholate treatment, are suitable for preparing compositions of the invention. 
         [0066]    The invention also provides a method of stimulating an immune response in a subject, comprising administering to the subject a composition of the invention, which may optionally further comprise a pharmaceutically acceptable carrier. In one embodiment, said composition comprises  Neisseria meningitidis  outer membrane vesicles (OMVs) and at least one antigenic component. In one embodiment, said composition comprises  Neisseria meningitidis  outer membrane vesicles (OMVs) that are enriched with at least one antigenic component. 
         [0067]    The invention also provides a method of preventing  Neisseria meningitidis  infection in a subject, comprising administering to the subject a composition as described above, which may optionally further comprise a pharmaceutically acceptable carrier. In one embodiment, said composition comprises  Neisseria meningitidis  outer membrane vesicles (OMVs) and at least one antigenic component. In one embodiment, said composition comprises  Neisseria meningitidis  outer membrane vesicles (OMVs) that are enriched with at least one antigenic component. 
         [0068]    Said subject is typically a mammal such as a human. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0069]    The embodiments of the invention are discussed in more detail by means of the Example described below. The results referred to in the Example are illustrated by the accompanying drawings, in which: 
           [0070]      FIG. 1  shows immunisation of mice with TbpA+B and outer membrane vesicles; 
           [0071]      FIG. 2  shows immunisation of mice with TbpA+B; 
           [0072]      FIG. 3  shows protection of mice against IP infection after immunisation with TbpA+B, isolated TbpA or isolated TbpB; 
           [0073]      FIGS. 4A and 4B  show protection of mice against meningococcal infection following immunisation with either neisserial TbpA+B, TbpB or TbpA (nTbps) or recombinant Tbps (rTbps); 
           [0074]      FIGS. 5A and 5B  show protection of mice against challenges of 10 6  and 10 7  organisms/mouse of  N. meningitidis  strain K454 respectively, following immunisation with recombinant TbpA+B, TbpB or TbpA; 
           [0075]      FIG. 6  shows protection against challenge with heterologous serogroup; 
           [0076]      FIG. 7  shows protection against challenge with B16B6; 
           [0077]      FIG. 8  shows insertion of a promoter construct into the  N. meningitidis  genome for up regulation of endogenous TbpB in  N. meningitidis  by homologous recombination; 
           [0078]      FIG. 9 . shows insertion of an expression cassette into the  N. meningitidis  genome by homologous recombination; 
           [0079]      FIG. 10  illustrates IgG surface labelling of  N. meningitidis  strains NZ98/254 and M01-240149. Total IgG binding to both strains NZ98/254 and M01-240149 was increased when mice were vaccinated with OMV+PorA, as compared to vaccination with OMVs alone. The symbol ** indicates a greater than 95% certainty of a significant difference between these values, determined using a z-test; and 
           [0080]      FIG. 11  illustrates the OPA response against  N. meningitidis  strains NZ98/24 and M01-240149. Mice immunised with OMV+PorA elicited a greater OPA response against strains NZ98/254 and M01-240149, as compared to the OPA response to immunisation with OMVs alone. The symbol ** indicates a greater than 95% certainty of a significant difference between these values, determined using a z-test. 
       
    
    
     DETAILED DESCRIPTION 
       [0081]    The invention is further illustrated by the following non-limiting examples. 
       EXAMPLES 
     Example 1 
       [0082]    Mouse Protective Data 
         [0083]    Mice (CAMR-NIH) were immunised with Tbps and/or outer membrane vesicles and challenged with either the homologous or a heterologous meningococcal strain. Survivors per group of mice immunised with Tbps from strain K454 and the outer membrane vesicle vaccine following challenge by strain K454 are shown in  FIG. 1 . Compared to Tbps, the outer membrane vesicle vaccine gave reduced protection but this may be because it is produced from a different group B strain (H44/76). Animals immunised with TbpA+B isolated from strain K454 were also protected against challenge with other serogroup B organisms ( FIG. 1 ), with greater protection seen with the homologous strain and strains expressing a TbpB with a similar molecular weight. Little or no protection was observed against challenge with meningococci possessing TbpB with a very different molecular weight (strain B16B6). With the heterologous challenge strains, there is a slightly greater number survivors in the groups vaccinated with the combination of Tbps+outer membrane vesicles. However, the numbers involved are small and no definite conclusions can be reached. It is interesting to observe that mice immunised with TbpA+B from strain K454 were also protected against infection with a serogroup C but not a serogroup A strain ( FIG. 2 ). 
         [0084]    Protection against challenge with strain K454 with mice immunised with co-purified TbpA+B and isolated TbpA and TbpB is shown in  FIG. 3 . It can be seen that TbpB is the predominant antigen responsible for protection, with little protection afforded by TbpA alone. 
         [0085]    Recombinant TbpA and TbpB 
         [0086]    TbpA and TbpB from  N. meningitidis  strain K454 were cloned and overexpressed in  E. coli . The proteins were purified using affinity chromatography and used to determine their protective potency in a mouse model of meningococcal disease. Recombinant Tbps showed equivalent protection to that provided by Tbps isolated from iron-stressed  N. meningitidis  ( FIGS. 4A and 4B ). These recombinant Tbps were also utilised in two further larger IP challenge experiments ( FIGS. 5A-B ). 
         [0087]    The strong and consistent protective potency of Tbps against mouse I.P. infection with  N. meningitidis  is probably the most compelling evidence for their vaccine potential. 
         [0088]    Interestingly, the results shown in FIGS.  4 B and  5 A-B, although arising from experiments of similar design, show the surprising variability associated with vaccine compositions that depend solely upon either TbpA or TbpB alone. In  FIG. 4B  the recombinant TbpA composition displayed low levels of protection compared to TbpB or TbpA+B. However, in  FIGS. 5A and 5B  the recombinant TbpA vaccine composition that showed high levels of protection comparable to the TbpA+B complex and the TbpB alone composition demonstrated poor protection to infection. This latter result is completely contrary to the teaching of the prior art. 
         [0089]    Human Immune Response to TbpA and TbpB in Convalescent Sera 
         [0090]    We have undertaken a number of studies looking at the antibody response in humans to TbpA and TbpB following meningococcal disease. The general conclusions are that both TbpA and TbpB are expressed during meningococcal disease and that an immune response is raised against them. The response is functional (opsonic) and is more cross-reactive between different meningococcal strains than is the response induced by immunisation of animals with Tbps. The immune response to TbpA appears to be stronger and more cross-reactive than that to TbpB, confirming the importance of the vaccine potential of TbpA. 
         [0091]    TbpA and TbpB Form a Transferrin Receptor 
         [0092]    Our structural studies indicate that the transferrin receptor on the meningococcal surface consists of two TbpA molecules and one TbpB molecule that act together. 
         [0093]    Effect of a Vaccine Containing A+B Tbps 
         [0094]    We carried out further tests of the efficacy of recombinant TbpB versus recombinant TbpA+B containing vaccine formulations against challenge from  N. meningitidis  strains L91 705 and B16B6, the results of which are illustrated in  FIGS. 6 and 7  respectively. In both cases some improved protection was conferred by A+B compared to B alone. 
       Example 2 
     Up Regulation of Genes in Meningococcal Species 
       [0095]    These methods can be used to enhance expression of TbpB but can equally be applied to other meningococcal outer membrane proteins with known sequence such as NspA (Martin et al., 1997), OMP85 (Manning et al., 1998) or FrpB (Pettersson et al., 1995). Any of the antigenic component sequences disclosed herein (SEQ ID NOS: 1; 3; 5; 7; 9; 11; 13; 15; 17; 19; 21; 23; 25; 27; 29; 31; 33; 35; 37; 39; 41; 43; 45; 47; 49; 51; 53; 55; 57; 59; 61; 63; 65; 67; 69; and 71) would also be suitable for over expression in this system as would any immunogenic meningococcal sequence. 
         [0096]    In the following methods primers are designed using the primer select program from the &gt;DNA star=software package (www.dnastar.com). Primers to flanking sequences are designed following sequencing from the known coding region. 
         [0097]    Up Regulation of TbpB in  N. meningitidis  by Promoter Delivery 
         [0098]    Using homologous recombination (Frosch et al., 1990, van der Ley et al., 1995) a strong promoter such as the porA promoter is inserted into the  N. meningitidis  genome upstream of the tbpB coding region (Legrain et al., 1993). A selectable marker, encoding resistance to kanamycin, is also included in the delivery cassette upstream from the promoter. The integration locus is selected by sequencing upstream from the tbpB coding region. 
         [0099]    PCR primers are designed to the regions flanking the porA promoter and incorporate restriction endonuclease sites for ligation into this amplified sequence. This is then inserted into the multiple cloning site of a vector containing the kanamycin resistance gene. Also inserted through PCR produced products are integration loci that complement a region directly before the coding region for tbpB in the  N. meningitidis  genome. Through homologous recombination the construct shown in  FIG. 8  will be produced. 
         [0100]    From the published sequence of porA (McGuinnes et al. 1990) (SEQ ID NO: 1) a primer is constructed at the N-terminal region in order to sequence up steam of the coding region. This sequence data yields the promoter region of the highly expressed porA gene. Primers are designed to this sequence so as to amplify any conserved hexamers centred around −35 and −10 the consensus sequences of TTGACA and TATAAT with a 16-18 bp gap between them and up to 67 bp upstream of the start of transcription (ATG). The designed primers have restriction endonuclease sites incorporated in order to insert the amplified region into a suitable vector. 
         [0101]    In order to identify a suitable integration locus in front of the tbpB coding region a primer is designed to the start of the tbpB coding region, using the published sequence (Legrain et al. 1993), running up stream into the tbpB promoter region. The promoter region itself as well as some sequence upstream is suitable as the integration locus. This section is amplified in two halves using 4 primers and restriction endonucleases are incorporated in order to ligate the sections into a suitable vector. 
         [0102]    The vector used already contains an antibiotic resistance marker (such as kanamycin) and this is suitably positioned between the integration loci but upstream from the incorporated porA promoter, as illustrated in  FIG. 8 . This means that the vector used has cloning sites either side of the selectable marker. 
         [0103]    Up Regulation of TbpB in  N. meningitidis  by Gene Delivery 
         [0104]    A construct is produced containing a selectable marker, such as kanamycin resistance, up stream from a strong promoter (porA) which is in turn upstream from the coding region for tbpB. Using homologous recombination (Frosch et al., 1990, van der Ley et al., 1995), the cassette is inserted into the  N. meningitidis  genome. The integration locus is selected by sequencing upstream from a known sequenced area that is also known to actively expressed. 
         [0105]    PCR is used to amplify the tbpB coding region and porA promoter region with suitable restriction endonucleases incorporated so they can be inserted in series, into a vector containing kanamycin resistance. Also inserted into the construct are two flanking integration loci complementing a region of the  N. meningitidis  genome (see  FIG. 9 ). 
         [0106]    From the published sequence of porA (McGuinness et al. 1990) (SEQ ID NO: 1) a primer can be constructed to complement the N-terminal region in order to sequence up steam of the coding region. This sequence data yields the promoter region of the highly expressed porA gene. Primers are designed to this sequence so as to amplify any conserved hexamers centred around −35 and −10 the consensus sequences of TTGACA and TATAAT with a 16-18 bp gap between them and up to 67 bp upstream of the start of transcription (ATG). The reverse primer is designed to be as close as possible to the start of the coding region, but does not actually include any of it. This ensures that when it is positioned in series with any inserted DNA it acts as an efficient promoter. The designed primers have restriction endonuclease sites incorporated in order to insert the amplified region into a suitable vector. 
         [0107]    From the published sequence of tbpB (Legrain et al. 1993) primers are constructed to complement the C-terminal and N-terminal regions in order to sequence the flanking regions of the gene. From this sequence primers are constructed which amplify the entire gene. The forward primer incorporates the first coding triplet (ATG) of the gene and does not include the full native promoter region. The reverse is as close to the end of the coding region as possible while still being a good partner to the forward primer. It is accepted that this means the reverse primer may be some distance downstream of the gene of interest. 
       Example 3 
       [0108]    A combination of outer membrane vesicles (OMV)+PorA extends the cross-reactivity of the antibody response. 
         [0109]    We have demonstrated that the addition of recombinant PorA from strain NZ98/254 (PorA serosubtype P1.7-2, 4) to OMVs isolated from strain 44/76 (PorA serosubtype P1.7,16) broadens the protective response elicited by OMVs from strain 44/76. 
         [0110]    Serum bactericidal antibody (SBA) is a correlate of protection (Frasch et al., Vaccine, 2009; 27 Suppl 2:B112-6). 
         [0111]    We have shown that the SBA titre against strain NZ98/254 in serum from mice vaccinated with 44/76 OMVs alone was 16 (Table 1). However, the SBA titre obtained against this strain with OMV+PorA was 256. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 SBA titres: 
               
             
          
           
               
                   
                   
                 SBA titre against 
               
               
                   
                 Vaccine 
                 strain NZ98/254 
               
               
                   
               
             
          
           
               
                   
                 OMVs from 44/76 
                 16 
               
               
                   
                 PorA from NZ98/254 
                 4 
               
               
                   
                 OMVs from 44/76 + 
                 256 
               
               
                   
                 PorA from NZ98/254 
               
               
                   
               
             
          
         
       
     
         [0112]    Binding of IgG antibody to the surface of intact  N. meningitidis  can be determined by flow cytometry. Antibody binding to the organism is essential for bactericidal activity and also opsonophagocytic activity.  FIG. 10  shows that the total IgG binding to both strains NZ98/254 and M01-240149 was increased when mice were vaccinated with OMV+PorA, as compared to vaccination with OMVs alone. 
         [0113]    Opsonophagocytosis (OPA) is also important for protection against meningococcal disease (Granoff, Vaccine, 2009; 27 Suppl 2:B117-25).  FIG. 11  shows that mice immunised with OMV+PorA elicited a greater OPA response against strains NZ98/254 and M01-240149, as compared to the OPA response to immunisation with OMVs alone. 
         [0114]    In summary, the SBA, SLA and OPA assays all confirm that the OMV+PorA vaccine elicits greater cross-strain protection than OMV alone. 
         [0115]    Methods: 
         [0116]    Animal Vaccination 
         [0117]    NIH mice (six to eight weeks old, Harlan, UK) were immunized on days 0, 21, and 28 by subcutaneous injection (0.2 ml), with: 
         [0118]    MPL adjuvant only; 
         [0119]    MPL and 10 μg  Neisseria meningitidis  44/76-SL OMVs; 
         [0120]    MPL and 10 μg PorA (P1.7-2,4); or 
         [0121]    MPL with 10 μg  Neisseria meningitidis  44/76-SL and 10 μg PorA (P1.7-2,4) 
         [0122]    On Day 35 terminal sera were collected from mice. 
         [0123]    Serum bactericidal assay (SBA): 
         [0124]    SBA assays were performed using a standardized assay with a starting dilution of 1:2. Briefly, human serum, at 25%, was used as an exogenous source of human complement, with titers expressed as the reciprocal of the final dilution giving ≧50% SBA killing at 60 min compared with the level for the control (inactive complement and no test serum). Single determinations were made using each serum sample, the human complement used had previously been screened for lack of intrinsic bactericidal activity. 
         [0125]    Surface Labelling and Opsonophagocytosis Assays 
         [0126]    Bacteria: Bacteria used were killed using a protocol designed to minimise chemical alteration of surface epitopes. Briefly, live meningococci were incubated with 0.2% (w/v) sodium azide and 17 μg.ml −1  phenylmethylsulphonyl fluoride for 48 hours at 37° C. Bacteria used in the opsonophagocytosis assay were first stained for 1 hour with 20 μM of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonic acid (DiIC 18 (5)-DS) (Invitrogen, UK). 
         [0127]    Complement: Human plasma was used as a complement source and was IgG depleted using a protein G Sepahrose column immediately before use. Briefly, the column was equilibrated with three column volumes of hanks buffered saline solution before one column volume of plasma was added. This was incubated for 5 minutes at 4° C. before one column volume of hanks buffered saline solution was used to displace the complement. 
         [0128]    Opsonophagocytosis assay (OPA): Opsonophagocytosis (OP) of meningococci by HL-60 cells (American Type Culture Collection, Rockville Md., USA) differentiated to granulocytes with 0.8% N,N dimethyleformamide (Sigma, UK) for 5 days, was measured using a flow cytometric assay performed in U-bottom 96-well microtitre plates (Sterilin, UK). 20 μl of each test serum diluted 1:10 in OP buffer (Hanks balanced salts solution (Sigma, UK) containing 2% skimmed milk powder (Marvel, Premier International Foods, UK), 1.2 mM CaCl 2 , and 1 mM MgSO 4  (Sigma, UK)) was added to 10 μl of target bacteria at 6.25×10 8  ml −1  in OP buffer, followed by 10 μl of IgG depleted Human plasma as a complement source, and then incubated for 7.5 min with shaking (900 rpm) at 37.0° C. 
         [0129]    Differentiated HL60 cells at 2.5×10 7  ml −1  in OP buffer (50 μl) were added and incubation continued, with shaking, at 37.0° C. for 7.5 min. Opsonophagocytosis was stopped by addition of 80 μl of ice-cold Dulbecco phosphate-buffered saline (PBS; Sigma, UK) containing 0.02% EDTA (Sigma, UK). 
         [0130]    Horizontal gates in the DiIC 18 (5)-DS channel of a flow cytometer (Beckman Coulter FC500) were set against a complement-only, no-antibody control to include approximately 10% of the population. For each sample, 7,500 live HL60 cells were measured, and the percentage of cells showing DilC 18 (5)-DS fluorescence in the appropriate gate (% gated) was multiplied by the mean fluorescence of the gated population (X−mean) to calculate a fluorescence index (FI). The FI of each test was subtracted from the FI of the complement-only no antibody control to give FI minus complement control (FI−C′). 
         [0131]    Total antibody binding assay (SLA): IgG binding to the surface of meningococci was measured using a flow cytometric assay performed in U-bottom 96-well microtitre plates (Sterilin, UK). 
         [0132]    For the assay, 2 μl of each test serum was added to 198 μl of target bacteria at an O.D 600 0.1 in a blocking buffer of 1% bovine serum albumin (BSA) in PBS and the mixture incubated for 30 min with shaking (900 rpm) at 25.0° C. This was then centrifuged at 3050 g for 5 minutes and the supernatant removed and the pellet was then washed with 200 μl of blocking buffer. The process was repeated twice before the addition of 200 μl FITC-labelled goat anti-human IgG (Biodesign, UK) at 1:500 in blocking buffer, followed by incubation for 20 minutes at 4° C., before being washed twice more with blocking buffer. 
         [0133]    Horizontal gates in the FITC channel of a flow cytometer (Beckman Coulter FC500) were set against a conjugate-only, no-antibody control to include approximately 10% of the population. For each sample, 7,500 bacteria were measured, and the percentage of cells showing FITC fluorescence in the appropriate gate (% gated) was multiplied by the mean fluorescence of the gated population (X−mean) to calculate a fluorescence index (FI).; the FI of the conjugate only control was subtracted from the FI of each test sera to give FI minus complement control (FI−Conj). 
         [0134]    From the Foregoing, it will be appreciated that although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.