WO2007110602A1

WO2007110602A1 - Immunogenic compositions

Info

Publication number: WO2007110602A1
Application number: PCT/GB2007/001055
Authority: WO
Inventors: Christoph Marcel Tang; Dlawer Ala'aldeen; Karl G. Wooldridge; Yanwen Li
Original assignee: The University Of Nottingham
Priority date: 2006-03-28
Filing date: 2007-03-23
Publication date: 2007-10-04

Abstract

An immunogenic composition comprising one or more proteins secreted by one or more strains of one or more pathogenic organisms, wherein the composition is capable of eliciting an immune response directed to one or more of the pathogenic organisms from which one or more of the secreted proteins are derived.

Description

IMMUNOGENIC COMPOSITIONS

The present invention relates to immunogenic compositions for use in eliciting immune responses to pathogenic organisms, and in particular, to immunogenic compositions capable of eliciting protective immune responses.

Neisseria meningitidis (meningococcus) is an encapsulated gram-negative diplococcus bacterium that inhabits the nasopharynx of humans. Carriage rates in the general population are usually around 10% and in overcrowded populations, such as university students, rates can reach up to 30% (Ala'Aldeen, D. A. et al. (2000) J Clin Microbiol 38:2311-2316; Neal, K. R. et al (2000) BMJ 320:846-849) . The complex host-pathogen relationship is usually of a commensal nature. Occasionally, however, meningococcal carriage can lead to invasive disease. This phenomenon is usually associated with strains belonging to a small number of hypervirulent clonal lineages (Caugant, D. A. et al. (1986) Proc Natl Acad Sci U S A 83:4927-4931; Caugant, D. A. et al. (1987) J Bacteriol 169:2781-2792) . N. meningitidis is the leading cause of pyogenic meningitis worldwide and is the only bacterium capable of generating outbreaks of meningitis and septicaemia. Attack rates vary between 1-3 per 10^s population, depending on the endemic or epidemic prevalence of disease in any one geographical location. There is a need, therefore, to develop preventative and therapeutic strategies to reduce the incidence, mortality and morbidity of invasive meningococcal disease.

The Neisseria meningitidis organism is sensitive to several front line antibiotics, however, despite this, a significant number of patients diagnosed with meningococcal infection die of overwhelming disease or suffer serious complications. Mortality rates vary from 2-3% in cases of uncomplicated meningitis to 50% or more in cases of septic shock (Cartwright, K. A. and D. A. Ala'Aldeen (1997) J Infect 34:15-19) .

Thirteen serogroups of Neisseria meningitidis have been identified based on the chemical and antigenic differences of their capsular polysaccharides. Only five of these, A, B, C, Y and W-135, are commonly associated with human disease. There are currently vaccines available directed to four of the five (A, C, Y and W-135), whose polysaccharide capsules are capable of inducing protective immunity (Pichichero, M. et al (2005) Pediatr Infect Dis J 24:57-62) . However, no effective vaccine has yet been developed directed to serogroup B strains: this is the group responsible for the majority of meningococcal disease in many developed countries. The capsular polysaccharide of bacterium of serogroup B is not immunogenic. Therefore approaches to develop vaccines directed to this serogroup have focussed on surface-exposed non- capsular antigens, including lipooligosaccharides (LOS) and outer membrane proteins (OMP) either individually or in complex preparations such as outer membrane vesicles (OMV) . Several OMV vaccines have undergone extensive clinical trials in Scandinavia and South America (Bjune, G. E. A. et al. (1991) Lancet 338: 1093-1096; Sierra, G. V. et al (1991) NIPH Ann 14:195-207; Tappero, J. W. et al (1999) Jama 281:1520-1527) . The results, however, were largely disappointing, particularly among children under the age of four: the most vulnerable age group.

The present invention relates to novel compositions comprising the secreted proteins of pathogenic organisms, and to the use of these compositions to elicit an immune response to the pathogenic organism from which the secreted proteins are derived. One aim of this invention is to provide one or more compositions which can be used to elicit a protective immune response to Neisseria meningitidis , and in particular to N. meningitidis of serogroup B.

According to a first aspect, the present invention provides an immunogenic composition comprising one or more proteins secreted by one or more strains of one or more pathogenic organisms, wherein the composition is capable of eliciting an immune response, when administered to a human or non-human animal, directed to one or more of the pathogenic organisms from which one or more of the secreted proteins are derived.

An immunogenic composition is a composition that is capable of eliciting an immune response to an antigen in the composition when the composition is administered to a subject. Preferably the subject is a human or non-human animal, more preferably a human or non-human mammal. The antigen in the immunogenic composition of the invention is the one or more secreted proteins.

An "immune response directed to" one or more pathogenic organisms means that the immune response elicited can, as well as recognising the secreted protein, also affect the pathogenic organism. Preferably the pathogenic organism is affected in that the ability of the organism to infect the human or non-human animal is impeded or prevented. This may be achieved in a number of ways. The immune response elicited may recognise and destroy the pathogenic organism. Alternatively, or additionally, the immune response elicited may impede or prevent replication of the pathogenic organism. Alternatively, or additionally, the immune response elicited may impede or prevent the pathogenic organism causing disease in the human or non-human animal. Preferably, the immune response elicited by the composition is also capable of being directed to organisms related to the pathogenic organism from which the secreted proteins are derived. Related organisms may be from the same, or a different, clonal lineage. Related organisms may be from the same, or a different, serogroup.

The term "secreted protein" refers to one or more proteins which can be found in the supernatant of cultured pathogenic organisms. The proteins may be actively secreted from the organism and/or they may be shed from the surface of the organism as part of an outer membrane vesicle.

The one or more secreted proteins may be naturally produced (e.g. purified from the pathogenic organism, or from the culture medium/supernatant of the pathogenic organism) , recombinantly produced (e.g. from a genetically-engineered expression system) or be a synthetic product. One or more of the secreted proteins may be functional analogues, or modified variants, based on, or derived from, proteins secreted from a pathogenic organism but may include modifications such as deletions, insertions, inversions, additions and substitutions, provided that the immune response elicited would also recognise the pathogen from which the modified secreted protein or functional analogue is derived or based.

The skilled man will appreciate that all reference made herein to secreted proteins refers to proteins secreted from a pathogenic organism and recovered from the supernatant, and also to recombinant or synthetic versions of the naturally secreted proteins. Reference to secreted proteins also includes functional analogues, or modified variants, of the naturally secreted proteins which are capable of eliciting an immune response directed to the pathogenic organism which secretes the protein on which the analogues or variants are based. The composition may comprise a combination of two or more proteins selected from the group comprising actually secreted proteins, recombinant proteins, synthetic proteins and modified versions or functional analogues thereof.

To export and secrete proteins across the inner and outer membranes, gram-negative bacteria, such as Neisseria meningitidis, have evolved several protein secretion mechanisms, including the well-characterised secretion pathways labelled types I- V. Not every pathway is functional in every gram-negative bacterium but, where present, they tend to be highly conserved across species and genera. This has enabled the identification of protein secretion pathways in the meningococcus by analysis of the meningococcal genome sequence databases (Wooldridge, K. G. et al. (2005) Infect Immun 73:5554-5567; Hadi, H. A. et al. (2001) MoI Microbiol 41:611-623; Turner, D. et al (2006) Infect Immun 74:2957- 2964; Turner, D. P. et al. (2002) Infect Immun 70:4447-4461) .

The composition may comprise all the proteins secreted by one or more strains of one or more pathogenic organisms, or it may comprise only some of the proteins secreted by one or more strains of one or more pathogenic organisms. The composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more proteins secreted by one or more strains of one of more pathogenic organisms. Where only some of the secreted proteins are used in the composition, the proteins may be selected on the basis of their size, their charge, their function, their conservation between strains and/or they may be randomly selected.

The composition may comprise secreted proteins from only one strain of one pathogenic organism. Alternatively, the composition may comprise proteins secreted from more than one strain of a pathogenic organism and/or more than one type of pathogenic organism. In one embodiment the secreted proteins in the composition are meningococcal secreted proteins (MSPs) from the pathogenic bacteria Neisseria meningitidis. Preferably when a composition comprising meningococcal secreted proteins is administered to a subject it is capable of eliciting a protective immune directed to Neisseria meningitidis . MSPs from one strain of Neisseria meningitidis may be capable of eliciting an immune response to the same strain and/or to other strains of Neisseria meningitidis. The other strains of Neisseria meningitidis may be of the same, or a different, serogroup as the strain used to prepare the MSPs. The other strains of Neisseria meningitidis may be of the same, or a different, clonal lineage as the strain used to prepare the MSPs. The MSPs may be from an ET-5, an ET-37, an A4 and/or a lineage III strain of Neisseria meningitidis, and may be capable of eliciting an immune response reactive to the same, and optionally other, ET-5, ET-37, A4 and/or lineage III strains of Neisseria meningitidis . The MSPs may also be capable of eliciting an immune response reactive to non-ET-5 , non-ET- 37, non-A4 and/or non-lineage III strains of Neisseria meningitidis. Preferably the composition of the invention comprises one or more MSPs from one or more serogroup B strains of Neisseria meningitidis which is/are capable of eliciting an immune response reactive to one or more serogroup B strains of Neisseria meningitidis. In order to produce a composition which provides effective immune protection from a pathogenic organism, such as Neisseria meningitidis serogroup B, the composition may comprise MSPs from more than one strain of the pathogenic organism. The MSPs may be isolated from the supernatant of one or more pathogenic organisms and/or they may be recombinantly and/or synthetically produced.

The composition may comprise one or more proteins secreted from one or more strains of Neisseria meningitidis. Alternatively, or additionally, the composition may comprise one or more proteins secreted from one or more strains of Neisseria gonorrhoeae, Haemophilus influenzae, ActinohacilluR pleuropneumonia^ and/or any similar organism. The composition may comprise proteins based on one or more proteins secreted from one or more strains of Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Actinobacillus pleuropneumoniae and/or any similar organism.

Reference to a similar organism may refer to an organism that has at least 40% homology to the genomic sequence of a strain of Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenza or Actinobacillus pleuropneumoniae. Preferably similar organisms have at least 50%, 60% 70%, 80%, 90% or 95% homology to Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae or Actinobacillus pleuropneumoniae. Alternatively, reference to a similar organism may refer to an organism that has at least 40% homology in the genomic sequence when one or more genes from the related organisms are aligned. Preferably the degree of homology is at least 50%, 60% 70%, 80%, 90% or 95%. Preferably a similar organism has one or more genes which have at least 40%, and preferably at least 50%, 60% 70%, 80%, 90% or 95%, homology to one or more genes from at least one strain of Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenza or Actinobacillus pleuropneumoniae .

Reference to percentage homology relates to the percent identity between two aligned sequences. The percent identity refers to the residues in two genomes or two genes which are the same, when the genomes or genes are aligned for maximum correspondence and when inversions and translocations are accounted for. Preferably the percent identity ignores any conservative differences between the aligned sequences which do not affect function. The percent identity between aligned sequences can be established by using well-established tools (such as the BLAST algorithm - Basic Local Alignment Search Tool; Altschul et al.₅ (1990) J MoI Biol. 215:403-10)

Neisseria gonorrhoeae is a leading cause of sexually transmitted disease. There is currently no vaccine available for the treatment or prevention of gonococcus. Neisseria gonorrhoeae is closely related to Neisseria meningitidis, both organisms share many antigens, including Opas and

LPS. At the genetic level, the degree of sequence identity between homologues in the meningococcal and gonococcal genomes is around 90%.

The composition may also comprise a further one or more antigens, in addition to the one or more secreted proteins. The further antigens may also be capable of eliciting an immune response directed to the pathogenic organism from which they are derived. The further antigens may be derived from the same or a different pathogenic organism to the secreted proteins. The further antigens may comprise one or more proteins expressed on the surface of one or more pathogenic organisms.

The composition may comprise a mixture of antigens only some of which are secreted proteins. The composition may be capable of eliciting an immune response, preferably a protective immune response, directed to one or more pathogenic organism. Preferably the composition is capable of eliciting a protective immune response directed to more than one pathogenic organism, or at least more than one strain of the same pathogenic organism. For example, a composition may comprise antigens capable of eliciting an immune response in a host organism directed to meningococcus of one or more of the serogroups A, B, C, W135 and Y. A composition capable of eliciting an immune response in a host organism directed to meningococcus of one or more of the serogroups A, B, C, Wl 35 and Y, may comprise proteins secreted from one or more strains of meningococcus together with other antigens, such as, outer membrane vesicles (OMVs) derived from one or more strains of meningococcus.

The composition may be used to elicit/produce a protective immune response when administered to a subject. The protective immune response may cause pathogenic organisms, or related pathogenic organisms, to be killed upon infecting the subject, or it may prevent or inhibit the pathogenic organisms from replicating and/or from causing disease.

The composition may be used as a prophylactic or a therapeutic vaccine directed to the pathogenic organism, or a related organism, from which the secreted proteins are derived.

The composition may be used as a prophylactic or a therapeutic vaccine directed to infection by Neisseria meningitidis, Neisseria gonorrhoeae,

Haemophilus influenzae, Actinobacillus pleuropneumoniae or a similar organism. Preferably if the composition is a vaccine for Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae,

Actinobacillus pleuropneumoniae or a similar organism, it will comprise one or more proteins secreted by Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Actinobacillus pleuropneumoniae or a similar organism respectively.

According to a further aspect, the invention provides a pharmaceutical composition comprising proteins secreted by a pathogenic organism in combination with a pharmaceutically acceptable carrier or excipient.

Preferably the pharmaceutical composition comprises a composition according to the first aspect of the invention. Preferably the proteins are secreted by a pathogenic organism such as Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Actinobacillus pleuropneumoniae and/or a similar organism.

Preferably the pharmaceutical composition is capable of producing a protective immune response to the pathogenic organism from which the secreted proteins are derived, such as Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Actinobacillus pleuropneumoniae or any similar organism.

The phrase "producing a protective immune response" as used herein means that the composition is capable of generating a protective response in a host organism, such as a human or a non-human mammal, to whom it is administered. Preferably a protective immune response protects against subsequent infection by the pathogenic organism and/or a related pathogenic organism from which the secreted proteins are derived. The protective immune response may eliminate or reduce the level of infection by reducing replication of the pathogenic organism or by affecting the mode of action of the infecting pathogenic organism to reduce disease.

Suitable acceptable excipients and carriers will be well known to those skilled in the art. These may include solid or liquid carriers. Suitable liquid carriers include water and saline. The proteins of the composition may be formulated into an emulsion or they may be formulated into biodegradable microspheres or liposomes.

The composition may further comprise an adjuvant. Suitable adjuvants will be well known to those skilled in the art, and may include Freund's Incomplete Adjuvant (for use in animals) , and metal salts, such as aluminium or calcium salts, The composition may also comprise polymers or other agents to control the consistency of the composition, and/or to control the release of the antigen/secreted protein from the composition.

The composition may also comprise other agents such as diluents, which may include water, saline, glycerol or other suitable alcohols etc; wetting or emulsifying agents; buffering agents; thickening agents for example cellulose or cellulose derivatives; preservatives; detergents, antimicrobial agents; and the like.

Preferably the active ingredients in the composition are greater than 50% pure, usually greater than 80% pure, often greater than 90% pure and more preferably greater than 95%, 98% or 99% pure. With active ingredients approaching 100% pure, for example about 99.5% pure or about 99.9% pure, being used most often.

The composition of the present invention may be used as vaccine against infections caused by the pathogenic organism, or a pathogenic organism related to the pathogenic organism, from which the secreted proteins are derived. For example, if the secreted proteins are derived from Neisseria meningitidis the composition may be used as a vaccine directed to meningitis or other invasive meningococcal diseases including septicaemia or septic shock. The vaccine may be administered prophylactically to those at risk of exposure to the pathogen, and/or therapeutically to persons who have already been exposed to the pathogen.

Preferably, if the composition is used as a vaccine, the composition comprises an immunologically effective amount of antigen (comprised of secreted proteins). An "immunologically effective amount" of an antigen is an amount that when administered to an individual, either in a single dose or in a series of doses, is effective for treatment or prevention of infection by the pathogenic organism. This amount will vary depending upon the health and physical condition of the individual to be treated and on the antigen. It is expected that the amount will fall in a relatively broad range that can be determined by routine trials.

The route of administration of the composition may vary depending on the formulation of the proteins in the composition. The composition may be arranged to be administered intramuscularly, intradermally, subcutaneously, intraperitonealy or intravenously. Alternatively the composition may be arranged to be administered parenterally, such as by intranasal, oral, buccal, inhalation, epidermal, transcutaneous, topical, vaginal or rectal administration.

The composition may be arranged to be administered as a single dose or as part of a multiple dose schedule. Multiple doses may be administered as a primary immunisation followed by one or more booster immunisations. Suitable timing between priming and boosting immunisations can be routinely determined.

Compositions of the invention may be able to induce a serum bactericidal antibody response after being administered to a subject. These responses are conveniently measured in mice and the results are a standard indicator of vaccine efficacy.

The compositions of the invention may also, or alternatively, be able to elicit an immune response which neutralises proteins secreted by the pathogenic organisms, thereby preventing them from having their normal function and preventing or reducing disease progression without necessarily destroying the pathogenic organism/bacteria. The compositions of the invention may also, or alternatively, be able to elicit an immune response which effects proteins on the host cells to defend against infection by a pathogenic organism, without necessarily destroying the pathogenic organism/bacteria.

According to a further aspect, the present invention provides the use of one or more secreted proteins from one or more pathogenic organisms in the preparation of a medicament for eliciting an immune response. The medicament may be used for the prophylactic or therapeutic vaccination of subjects against the pathogenic organism, or a related pathogenic organism, from which the secreted proteins are derived. The medicament may be a prophylactic or a therapeutic vaccine. The pathogenic organism may be Neisseria meningitidis, and the secreted proteins may be used in the preparation of a vaccine for meningitis, septicaemia and/or septic shock caused by Neisseria meningitidis.

The pathogenic organism may be Neisseria gonorrhoeae and the secreted proteins may be used in the preparation of a vaccine for gonorrhoea. Alternatively, the pathogenic organism may be Haemophilus influenzae, Actinobacillus pleuropneumoniae or a similar organism.

According a still further aspect, the present invention provides a method of protecting a human or non-human animal from the effects of infection by a pathogenic organism comprising administering to the human or non- human animal a composition according to any other aspect of the invention, wherein the one or more secreted proteins are derived from a pathogenic bacteria, or a related pathogenic bacteria, to which the method is to provide protection from the effects of infection. The composition may be a vaccine. Preferably the method of the invention provides protection against the effect of infection by Neisseria meningitidis, comprising administering a composition comprising proteins secreted by Neisseria meningitidis .

Alternatively the pathogenic organism may be Neisseria gonorrhoeae, Haemophilus influenzae, Actinobacillus pleuropneumoniae or a similar organism, and the one or more secreted proteins are derived from the respective organism.

According to another aspect, the invention provides a method for raising an immune response in a human or non-human animal comprising administering a pharmaceutical composition according to the invention to the human or non-human animal. The immune response is preferably protective. The method may raise a booster response in a patient that has already been primed. The immune response may be prophylactic or therapeutic.

The uses and methods of the invention are preferably for the prevention and/or treatment of a disease caused by N. meningitidis such as bacterial (or, more specifically, meningococcal) meningitis, septicaemia or septic shock.

Alternatively, the uses and methods of the invention may be for the prevention and/or treatment of disease caused by one or more of Neisseria gonorrhoeae, Haemophilus influenzae, Actinobacillus pleuropneumoniae or a similar organism

One way to check the efficacy of a therapeutic treatment comprising administration of proteins secreted by Neisseria meningitidis involves monitoring for N. meningitidis infection after administration of a composition of the invention comprising MSPs. One way to check the efficacy of a prophylactic treatment comprising administration of proteins secreted by Neisseria meningitidis involves monitoring immune responses to Neisseria meningitidis after administration of the composition.

These methods to check efficacy may be used with any other pathogenic organism, and could readily be performed by the skilled man using routine procedures.

According to another aspect, the invention provides the use of one or more proteins secreted by a pathogenic organism in the preparation of a medicament for use in the immunisation of human or non-human mammals against infection by the pathogenic organism or a related pathogenic organism.

The pathogenic organism in this method may be Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Actino bacillus pleuropneiimoniae or a similar organism

The skilled man will appreciate that any of the preferable features discussed above can be applied to any of the aspects of the invention. The skilled man will also appreciate that pathogen and pathogenic organism have the same meaning and are used interchangeably.

Preferred embodiments of the present invention will now be described, merely by way of example, with reference to the following figures and examples .

Figure 1 - is a table showing the results of serum bactericidal assays (SBA) against a range of meningococcal strains. The results are given as the reciprocal of the maximum dilution of anitsera, raised to the strains indicated in the top row, needed to achieve

> 50% bacterial killing of the strains indicated in the left hand column belonging to the electropherotypes/serogroups indicated in the second and third columns. AU assays were performed in duplicate. Key: ET = electrophoretic type (or clonal lineage); Unknown = do not belong to any known ET or clonal lineage; > and < = titres higher or lower than stated but serial dilutions were not carried out;

Figure 2 - is a silver-stained 2D-gel of MSPs obtained from N. meningitidis strain MC58 grown under conditions of limited iron availability;

Figure 3 - is a graph illustrating the effects of the immunisation of mice with MSPs on the susceptibility of the mice to infection by Neisseria meningitidis strain MC58. Three groups of 15 mice were immunised on days 0, 14 and 21 with (1) 25 μg MSPs/dose (test) ,

(2) live attenuated strain of MC58 (positive control) or (3) adjuvant only (negative control), and challenged 2 weeks later with 10⁷ cfu of live bacteria (strain MC58) . The test and positive control mice produced identical results, hence their overlap on the single line;

Figure 4 - shows the results of a whole cell ELISA of post- immunisation sera from mice receiving MSPs (Rl -4) or live attenuated bacteria. Pooled pre-immune sera from the same mice (negative control) is also included. The secondary antibodies used were goat anti-mouse antibodies conjugated to horse-radish peroxidase;

Figure 5A - shows the results of FACS analysis of surface bound antibodies on strain MC58 using pooled anti-MSP sera from mice before (negative control, light grey) or after (black) immunisation with MSPs. Positive control (dark grey) consisted of antiserum to the live attenuated strain. Antibody binding was detected with donkey anti-mouse antibodies conjugated to FITC. Surface labelled bacteria induce a shift to the right. Figure 5B - is a summary of triplicate FACS experiments (as in Figure 5A) expressed in columns and error bars. The relative fluorescence index is the percentage of positive cells multiplied by the geometric mean fluorescence;

Figure 6 - is an immunoblot of whole-cell lysates from strains reacted with pooled sera from animals immunised with MSPs derived from N. meningitidis MC58 (ET-5) . The strains are indicated above each lane;

Figure 7 - is an immunoblot of whole-cell lysates from strains reacted with pooled sera from animals immunised with MSPs derived from N. meningitidis Z4262 (ET-37). Strains are indicated above each lane; and

Figure 8 - shows the results of a whole cell ELISA, to the indicated strains of N. meningitidis , with pooled, post- immunisation sera from mice receiving MSPs derived from N. meningitidis Z4262 (ET-37). Pooled pre-immune sera from the same mice (negative control) is included. The order of the lines on the graph from the uppermost line to the lowermost line at the marker on the X-axis to the left of 16384 is as follows: BZ169,

1000, A22, NG6/88, Z2941, MC58, H4476 and pre-immune sera. Whole cells of the indicated strains were used to coat ELISA plates and probed with sera after immunisation with strain Z4262 or with pre-immune sera. The graph shows that the serum recognises a number of (one or more) surface-exposed antigens on each of the strains tested, which belong to a number of serogroups (B and W- 135) and clonal lineages (including ET-37 and IV-I) .

METHODS

Bacterial strains, growth conditions and secreted protein preparation

In all experiments, with the exception of MSP preparation, N. meningitidis was grown on Brain Heart Infusion (BHI) agar with Levanthal's supplement in 5% CO₂ at 37⁰C overnight and collected at about 16 hours for the experiments.

Strain MC58 of N. meningitidis was isolated from a case of meningitis and is probably the most widely used strain. The genome of MC58 has been completely sequenced (Pohlner et al. , (1991) Nature 325:458-462.) . MC58 is a serogroup B isolate belonging to electropherotype or clonal lineage ET-5.

Z4262 is also a serogroup B isolate of N. meningitidis. Z4262 is from the clonal lineage ET-37 and is described by Maiden M. C. J et al., (1998) PNAS 95:3140-3145.

Strain YH102 is a derivative of strain MC58 in which the saiD gene (essential for production of the polysaccharide capsule) and rfaF (encodes an ADP-heptose heptosyltransferase required for biosynthesis of lipooligosaccharide) have been ablated. The strain is attenuated for virulence (Infect Immun. 2004 January; 72(1): 345-351) .

Meningococcal secreted proteins (MSPs) were prepared by the following method, as described previously by Robinson, K. et al. in Cell Microbiol (2004) 6:927-938. Meningococci were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) for 18 hours, before centrifugation at 20 000 g for 20 minutes at 4°C. The supernatants were then passed through 0.2 μm syringe filters. The supernatants were concentrated by approximately 50-fold using centrifugal concentrators with a 30 kDa molecular weight cut-off (VivaScience) . Lipooligosaccharide (LOS) was adsorbed out by incubation for 1 hour at room temperature with polymyxin B-conjugated agarose (Sigma- Aldrich) . After centrifugation to remove the agarose, the LOS content was tested by limulus assay using an E-TOX ATE^® kit (Sigma) and the protein concentration was estimated by Bradford assay (Bio-Rad) . Typical preparations contained 500 μg ml^"1 protein and residual LOS was below detectable levels. A minor modification was made to this method to further minimise cell lysis, this modification involved culturing the strains in DMEM for 12 hours, instead of 18 hours, before centrifugation.

The protein content of the MSP preparations was analysed by immunoblotting (Western blotting) as described by Robinson, K. et al. in Cell Microbiol (2004) 6:927-938. Samples of whole meningococci and MSPs were loaded onto 10% SDS polyacrylamide gels and electrophoretically separated using a Bio-Rad™ Mini-Protean 2 system. Proteins were transferred onto nitrocellulose membranes (Schleicher and Schuell BioScience GmbH) , which were blocked for 1 hour with PBS/0.05% Tween 20/2% dried skimmed milk powder. The blots were probed with a rabbit polyclonal antiserum raised to MSPs and also anti- PorA monoclonal antibody (NIBSC) to check for contaminating outer membrane proteins. Anti-rabbit and anti-mouse IgG-peroxidase conjugates (Sigma) were applied as appropriate, before developing the blots with a chromogenic 4-chloro-l-naphthol substrate solution (Sigma) . Immunisation of mice with MSPs from MCS8(ET-5) & Z4262(ET-37)

For active immunisation studies, 45 female adult mice (six week old, Balb/C) received a subcutaneous (s.c.) injection of immunogen on days 1, 21 and 28 (Li, Y. et al (2004) Infect Imniun 72:345-351). The immunogen comprising MSPs in a 20 μl volume were emulsified with Freund's incomplete adjuvant (1 :1 vol/vol) prior to injection. On day 42, animals received a challenge with 10⁷ colony forming units (cfu) of live wild-type MC58 strain, given by intraperitoneal (i.p.) route. Serum was collected from four individual mice (R1-R4) on day 35 by cardiac puncture. To prepare the innoculum, bacteria for use in the challenge were grown overnight on solid media and re-suspended in 400 μl of PBS, and the number of cfu was estimated by measuring the O. D. A260 of a lysate of the suspension in 1%SDS/O.1M NaOH; the results were confirmed by plating bacteria on solid media. The bacteria were re-suspended in BHI/0.5% iron dextran (Sigma, Poole, United Kingdom) prior to administration. Survival of animals was compared using a one tailed Student's T-test. All animal experimental protocols were reviewed and approved by the Home Office, UK.

Immunologic assays

Serum bactericidal assays (SBA) were performed according to CDC (Center For Disease Control, Altanta, USA) protocol Romero-Steiner, S. et al (2001) Clin. Diagn Lab. Immunol. 8:1115-1119) using baby rabbit sera as a complement source (Pel-freeze) . Approximately 1,000 cfu of each strain in a final volume of 100 μl was incubated in the wells of a microtitre dish with complement and serial dilutions of serum for 1 hr at 37° C. The number of viable bacteria before and after exposure to serum was measured by plating on solid media. Assays were performed in duplicate. The results presented in Figure 1 are calculated as the reciprocal of the dilution of serum that gave > 50% bacterial killing. Negative controls include both pre-immune sera from homologous mice and sera from mice injected with adjuvant-only.

Whole cell ELISA was used to detect Neisseria-specific antibodies in sera using a method previously described by Scholten, R. et al (1994) J Med Microbiol 41:236-243.

For fluorescent-activated cell sorting (FACS) analysis, bacteria were harvested after overnight growth, collected into PBS, and washed before fixation with paraformaldehyde (4%) . Approximately 10⁷ cells were incubated with sera (1 :50 dilution) on ice for 30 min. The samples were pelleted by centrifugation and washed with PBS containing 0.1% Tween-20, before incubation with a donkey anti-mouse IgG conjugated to R-phycoerythrin (Jackson Laboratories at a 1:200 dilution) . The cells were washed prior to analysis in a FACSCalibur flow cytometer (BD) . Results are presented as geometric mean fluorescence after values from the pre-immune sera had been subtracted.

Bacterial lysates preparation and western blotting

The bacteria grown on BHI plates were collected and washed with PBS. The bacteria were adjusted to an O. D. A600 of 6, then boiled with loading buffer for ten minutes. The samples were resolved by polyacrylamide gel electrophoresis on 12% SDS-polyacrylamide gels and electro-transferred to polyvinylidene difluoride membranes (Millipore Corporation) . After blocking in 0.1% Tween 20 /PBS containing 5% dry milk at 4⁰C overnight, membranes were incubated with MSPs raised sera for two hours at room temperature. Membranes were briefly washed then incubated with horseradish peroxidase-conjugated secondary antibodies for another two hours at room temperature. Following washing, immunoreactivity was detected using chemiluminiscence detection kit ECL™ ( Amersham Pharmacia Biotech) .

2D-gel Electrophoresis

Concentrated meningococcal secreted protein (MSP) samples (lOOμl) were initially solubilised by mixing in 50O_jWl lysis buffer containing 7M urea, 2M thiourea, 2% CHAPS, 10OmM dithiothreitol (DTT) (all Sigma) and 2% IPG buffer 3-11 NL (Amersham Biosciences) ; it was shaken for 50 minutes at room temperature. Samples were then concentrated to 50-60 μl in Microcon^® concentrators and excessive DTT was removed by desalting with 2-D clean-up kit (Amersham Biosciences) according to manufacturer's protocol. The resulting pellet was solubilised in lOOμl solubilising buffer (7M urea, 2M thiourea, 2% CHAPS, 1OmM DTT (all Sigma) and 1% IPG buffer 3-11NL; Amersham Biosciences) by sonicating on ice for 4 x 5 seconds and finally incubated for 50 minutes on rotary shaker at room temperature. Protein concentration of the samples was measure using a BIO -RAD^® Protein Assay Kit according to the manufacturer's instructions.

Isoelectric Focusing (IEF) was performed using an Ettan IPGphor I with 13cm Immobiline DryStrips pH 3-11 NL (Amersham Biosciences) as described by Hopkinson, A. et al. (2005) Proteomics 5:1967-1979. Electrophoresis in the second dimension was carried out in a 10% polyacrylamide gel and the gel sliver stained, as described by Hopkinson, A. et al. (2005) Proteomics 5:1967-1979 and Yan, J. X. et al. (2000) Electrophoresis 21:3666-3672. RESULTS

The results below demonstrate the use of MSPs to elicit protective immunity to meningococcal disease. The murine challenge model used demonstrates the ability of MSPs to generate immunity to meningococcal infection, and the bactericidal activities of murine antisera induced by immunisation with MSPs is shown to be effective against pathogens of the same clonal lineage and against pathogens of other clonal lineages.

Meningococci secrete large numbers of proteins

SDS-PAGE and immunoblotting techniques have been used to show that meningococci bacteria secrete a large numbers of proteins (Robinson, K. et al. (2004) Cell Microbiol 6:927-938; Wooldridge, K. G. et al. (2005) Infect Immun 73:5554-5567). To undertake a qualitative assessment of the number of proteins secreted/released by the growing organism, concentrated MSP preparations from meningococcal strain MC58 were fractionated using 2D-gel electrophoresis. Figure 2 shows a silver- stained 2D-gel of MSP from MC58, illustrating a relatively large number of proteins, and thus potential antigens, with diverse molecular sizes and isoelectric foci. The MSP preparations used were free from integral outer membrane components, as confirmed by probing immunoblots with anti-PorA antibodies (as described in Robinson, K. et al. (2004) Cell Microbiol 6:927-938 - data not shown) . Furthermore, the MSP preparations were controlled for cell lysis, as described in Wooldridge, K. G. et al. (2005) Infect Immun 73:5554-5567.

MSP preparations from the wild type MC58 strain were compared with a mutant lacking the protein HIyB, which is an essential component of the type I secretion pathway and required for the secretion of FrpC. FrpC was readily detected by immunoblotting in the intact cells of both strains and in MSP preparations of the wild type strain but could not be detected in MSP preparations of the mutant strain (as shown in Wooldridge, K. G. et al. (2005) Infect Immun 73:5554-5567), demonstrating that the method used did not contain the product of lysed cells.

Of the five major gram-negative protein secretion pathways, N. meningitidis appears to utilise only the Type I and the Type V (autotransporter) secretion pathways for secretion of soluble proteins (Ulsen P. and Tommassen J. (2006) FEMS Microbiol Rev 30:292-319. ; Wooldridge, K. G. et al. (2005) Infect Immun 73 :5554-5567) . The secretory components of the Type I pathway have been identified and demonstrated to be required for the secretion of the meningococcal RTX toxin-like proteins FrpC, FrpC2 and related molecules (Wooldridge, K. G. et al. (2005) Infect Immun 73:5554-5567) . At least seven meningococcal autotransporter proteins have been identified: the IgAl protease (Pohlner, J. et al. (1987) Nature 325:458-462); neisserial haemagglutinin homologue A (NhhA) (Peak, I. R. et al (2000) FEMS Immunol Med Microbiol 28:329-334); adhesion and penetration protein (App) (Hadi, H. A. et al (2001) MoI Microbiol 41:611-623) ; autotransporter serine protease A (AspA, also known as NaIP) (Turner, D. P. et al (2002) Infect Immun 70:4447-4461; van Ulsen, P. et al. (2003) MoI Microbiol 50:1017-1030); autotransporter protein A (AutA) (Ait-Tahar, K. et al (2000) MoI Microbiol 37:1094-1105); and meningococcal serine protease A (MspA) (Turner, D. et al. (2006) Infect Immun 74:2957-2964) . Finally, a two-partner secretion system (a subset of the Type V secretion pathway), homologous to the secreted Bordetella pertussis filamentous haemagglutanin and its secretion partner (FhaB and FhaC, respectively), has also been identified in the meningococcal genome sequence (Parkhill, J. et al (2000) Nature 404:502-506) . MSPs protect mice against meningococcal challenge

The utility of proteins secreted from a pathogenic organism to stimulate an immune response to the pathogenic organism was unexpected and surprising. Such proteins might be assumed to be poor antigens for bactericidal and protective antibodies as they may not be retained on the bacterial surface.

To demonstrate that MSPs do contain antigens capable of generating protective immunity from infection by Neisseria meningitidis, three groups of six-week old Balb/c mice were immunised and challenged. A test group of 15 mice was immunised on days 0, 14 and 21 with 25 μg MSPs per dose (mixed with Fruend's incomplete adjuvant) and challenged 2 weeks later with 10⁷ cfu of live Neisseria meningitidis bacteria (strain MC58) . The MSPs were derived from Neisseria meningitidis MC58 (ET-5) . A negative control group consisting of 15 mice was injected with adjuvant only and a positive control group of 15 mice was immunised with a previously characterised live attenuated strain YH102, derived from the MC58 strain of Neisseria meningitidis (Li, Y. et al. (2004) Infect Immun 72:345-351) . 7 of the 15 (46.7%) mice from the negative control group died after 72 h of challenge, whereas none of the mice immunised with either live attenuated bacteria or with the MSPs died (Figure 3) .

Murine anti-MSPs are bactericidal

Post-immunisation sera from four mice (Rl-4) immunised with MSPs from MC58, and post-immunisation sera from a mouse immunised with MSPs from Z4262, was individually tested for its ability to kill the homologous strain of N. meningitidis in the presence of a complement source. As demonstrated in Figure 1 all five murine sera showed extremely high serum bactericidal titres ( > 1 : 2048) against the strain of N. meningitidis from which the MSPs were prepared. Pre-immune sera from the same mice, and those from the negative control group (injected with adjuvant only) failed to kill the same strain (titres < 1:32) . Sera from the positive control mice (immunised with live attenuated bacteria) killed the organism with similar bactericidal titres as the test group ( > 1 :2048) .

Antibodies to MSPs recognise antigens on the meningococcal cell surface

The SBA (serum bactericidal assay) results suggest that the protection observed in the murine model may (at least in part) results from the bactericidal activity of antibodies generated after immunisation with MSPs. These results also show that the anti-MSP sera, which were raised to extracellular secreted proteins, contain antibodies that bind to surface-bound bacterial antigens.

To confirm the ability of anti-MSP sera to bind to antigens on the surface of bacteria, anti-MSP sera (from Rl -4) was added to live and killed whole meningococci (strain MC58) and binding was examined in a whole cell ELISA. Pooled pre-immune sera from these mice (negative control) and an antiserum from the positive control group (immunised with the live attenuated meningococci) were also included. Sera from MSP-vaccinated mice exhibited high titres of reactive antibodies specific to meningococci (Figure 4) . Antibody titres of all four sera from MSP-vaccinated mice were higher than those of the mice vaccinated with whole cells of the attenuated strain; negligible binding was observed with negative control serum.

To confirm the surface binding of anti-MSP antibodies, intact (fixed) meningococci were co-incubated with pooled Rl-4 anti-MSP antisera and the cell suspensions were subsequently incubated with fluorescent-labelled anti-mouse antibodies. The cell suspensions were then subjected to FACS analysis. A clear shift of the population of bacterial cells was observed in suspensions of bacteria probed with test serum, compared with cells probed with pooled pre-immune sera from the same mice (negative control) (Figure 5) . Cells probed with antiserum to the live attenuated strain were included as a positive control and were also shifted by comparison to cells probed with pre-immune serum.

Murine anti-MSPs cross-kill strains from homologous clonal lineages

To demonstrate the ability of anti-MSP antibodies to elicit cross- protection directed to heterologous meningococcal isolates, a selection of 17 isolates, representing a number of different clonal lineages, were tested in serum bactericidal assays (SBAs) against antisera Rl-4 from mice immunised with the MC58 strain of Neisseria meningitidis and antisera from mice immunised with from the Z4262 strain of Neisseria meningitidis (Figure 1).

The sera from four mice immunised with MC58 showed extremely high bactericidal titres to the homologous serogroup B strain MC58. The sera from four mice immunised with MC58 showed extremely high bactericidal titres to heterologous serogroup B strains H44/76, BZ169, BZ83 and C311 which belong to the same ET-5 clonal lineage as strain MC58 (Figure 1) . The sera from a mouse immunised with Z4262 showed high bactericidal titres to its homolgous serogroup B strain, as well as against a number of heterologous strains - some from the same clonal lineage/electrophoretic type (ET) and some from different clonal lineages/electrophoretic types. Some of the strains which gave a positive result were also from a different serogroup. More specifically, MSPs from Z4262 were able to elicit an immune response directed to bacteria belonging to (i) another serogroup B ET-37 strain, (ii) serogroup C ET-37 strains, (iii) serogroup B ET-5 strains and (iv) a serogroup A IV-I strain. These results demonstrate the cross-reactivity of the antisera raised by some MSPs.

Representative sera from the positive control group of mice were included in these experiments. The positive control group were vaccinated with whole live bacteria of the attenuated strain YH102. One of these sera, for example, showed SBA titres of 8192, 2048, 1024 and < 64 against strains MC58, C311, Z2491 and FAM18, respectively. The results show that the sera were active against each of the strains tested apart from FAM18 (a serogroup C strain, of ET -type 37) . The positive control was added for comparison with the test group and to show that the assays were working correctly.

Immunoblots were performed on whole cell lysates from various bacterial strains using antisera from mice immunised with MSPs from MC58 (Figure 6) or Z4262 (Figure 7) . The antisera from mice R1-R4 immunised with MC58 was pooled for use in this experiment. Figure 6 and 7 both show that immunisation with MSPs elicits broad cross-reactive antibodies. Cell lysates from all bacterial strains show a similar Western blot profile with both antisera. That is, the MSPs used produce antibodies that will react with a large range of antigens across a range of bacterial strains.

The data shows that protective immunity in a host to a pathogenic organism may be conferred by immunising the host with proteins secreted by the pathogenic organism. The immunity may be provided by complement-mediated lysis of the pathogen, via surface-bound antigens, or by opsonisation, mediated by antibodies to surface bound molecules.

The secreted proteins may generate antibodies which neutralise receptors on the surface of the pathogen and/or on host cells which would normally recognise virulence factors that are important for disease, thereby reducing the effective virulence and/or survival of the pathogen.

Alternatively, or additionally, antibodies directed to non-surface bound secreted proteins may neutralise the binding, modulating or damaging effects of secreted virulence factors to host cells. For example, during invasive disease, the pathogenic organism may employ secreted proteins in survival and virulence functions such as adhesion, invasion, acquisition of nutrition, cytopathogenesis and cytotoxicity, by interrupting these functions the survival and/or invasive capability of the organism in the host environment may be impeded. Secreted proteins may cause either or both of the following effects: a) the generation of a population of antibodies that prevent or reduce disease (by preventing invasion) while not affecting colonisation or survival of the pathogen; and b) the generation of a second population of pathogen specific antibodies that protect against disease and possibly colonisation.

Claims

1. An immunogenic composition comprising one or more proteins secreted by one or more strains of one or more pathogenic organisms, wherein the composition is capable of eliciting an immune response directed to one or more of the pathogenic organisms from which one or more of the secreted proteins are derived.

2. A composition according to claim 1 which is capable of eliciting an immune response directed to an organism related to the pathogenic organism from which one or more of the secreted proteins are derived.

3. A composition according to any claim 1 or 2 wherein the immune response is a protective immune response.

4. A composition according to any preceding claim comprising at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the proteins secreted by one or more strains of one or more pathogenic organisms.

5. A composition according to any preceding claim wherein the secreted proteins are from only one strain of one pathogenic organism.

6. A composition according to any preceding claim wherein the secreted proteins are meningococcal secreted proteins (MSPs) secreted by the pathogenic bacteria Neisseria meningitidis.

7. A composition according to claim 6 comprising MSPs from one strain of Neisseria meningitidis.

8. A composition according to claim 6 comprising MSPs from more than one strain of Neisseria meningitidis.

9. A composition according to any of claims 6 to 8 wherein the composition is capable of eliciting a protective immune response directed to Neisseria meningitidis.

10. A composition according to any of claims 6 to 9 comprising MSPs derived from one or more serogroup B strains of Neisseria meningitidis .

11. A composition according to claim 10 wherein the composition is capable of eliciting a protective immune response directed to one or more serogroup B strains of Neisseria meningitidis.

12. A composition according to any of claims 6 or 11 wherein the composition is capable of eliciting a protective immune response directed to one or more serogroups of Neisseria meningitidis selected from serogroup A, C, W135 and Y.

13. A composition according to any of claims 6 to 12 wherein the composition is capable of eliciting a protective immune response directed to more than one strain of Neisseria meningitides from one or more clonal lineages.

14. A composition according to any preceding claim comprising proteins secreted by one or more strains of Neisseria gonorrhoeae, Haemophilus influenzae, Actinobacillus pleuropneumoniae or a similar organism.

15. A composition according to any preceding claim for use as a prophylactic and/or a therapeutic vaccine against one or more pathogenic organisms, or related pathogenic organisms, from which the one or more secreted proteins are derived.

16. A pharmaceutical composition comprising a composition according to any preceding claim in combination with a pharmaceutically acceptable carrier or excipient.

17. A composition according to claim 16 comprising proteins secreted by one or more strains of Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Actinobacillus pleuropneumoniae or a similar organism.

18. A composition according to any preceding claim which is capable of inducing a serum bactericidal antibody response after administration to a subject.

19. A composition according to any preceding claim which is capable of eliciting an immune response which neutralises proteins secreted by the pathogenic organism.

20. A composition according to any preceding claim which is capable of eliciting an immune response directed to proteins on the host cells to defend against infection by a pathogenic organism.

21. A vaccine composition comprising a composition according to any preceding claim.

22. A vaccine composition according to claim 21 where the vaccine is a therapeutic or a prophylactic vaccine.

23. Use of a composition according to any preceding claim in the preparation of a medicament for eliciting an immune response.

24. Use of a composition according to any preceding claim in the preparation of a medicament for ameliorating disease.

25. Use of a composition according to any preceding claim in the preparation of a prophylactic or a therapeutic vaccine directed to one or more the pathogenic organisms, or related pathogenic organisms, from which the one or more secreted proteins are derived.

26. The use of any of claims 23, 24 or 25 wherein the composition comprises proteins secreted from Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Actinobacillus pleuropneumoniae or a similar organism.

27. The use of claim 25 wherein the proteins are secreted from Neisseria meningitidis and the vaccine is directed to meningitis, septicaemia and/or septic shock caused by Neisseria meningitidis.

28. The use of claim 25 wherein the vaccine is directed to meningitis, septicaemia or septic shock caused by serogroup B Neisseria meningitidis.

29. A method of protecting a human or non-human animal from the effects of infection by a pathogenic organism comprising administering to the human or non-human animal a composition according to any of claims 1 to 22.

30. A method for raising an immune response in a human or non- human animal comprising administering a composition according to any of claims 1 to 22 to the human or non-human animal.

31. A composition substantially as herein described with reference to the examples.

32. A use substantially as herein described with reference to the examples .