WO2011033319A1

WO2011033319A1 - Commensal neisserial stress protein preparations

Info

Publication number: WO2011033319A1
Application number: PCT/GB2010/051581
Authority: WO
Inventors: Andrew Gorringe; Shaun Mcnulty
Original assignee: Health Protection Agency; Immunobiology Limited
Priority date: 2009-09-21
Filing date: 2010-09-21
Publication date: 2011-03-24
Also published as: GB0916557D0

Abstract

There is provided an immunogenic preparation, comprising a commensal Neisserial stress protein and an antigenic polypeptide.

Description

COMMENSAL NEISSERIA!. STRESS PROTEIN PREPARATIONS

The present invention relates to preparations obtained from commensal Neisseria, which preparations comprise stress polypeptides and antigens; to methods of preparing said preparations; to compositions comprising said preparations, and to uses of these preparations in the prevention of Neisserial disease.

Neisseria meningitidis (N. meningitidis) is the causative agent of meningococcal meningitis and meningococcal septicaemia, and is of particular importance as a worldwide health problem. Neisseria gonorrhoeae {N. gonorrhoeae) is the causative agent of gonorrhoea.

Commensal Neisseria include species such as Neisseria lactamica (N. lactamica), which closely resembles N. meningitidis and is also a common inhabitant of the human nasopharynx. However, unlike N. meningitidis, commensal Neisseria such as N. lactamica lack the key genes required for invasive disease and as such are limited to a commensal existence in the nasopharynx. Natural colonisation by N. lactamica is thought to reduce the risk of invasive meningococcal disease, probably by inducing a cross-protective immune response.

Commensal Neisseria such as N. lactamica are important as the basis for experimental live vaccines and outer-membrane vesicle (OMV) vaccines against meningococcal disease caused by N. meningitidis. For example, N. lactamica OMVs have been demonstrated to protect against lethal challenge in a mouse model of meningococcal disease. OMVs and live commensal Neisseria such as N. lactamica have also been advocated as delivery vehicles for antigens intended to confer protective immunity against other diseases (WO 00/50074).

There is an on-going need in the art to provide further, improved, vaccines that give protective immunity to Neisserial diseases.

An object of the present invention is to provide compositions containing immunostimulating components, and vaccines based thereon, which meet this need in the art. In a first aspect, the present invention provides an immunogenic preparation, comprising a commensal Neisserial stress protein and an antigenic polypeptide. In one embodiment, the immunogenic preparation is a mixture of polypeptides comprising a commensal Neisserial stress protein and an antigenic polypeptide).

In one embodiment, the immunogenic preparation (eg. mixture of polypeptides) is enriched with (ie. comprises an elevated amount or concentration of) commensal Neisserial stress protein, as compared with a preparation comprising antigenic polypeptide.

In one embodiment, the immunogenic preparation comprises or consists of an immunogenic complex comprising the commensal Neisserial stress protein and antigenic polypeptide.

In one embodiment, at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 75% by weight of protein in the immunogenic preparation (eg. immunogenic complex) is commensal Neisserial stress protein. In one embodiment, up to 5, 10, 15, 20, 25, 30, 40, 50 or 75% by weight of protein in the immunogenic preparation (eg. immunogenic complex) is commensal Neisserial stress protein.

The present invention also provides a method of preparing said immunogenic preparation (eg. immunogenic complex), comprising the steps of:

(i) subjecting a cell to a stress-inducing stimulus, wherein said cell is capable of expressing a commensal Neisserial stress protein and an antigenic polypeptide; and

(ii) recovering a preparation comprising said commensal Neisserial stress protein and said antigenic polypeptide from the stress-treated cell.

In one embodiment, the preparation recovered from the cell comprises or consists of a mixture of the commensal Neisserial stress protein and antigenic polypeptide. In one embodiment, the commensal Neisserial stress protein and antigenic polypeptide are present in the preparation in the form of an immunogenic complex. The present invention is based on the unexpected finding that commensal Neisserial stress proteins are surprisingly highly effective for eliciting an immune response against antigenic polypeptides.

As such, preparations (eg. mixtures or complexes) comprising a commensal Neisserial stress protein and an antigenic polypeptide are surprisingly useful for immunostimulation - for example, as vaccines against infectious diseases.

Preparations (eg. mixtures or complexes) comprising antigenic peptides and stress proteins derived from pathogens or from pathogen-infected cells are known to be efficacious as vaccines against infectious diseases (see for example WO 00/10597 and WO 01/13944, WO 02/20045 and PCT/GB2009/051133, published as WO 2010/026432, incorporated herein by reference).

However, none of these prior publications provides any suggestion of the advantageous immunogenic properties of the immunogenic preparations (eg. mixtures or complexes) described herein, which comprise a commensal Neisserial stress protein.

The present Applicants have unexpectedly identified that preparations (eg. mixtures or complexes) comprising a commensal Neisserial stress protein and an antigenic polypeptide are surprisingly more immunogenic than preparations (eg. mixtures or complexes) comprising a pathogenic Neisserial stress protein and an antigenic polypeptide. Most surprisingly, when used as vaccines, preparations (eg. mixtures or complexes) isolated from a commensal Neisserial strain induce better protective immunity against a pathogenic Neisserial strain than preparations (eg. mixtures or complexes) isolated from the same pathogenic strain. The unexpected immunogenic efficacy of the preparations of the present invention (as compared with preparations comprising pathogenic Neisserial stress proteins) is particularly surprising in view of the very high sequence identity between commensal Neisserial stress proteins and the equivalent pathogenic Neisserial stress proteins.

In this regard, the amino acid sequence of the commensal Neisserial stress protein GroEL of Neisseria lactamica strain Y92-1009 (shown herein as SEQ ID NO: 1) has over 99% amino acid sequence identity to the GroEL stress proteins of four Neisseria meningitidis strains (strains Z2491 , MC58, Fam18 and 052443).

The amino acid sequence of the commensal Neisserial stress protein DnaK of Neisseria lactamica strain Y92-1009 (shown herein as SEQ ID NO: 2) has over 98% amino acid sequence identity to the DnaK stress proteins of four Neisseria meningitidis strains (strains Z2491 , MC58, Fam18 and 052443).

A "commensal" organism coexists in an environment with another organism, such coexistence being beneficial to at least one of the organisms and generally not detrimental to either.

A number of different commensal Neisseria are known in the art and include Neisseria lactamica, Neisseria cinerea, Neisseria eiongata, Neisseria flavescens, Neisseria polysaccharea, Neisseria sicca and Neisseria subflava.

Thus, in one embodiment, the terms "commensal Neisseria^ or "commensal Neisserial" refer to a commensal Neisseria selected from Neisseria lactamica, Neisseria cinerea, Neisseria eiongata, Neisseria flavescens, Neisseria polysaccharea, Neisseria sicca and Neisseria subflava.

In one embodiment, the commensal Neisseria is Neisseria lactamica. Different commensal Neisserial species are known to colonise the buccal or nasal areas. Hence, different species of commensal Neisserial species may be selected depending on the known area of the body that it usually colonises.

In one embodiment, the commensal Neisseria is a recombinant commensal Neisseria {eg. a recombinant Neisseria lactamica) which expresses a polynucleotide sequence that is heterologous to said commensal Neisseria (eg. heterologous to said Neisseria lactamica).

A polynucleotide sequence that is 'heterologous' to a commensal Neisseria is a polynucleotide sequence that is 'not native to' or 'not normally present in' or 'not naturally occurring in' the commensal Neisseria. For example, said heterologous polynucleotide sequence does not naturally occur in the genome of the commensal Neisseria.

A heterologous polynucleotide sequence may comprise a (at least one) gene that is heterologous to said commensal Neisseria, optionally linked to non-coding sequence (eg. regulatory sequence such as promoter sequence or terminator sequence).

The non-coding sequence may be heterologous to said commensal Neisseria or may alternatively be native to (naturally occurring in) said commensal Neisseria. Heterologous genes encode heterologous gene products. Thus, in one embodiment, the heterologous polynucleotide sequence encodes a (at least one) gene product that is heterologous to said commensal Neisseria. A gene product that is heterologous to a commensal Neisseria is a gene product that is 'not native to' the commensal Neisseria, or is 'not normally present in' or 'not naturally occurring in' the commensal Neisseria. For example, the gene product is not naturally encoded by the genome of the commensal Neisseria. Thus, in one embodiment, the commensal Neisseria (eg. Neisseria lactamica) expresses a gene product encoded by the heterologous polynucleotide sequence, wherein said gene product is heterologous to said commensal Neisseria (eg, heterologous to said Neisseria lactamica).

Examples of heterologous gene products include peptides, polypeptides, proteins and fragments thereof that are heterologous to said commensal Neisseria. A peptide, polypeptide, protein or fragment thereof that is heterologous to the commensal Neisseria is 'not native to' the commensal Neisseria, or is 'not normally present in' or 'not naturally occurring in' the commensal Neisseria.

Thus, in one embodiment, said commensal Neisseria (eg. Neisseria lactamica) expresses a polypeptide that is heterologous to said commensal Neisseria (eg. heterologous to said Neisseria lactamica).

In one embodiment, said heterologous polypeptide comprises an antigen, or an epitope, that is heterologous to said commensal Neisseria. Thus, in one embodiment, said commensal Neisseria expresses an antigen or epitope that is heterologous to said commensal Neisseria.

In one embodiment, the heterologous polynucleotide encodes a bacterial protein, eukaryotic protein (eg. mammalian such as human) or viral protein such as a viral surface peptide.

Thus, in one embodiment, the commensal Neisseria recombinantly expresses a heterologous polypeptide selected from bacterial proteins, eukaryotic proteins, or viral proteins such as viral surface peptides. In one embodiment, said heterologous polynucleotide comprises a gene that encodes an immunostimulatory gene product, such as an immunostimulatory polypeptide. An immunostimulatory gene product may be immunostimulatory for treatment of non-infectious disease, for example allergy or cancer. For example, a heterologous gene product may comprise a nut antigen (eg. peanut antigen), or may comprise a tumour-specific antigen (eg. melanoma-associated antigen "MAGE" or prostate specific antigen "PSA").

In one embodiment, the heterologous polynucleotide comprises a nucleic acid sequence of a pathogenic or non-pathogenic (eg. commensal) organism. Thus, in one embodiment, the heterologous polypeptide comprises a polypeptide (such as an antigen) of a pathogenic or non-pathogenic (eg. commensal) organism.

In one embodiment, the commensal Neisseria (eg. Neisseria lactamica) expresses a heterologous polynucleotide comprising a gene of a different species or strain of commensal Neisseria. Thus, in one embodiment, the commensal Neisseria expresses a heterologous polypeptide comprising a polypeptide of a different species or strain of commensal Neisseria. By way of example, the different species of commensal Neisseria may be selected from Neisseria lactamica, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria polysaccharea, Neisseria sicca or Neisseria subflava. For example, the commensal Neisseria may be N. lactamica, and may express a heterologous polypeptide comprising a polypeptide of N. cinerea, N. elongata, N. flavescens, N. polysaccharea, N. sicca or N. subflava.

In one embodiment, the heterologous polynucleotide comprises a gene of a pathogenic organism, which encodes a gene product (eg. a polypeptide, such as an antigen) of said pathogenic organism. Thus, in one embodiment, the heterologous polypeptide comprises a polypeptide, such as an antigen, of a pathogenic organism. Thus, in one embodiment, said commensal Neisseria expresses a polypeptide (such as an antigen) of a pathogenic organism. Pathogenic organisms typically include bacteria, viruses, fungi and protozoa.

In one embodiment, said heterologous polynucleotide comprises a bacterial gene that encodes a bacterial protein (eg. comprising a bacterial antigen). For example, the heterologous polypeptide may be a polypeptide of a gram positive or gram negative bacteria. In one embodiment, the heterologous polypeptide is a polypeptide of Acinetobacter, Actinobacillus, Actinomycetes, Aeromonas, Bacillus, Bordetelia, Borrelia, Branhamella, Brucella, Calymmatobacterium, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterobacter, Erwinia, Erysipelothrix, Escherichia, Fancisella, Haemophilus, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Pasturella, Pseudomonas, Proteus, Rickettsia, Salmonella, Shigella, Spirillum, Staphylococcus, Streptobacilius, Streptococcus, Streptomycetes, Treponema, Vibrio or Yersinia.

In one embodiment, said heterologous polynucleotide comprises a viral gene that encodes a viral protein (eg. comprising a viral antigen). For example, the heterologous polypeptide may be a polypeptide of rabies virus, herpes simplex virus, Epstein-Barr virus, vesicular stomatitis virus, vaccinia virus, Human immunodeficiency virus (HIV), Hepatitis A virus (HAV), hepatitis B (HBV), hepatitis C (HCV), human papillomavirus (HPV), Kaposi's Sarcoma-Associated Herpesvirus (KSHV), Respiratory Syncytial Virus, Ebola virus, Marburg virus, West Nile virus (WNV), St Louis Encephalitis virus (SLEV), Rift Valley Fever virus (RVFV), Influenza viruses, coronaviruses, rhinovirus, adenovirus, SIV, rotavirus, arbovirus, measles virus, polio virus, rubella virus, mumps virus, papova virus, varicella- zoster virus, varicella virus, huntavirus and cytomegalovirus.

In one embodiment, said heterologous polynucleotide comprises a yeast or fungal gene that encodes a yeast or fungal protein (eg. comprising a yeast or fungal antigen). For example, the heterologous polypeptide may be a polypeptide of Acremonium, Alternaria, Amylomyces, Arthoderma, Aspergillus, Aureobasidium, Blastochizomyces, Botrytis, Candida, Cladosporium, Crytococcus, Dictyostelium, Emmonsia, Fusarium, Geomyces, Geotrichum, Microsporum, Neurospora, Paecilomyces, PenicHiium, Pilaira, Pityrosporum, Rhizopus, Rhodotorula, Saccharomyces, Stachybotrys, Trichophyton, Trichoporon or Yarrowia.

In one embodiment, said heterologous polynucleotide comprises a protozoan gene that encodes a protozoan protein (eg. comprising a protozoan antigen). For example, the heterologous polypeptide may be a polypeptide of Plasmodium falciparum, a leishmania sp., a trypanosome sp., or a Cryptosporidium sp.

In one embodiment, said heterologous polynucleotide comprises (eg. consists of) a gene sequence of a pathogenic Neisseria, such as N. meningitidis or N, gonorrhoeae. In one embodiment, said heterologous polypeptide comprises (eg. consists of) a polypeptide such as an antigenic polypeptide of a pathogenic Neisseria, such as N. meningitidis or N, gonorrhoeae.

Thus, in one embodiment, the commensal Neisseria (such as Neisseria lactamica) expresses a pathogenic Neisserial polypeptide, such as a polypeptide of N. meningitidis or N. gonorrhoeae.

In one embodiment, the pathogenic Neisserial polypeptide is selected from a transferrin binding protein, a factor H binding protein (fHbp), NadA, a superoxide dismutase ("SOD") such as a Cu,Zn-SOD, Neisserial surface protein A ("NspA"), a porin (eg. PorA or PorB), or any other pathogenic Neisserial outer membrane protein. Gene and polypeptide sequences for these antigens are known in the literature. In one embodiment, the pathogenic Neisserial (eg. N. meningitidis or N. gonorrhoeae) polypeptide is selected from a transferrin binding protein, a superoxide dismutase ("SOD") such as a Cu,Zn-SOD, or PorB.

Stress proteins are a family of highly conserved proteins originally identified in cells subjected to heat stress, but now known to be associated with many other forms of stress, such as the stress-inducing stimuli discussed below. Thus, although commonly referred to as "heat shock proteins" (HSPs) these proteins are better defined as "stress proteins" (SPs), because their expression is not solely induced by heat stress.

On the basis of their molecular weights, the major stress proteins are grouped into six different families: small (hsp20-30ka); hsp40; hsp60; hsp70; hsp90; and hsp100. Members of the hsp60 family include the major chaperone GroEL. These form multimeric complexes with co-chaperones such as GroES. Members of the hsp70 family include DnaK and also form multimeric complexes with co- chaperones such as DnaJ. Other major hsps include the AAA ATPases, the Clp proteins, Trigger factor, Hip, HtpG, NAC, Clp, GrpE, SecB and prefoldin. Stress proteins are ubiquitously expressed in prokaryotic cells, such as commensal Neisseria (eg. Neisseria lactamica). The stress proteins have a range of biological functions; many act as 'molecular chaperones' in numerous processes that involve the folding and unfolding of polypeptides. In response to a stress-inducing stimulus, cells induce or up-regulate the expression or synthesis of stress proteins.

For example, the rate of synthesis of a commensal Neisseria! stress protein may increase from 2-100 fold (such as 2, 5, 10, 25, 50, 75 or 100-fold) in response to a stress-inducing stimulus, as compared with the basal rate of synthesis in the absence of the stress-inducing stimulus.

For example, the increase in expression and synthesis of a commensal Neisserial stress protein may commence within a short interval following the commencement of the stress-inducing stimulus - for example, over a period of 1-10 minutes - and may last for at least 10, 20 or 30 minutes, or for up to one or several hours after commencement of the stress-inducing stimulus.

The induced/ upregulated commensal Neisserial stress proteins are understood to associate with (for example, form complexes with) polypeptides (eg. antigenic polypeptides) in the cytoplasm of the cell, resulting in the formation of "stress protein-enriched" polypeptide preparations (eg. "stress protein-antigenic polypeptide preparations"). In one embodiment, the upregulated commensal Neisserial stress proteins form complexes with polypeptides in the cytosol of the ceil (ie. "stress protein-polypeptide complexes", such as "stress protein-antigenic polypeptide complexes").

Hence, commensal Neisseria stress proteins useful in the present invention are commensal Neisserial proteins: (i) whose intracellular concentration increases when a cell is exposed to a stress-inducing stimulus, and (ii) that are capable of associating with (eg. mixing with or forming a complex with) an antigenic polypeptide.

A commensal Neisserial stress protein is native to a commensal Neisseria (ie. naturally expressed in commensal Neisseria). As such, a commensal Neisserial stress protein is not native to pathogenic Neisseria (ie. not naturally expressed in pathogenic Neisseria). A commensal Neisserial stress protein is not a pathogenic Neisserial stress protein.

In one embodiment, the commensal Neisserial stress protein is a stress protein that is capable of being captured by antigen presenting ceils (APCs) such as dendritic cells (DCs). In one embodiment, the commensal Neisserial stress protein is a stress protein that is capable of activating innate immune responses.

In one embodiment, the commensal Neisserial stress protein has chaperone activity and/ or is classed as a 'chaperone protein'. In one embodiment, the commensal Neisserial stress protein is a Neisseria lactamica stress protein.

In one embodiment, the commensal Neisserial stress protein may be selected from any one or more of the hsp10 family, hsp40 family, hsp60 family or hsp70 family.

In a preferred embodiment, the commensal Neisserial stress protein comprises a mixture of stress proteins from more than one family.

In one embodiment, the commensal Neisserial stress protein is a member of the Hsp60 family. Members of the commensal Neisserial hsp60 family include the 'chaperone' protein GroEL (for example, as shown in SEQ ID NO: 1 of the present application). This protein forms multimeric complexes with co-chaperones such as GroES (a member of the hsp10 family).

Thus, in one embodiment, the commensal Neisserial stress protein is GroEL, or a fragment thereof. In one embodiment, the commensal Neisserial stress protein comprises or consists of the polypeptide sequence shown in amino acid residues 1 -544 of SEQ ID NO: 1 , or a fragment thereof comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 consecutive amino acids thereof.

Said fragment may retain one or more activity of the full-length GroEL polypeptide shown in SEQ ID NO: 1. For example, said fragment may retain 'chaperone activity' and hence the ability to associate with (eg. mix with or form a complex with) an antigenic polypeptide. For example, said fragment may retain the ability to be captured by an antigen presenting cell (APC) such as a dendritic cell (DC).

In one embodiment, the stress protein complex of the invention comprises a commensal Neisserial GroEL stress protein, or a fragment thereof as described above. In one embodiment, the stress protein complex further comprises GroES. In one embodiment, the commensal Neisserial stress protein is a member of the Hsp70 family. Members of the commensal Neisserial hsp70 family include the 'chaperone' protein DnaK for example, as shown in SEQ ID NO: 2 of the present application). This protein forms multimeric complexes, in this case with co- chaperones such as DnaJ (a member of the hsp40 family).

Thus, in one embodiment, the commensal Neisserial stress protein is DnaK, or a fragment thereof. In one embodiment, the commensal Neisserial stress protein comprises or consists of the polypeptide sequence shown in amino acid residues 1-642 of SEQ ID NO: 2, or a fragment thereof comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 consecutive amino acids thereof.

Said fragment may retain one or more activity of the full-length DnaK polypeptide shown in SEQ ID NO: 2. For example, said fragment may retain chaperone activity' and hence the ability to associate with (eg. mix with or form a complex with) an antigenic polypeptide. For example, said fragment may retain the ability to be captured by an antigen presenting cells (APC) such as a dendritic cell (DC). In one embodiment, the stress protein preparation of the invention (eg. stress protein mixture or complex) comprises a commensal Neisserial DnaK stress protein, or a fragment thereof as described above. In one embodiment, the stress protein preparation further comprises DnaJ. Other Neisserial stress proteins identified in commensal Neisseria (eg. Neisseria lactamica) include the proteins identified as GrpE, HscA, HscB, Hsp15, Hsp33 and HtpX. In one embodiment, the commensal Neisserial stress protein is selected from any of these commensal Neisserial stress proteins. In one embodiment, the stress protein preparation of the invention comprises any one or more of these commensal Neisserial stress proteins (instead of, or in addition to, the GroEL, DnaK, GroES and/ or DnaJ stress proteins discussed above). In a preferred embodiment, the stress protein preparation comprises a mixture of stress proteins from more than commensal Neisserial stress protein families.

An antigenic polypeptide is any polypeptide that can be recognized by the immune system and induces an immune response in a host organism exposed to the antigenic polypeptide. For example, an antigenic polypeptide may stimulate a T- cell mediated immune response in the host organism and/ or may stimulate the generation of antibodies by the host organism. As such, an antigenic polypeptide of the invention is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or a T cell receptor.

In one embodiment, an antigenic polypeptide of the invention provides a cell- mediated response involving T cells (eg. CD4+ and/ or CD8+ T cells). For example, the antigenic polypeptide has the ability to induce the secretion of Th1- type cytokines such as IFN-γ (eg. from predominantly CD4+ T cells).

An antigenic polypeptide comprises at least one antigenic determinant. The terms "antigenic determinant" and "epitope" are synonymous, and mean a part of an antigenic polypeptide that is recognised and bound by an antibody (or B cell or T cell) and elicits an immune response. In one embodiment, an antigenic polypeptide induces a neutralizing antibody response. In one embodiment, an antigenic polypeptide provides protection (such as long term protection) against subsequent challenge.

The antigenic polypeptide may be native to (ie. is normally present in or naturally occurring in - eg. naturally encoded by the genome of) the cell in which the commensal Neisserial stress protein complex is produced. In one embodiment, the antigenic polypeptide is native to commensal Neisseria (ie. the antigenic polypeptide is a commensal Neisseria! polypeptide).

By way of example, the commensal Neisserial polypeptide may be from a commensal Neisseria selected from Neisseria lactamica, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria polysaccharea, Neisseria sicca or Neisseria subflava.

In one embodiment, the commensal Neisserial antigenic polypeptide is selected from any of the following polypeptides identified by reference to the N. lactamica strain sequenced by the Sanger Institute: NLA16810 (similar to "Omp64"), NLA5600 (similar to "OprC"), NLA15790 (similar to "NspA2"), NLA17780 (similar to Opa), NLA13360 (similar to Opa), NLA18150 (almost identical to UniProtKB/TrEMBL:C0F7G9, similar to FHbp).

See ftp://ftp.sanaer.ac.uk/Dub/pathoaens/nl/NL.qlimmer CURATED.tab.

Alternatively, the antigenic polypeptide may be 'heterologous' to (ie. 'not native toV 'not normally present in'/ 'not naturally occurring in') the host cell in which the commensal Neisserial stress protein preparation is produced. For example, the antigenic polypeptide may be expressed recombinantly in a host cell in which the commensal Neisserial stress protein preparation is produced.

In one embodiment, the antigenic polypeptide is heterologous to commensal Neisseria (ie. the antigenic polypeptide is not a commensal Neisserial polypeptide). In one embodiment, the antigenic polypeptide is heterologous to N. lactamica (ie. the antigenic polypeptide is not a N. lactamica polypeptide).

In one embodiment, the antigenic polypeptide comprises or consists of a bacterial protein, eukaryotic protein or viral protein such as a viral surface peptide. In one embodiment, the antigenic polypeptide is immunostimulatory for treatment of non- infectious disease, for example allergy or cancer. For example, the antigenic polypeptide may comprise a nut antigen, or may comprise a tumour-specific antigen (eg. melanoma-associated antigen "MAGE" or prostate specific antigen "PSA").

Thus, the antigenic polypeptide may comprise (or consist of) a polypeptide of a non-pathogenic (eg. commensal) organism, such as a commensal Neisseria.

In an alternative embodiment, the antigenic polypeptide comprises or consists of a polypeptide from a pathogenic (disease-causing) organism. Examples of pathogenic organisms include pathogenic bacteria, viruses, fungi (eg. yeast) and protozoa.

In one embodiment, the antigenic polypeptide comprises or consists of a polypeptide of a gram positive bacterium or gram negative bacterium (such as a pathogenic gram positive or gram negative bacterium). In one embodiment, antigenic polypeptide comprises or consists of a polypeptide of Acinetobacter, Actinobacillus, Actinomycetes, Aeromonas, Bacillus, Bordetella, Borrelia, Branhamella, Brucella, Calymmatobacterium, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterobacter, Erwinia, Erysipelothrix, Escherichia, Fancisella, Haemophilus, Klebsiella, Legionella, Leptospira, Listeria, Moraxelia, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Pasturella, Pseudomonas, Proteus, Rickettsia, Salmonella, Shigella, Spirillum, Staphylococcus, Streptobacillus, Streptococcus, Streptomycetes, Treponema, Vibrio or Yersinia.

In one embodiment, the antigenic polypeptide comprises or consists of a Neisserial polypeptide or Neisserial polypeptide antigen.

By way of example, the antigenic polypeptide may comprise or consist of a polypeptide or antigen of a pathogenic Neisseria, such as N. meningitidis or N. gonorrhoeae. In one embodiment, the antigenic polypeptide comprises or consists of a pathogenic Neisserial polypeptide (eg. a N. meningitidis or N. gonorrhoeae polypeptide) selected from a transferrin binding protein, a superoxide dismutase ("SOD") such as a Cu,Zn-SOD, Neisserial surface protein A ("NspA"), a porin (eg. PorA) or any other outer membrane protein, or an antigenic determinant thereof.

In one embodiment, the antigenic polypeptide comprises or consists of a polypeptide of a virus (such as a pathogenic virus), for example a surface polypeptide. In one embodiment, antigenic polypeptide comprises or consists of a polypeptide of rabies virus, herpes simplex virus, Epstein-Barr virus, vesicular stomatitis virus, vaccinia virus, Human immunodeficiency virus (HIV), Hepatitis A virus (HAV), hepatitis B (HBV), hepatitis C (HCV), human papillomavirus (HPV), Kaposi's Sarcoma-Associated Herpesvirus (KSHV), Respiratory Syncytial Virus, Ebola virus, Marburg virus, West Nile virus (WNV), St Louis Encephalitis virus (SLEV), Rift Valley Fever virus (RVFV), Influenza viruses, coronaviruses, rhinovirus, adenovirus, SIV, rotavirus, arbovirus, measles virus, polio virus, rubella virus, mumps virus, papova virus, varicella-zoster virus, varicella virus, huntavirus and cytomegalovirus.

In one embodiment, the antigenic polypeptide comprises or consists of a polypeptide of a yeast or fungus (such as a pathogenic yeast or fungus). In one embodiment, antigenic polypeptide comprises or consists of a polypeptide of Acremonium, Alternaria, Amyiomyces, Arthoderma, Aspergillus, Aureobasidium, Blastochizomyces, Botrytis, Candida, Cladosporium, Crytococcus, Dictyostelium, Emmonsia, Fusarium, Geomyces, Geotrichum, Microsporum, Neurospora, Paecilomyces, Penicillium, Pilaira, Pityrosporum, Rhizopus, Rhodotorula, Saccharomyces, Stachybotrys, Trichophyton, Trichoporon or Yarrowia. In one embodiment, the antigenic polypeptide comprises or consists of a polypeptide of a protozoan (such as a pathogenic protozoan). In one embodiment, antigenic polypeptide comprises or consists of a polypeptide of Plasmodium falciparum, a leishmania sp., a trypanosome sp., or a Cryptosporidium sp.

An immunogenic preparation (eg. immunogenic mixture, or immunogenic complex) of the present invention comprises a (at least one) commensal Neisserial stress protein and an (at least one) antigenic polypeptide. In a preferred embodiment, the immunogenic preparation (eg. immunogenic mixture, or immunogenic complex) comprises more than one commensal Neisserial stress protein and more than one antigenic polypeptide.

As used herein, the phrase "commensal Neisserial stress protein-antigenic polypeptide preparation" or "commensal Neisserial stress protein preparation" refers to an immunogenic preparation of the invention comprising a (at least one) commensal Neisserial stress protein and an (at least one) antigenic polypeptide.

As used herein, the phrase "commensal Neisserial stress protein-antigenic polypeptide mixture" or "commensal Neisserial stress protein mixture" refers to an immunogenic mixture of the invention comprising a (at least one) commensal Neisserial stress protein and an (at least one) antigenic polypeptide.

As used herein, the phrase "commensal Neisserial stress protein-antigenic polypeptide complex" or "commensal Neisserial stress protein complex" refers to an immunogenic complex of the invention comprising a (at least one) commensal Neisserial stress protein and an (at least one) antigenic polypeptide.

In one embodiment, the immunogenic preparation of the invention comprises or consists of an immunogenic complex, in which the commensal Neisserial stress protein is coupled to the antigenic polypeptide. As used herein, the terms "coupled to", "complexed to" and "complexed with" are synonymous. In one embodiment, the term "coupled to" means that the commensal Neisserial stress protein and antigenic polypeptide are bound to each other by one or more covalent bonds, by one or more non-covalent bonds, or by both one or more covalent bonds and one or more non-covalent bonds.

In one embodiment, the stress protein complex of the invention has a molecular weight in the range of 50 to 1000 kDa (eg. from about 100, 200, 300, 400, 500 kDa to about 500, 600, 700, 800 or 900 kDa).

Commensal Neisserial stress proteins are discussed above.

In one embodiment, the immunogenic preparation (eg. immunogenic mixture or immunogenic complex) comprises a commensal Neisserial stress protein that is a member of the Hsp60 family (such as GroEL) or a member of the Hsp70 family (such as DnaK). In one embodiment, the immunogenic preparation (eg. immunogenic mixture or immunogenic complex) comprises a commensal Neisserial stress protein that is an N. lactamica stress protein. Illustrative examples of N. lactamica stress proteins are shown in SEQ ID NOs: 1 and 2 of the present application (and fragments thereof as discussed above).

Antigenic polypeptides are discussed above.

In one embodiment, the antigenic polypeptide comprises a Neisserial antigen. In one embodiment, the antigenic polypeptide comprises a commensal Neisserial antigen. In one embodiment, the antigenic polypeptide does not comprise a commensal Neisserial antigen, (eg. does not comprise any Neisserial antigen) and is heterologous to commensal Neisseria. The immunogenic preparations of the present invention (eg. immunogenic mixtures or immunogenic complexes) are capable of inducing an immune response to the antigenic polypeptide.

The immunogenicity of commensal Neisserial stress protein-antigenic polypeptide preparations (eg. mixture or complexes) results from two different properties of stress proteins.

The first property is the ability of the stress proteins to chaperone antigenic polypeptides associated with them (eg. complexed with them) in the stress protein- antigenic polypeptide preparation (eg. mixture or complex), and to elicit T cell responses against the antigenic polypeptides. In this regard, stress proteins in the preparations (eg. in the stress protein mixtures or complexes) are captured by antigen presenting cells (APCs) such as dendritic cells (DCs) by receptor- mediated endocytosis, or via specific receptors such as toll-like receptors. The antigenic polypeptide is then loaded onto major histocompatibility complex I and II (MHC I and MHC II) molecules on the surface of these cells, for presentation to CD4+ T cells and CD8+ T cells (eg. cytotoxic T cells).

In addition, the stress proteins appear to have an immunomodulatory activity that is independent of the associated antigenic polypeptides. For example, some stress proteins have been shown to induce maturation of dendritic cells and to up- regulate surface expression of the MHC molecules. Stress proteins such as stress proteins of the hsp70 family may also activate natural killer (NK) cells, part of the innate immune system. As such, preparations (eg. mixtures or complexes) of antigenic polypeptides with commensal Neisserial stress proteins target multiple innate and antigen-driven immune pathways.

The invention also provides the use of a cell capable of expressing a commensal Neisserial stress protein and an antigenic polypeptide for generating an immunogenic preparation (such as an immunogenic mixture or complex) comprising said commensal Neisserial stress protein and said antigenic polypeptide.

The invention also provides a method of preparing an immunogenic preparation of the invention (such as an immunogenic mixture or complex), as described above, wherein the method comprises the steps of:

(i) obtaining a cell that has been subjected to a stress-inducing stimulus, wherein said cell expresses or is capable of expressing a commensal Neisserial stress protein and an antigenic polypeptide; and

(ii) recovering preparations (such as mixtures or complexes) comprising said commensal Neisserial stress protein and said antigenic polypeptide from the stress-treated cell.

Suitable commensal Neisserial stress proteins and antigenic polypeptides are discussed above.

In one embodiment of the above use and methods, the cell is a commensal Neisseria. Suitable commensal Neisseria are discussed above (eg. Neisseria lactamica). A commensal Neisseria cell is naturally capable of expressing a commensal Neisserial stress protein and a commensal Neisserial antigenic polypeptide. Thus, in one embodiment, the commensal Neisserial stress protein and the antigenic polypeptide are native to (ie. normally present in or naturally occurring in - eg. naturally encoded by the genome of) the commensal Neisseria cell in which the complex is prepared. Thus, in one embodiment, the stress protein preparation (eg. mixture or complex) is prepared by the method of the invention, in a commensal Neisseria cell, and comprises a commensal Neisserial stress protein and comprises an antigenic polypeptide that comprises or consists of a commensal Neisserial polypeptide, wherein the commensal Neisserial stress protein and antigenic polypeptide are both native to (ie. normally present in or naturally occurring in - eg. naturally encoded by the genome of) the commensal Neisseria cell in which the stress protein preparation is prepared. In a preferred embodiment, a stress protein preparation (eg. mixture or complex) is prepared by the method of the invention wherein the stress-inducing stimuli is heat.

In an alternative embodiment, the immunogenic preparation (eg. mixture or complex) is prepared in a recombinant commensal Neisserial cell that expresses a commensal Neisserial stress protein that is heterologous to (ie. is not native to or not normally present in or not naturally occurring in - eg. not naturally encoded by the genome of) the commensal Neisseria cell in which the preparation is prepared. In accordance with this embodiment, the commensal Neisserial stress protein is native to (ie. is normally present in or naturally occurring in - eg. naturally encoded by the genome of) a different commensal Neisseria, other than the commensal Neisseria in which the preparation is prepared. A stress protein preparation prepared in this recombinant commensal Neisserial cell may comprise commensal Neisserial stress proteins native to the commensal Neisseria and/ or one or more of said heterologous commensal Neisserial stress proteins. Thus, in accordance with this embodiment, the stress protein preparation (eg. mixture or complex) is prepared in a commensal Neisserial cell, but comprises one or more commensal Neisserial stress proteins that are heterologous to (ie. is not native to or not normally present in or not naturally occurring in - eg. not naturally encoded by the genome of) the commensal Neisseria cell in which the stress protein preparation is prepared.

Alternatively, or in addition, the immunogenic preparation (eg. mixture or complex) is prepared in a recombinant commensal Neisserial cell that expresses an antigenic polypeptide that is heterologous to (ie. is not native to or not normally present in or not naturally occurring in - eg. not naturally encoded by the genome of) the commensal Neisseria cell in which the preparation is prepared. A stress protein preparation prepared in this recombinant commensal Neisserial cell may comprise antigenic polypeptides native to the commensal Neisseria and/ or one or more of said heterologous antigenic polypeptides. In accordance with this embodiment, the stress protein preparation (eg. mixture or complex) is prepared in a commensal Neisserial cell, but comprises one or more antigenic polypeptides that is heterologous to (ie. is not native to or not normally present in or not naturally occurring in - eg. not naturally encoded by the genome of) the commensal Neisseria cell in which the stress protein preparation is prepared.

Thus, in one embodiment, the stress protein preparation (eg. mixture or complex) is prepared in a commensal Neisseria cell, but both the commensal Neisserial stress protein and the antigenic polypeptide are heterologous to (ie. not native to or not normally present in or not naturally occurring in - eg. not naturally encoded by the genome of) the commensal Neisseria cell in which the stress protein preparation is prepared. Recombinant commensal Neisseria that express heterologous polypeptides are discussed above.

In an alternative embodiment, the cell in which the immunogenic preparation is prepared is not a commensal Neisserial cell. Suitable host cells for expression of commensal Neisserial stress proteins include Escherichia coii, Lactococcus lactis, Pichia pastoris or in vitro translation expression systems. In accordance with this embodiment, the cell in which the immunogenic preparation (eg. mixture or complex) is prepared is a recombinant host cell other than a commensal Neisserial cell, which has been genetically modified to express a commensal Neisserial stress protein.

Thus, according to this embodiment of the invention, the commensal Neisserial stress protein is heterologous to (ie. not native to/ not normally present in) the host cell in which the stress protein preparation is prepared. A stress protein preparation (eg. mixture or complex) prepared in this recombinant host cell may comprise a stress protein native to the host cell in addition to the recombinantly expressed commensal Neisserial stress protein. Thus, in accordance with this embodiment, the stress protein preparation (eg. mixture or complex) is prepared in a host cell other than a commensal Neisserial cell, and comprises one or more commensal Neisserial stress proteins that are heterologous to (ie. is not native to or not normally present in or not naturally occurring in - eg. not naturally encoded by the genome of) the host cell in which the stress protein preparation is prepared.

A stress protein preparation (eg. mixture or complex) prepared in accordance with this embodiment may comprise an antigenic polypeptide that is native to (ie. normally present in or naturally occurring in - eg. naturally encoded by the genome of) the host cell in which the stress protein preparation is prepared. Alternatively, or in addition, the host cell may recombinantly express an antigenic polypeptide, in which case the stress protein preparation (eg. mixture or complex) may comprise an antigenic polypeptide that is heterologous to (ie. is not native to or not normally present in or not naturally occurring in - eg. not naturally encoded by the genome of) the host cell in which the preparation is prepared.

Thus, in one embodiment, the stress protein preparation (eg. mixture or complex) is prepared in a host cell other than a commensal Neisseria cell, wherein both the commensal Neisserial stress protein and the antigenic polypeptide are heterologous to (ie. not native to or not normally present in or not naturally occurring in - eg. not naturally encoded by the genome of) the host cell in which the stress protein preparation is prepared. In one embodiment, discussed in more detail below, the cell in which the stress protein preparation is prepared (ie. a commensal Neisserial cell or a recombinant host cell other than a commensal Neisserial cell) has been genetically modified to cause the constitutive expression of a commensal Neisserial stress protein gene. Constitutive expression of a commensal Neisserial stress protein gene may be induced, for example, via the inactivation of a repressor gene that suppresses the expression of commensal Neisserial stress proteins or expression of an inducer gene that upregulates expression of commensal Neisserial proteins. Suitable repressor genes encode proteins such as the MisR/S and suitable inducer genes encode proteins such as rpoH and the sigma factors.

In the embodiments described above, commensal Neisserial stress proteins that are "heterologous to" the cell in which the stress protein preparation is prepared may be recombinantly expressed in the cell by the introduction into the cell of a polynucleotide sequence encoding the commensal Neisserial stress protein (operably linked to regulatory control sequence capable of directing expression of the antigenic polypeptide in the cell) - eg. as part of a vector.

In the embodiments described above, antigenic polypeptides that are "heterologous to" the cell in which the stress protein preparation is prepared may be recombinantly expressed in the cell by the introduction into the cell of a polynucleotide sequence encoding the antigenic polypeptide (operably linked to regulatory control sequence capable of directing expression of the antigenic polypeptide in the cell) - eg. as part of a vector.

Thus, in one embodiment, the method of preparing an immunogenic preparation (eg. immunogenic mixture or immunogenic complex) comprises the initial step of recombinantly expressing the commensal Neisserial stress protein and/ or the antigenic polypeptide in the host cell. Said initial step may comprise introducing into the cell one or more nucleotide sequences encoding at least said one or more stress proteins or said one or more antigenic polypeptides. In one embodiment, said nucleotide sequence is a polycistronic sequence encoding multiple stress proteins, or multiple antigenic polypeptides. In one embodiment, said nucleotide sequence is a polycistronic sequence encoding both said stress protein(s) and said antigenic polypeptide(s). Alternatively, said stress protein(s) and said antigenic polypeptide(s) may be encoded by individual recombinant nucleotide sequences.

Said initial step may typically also include the step of culturing the cell under conditions that provide for expression of the stress protein or antigenic polypeptide. The nucleotide sequence may optionally operably linked to non- coding control sequences such as a promoter and/ or enhancer and other control sequences that direct and regulate the transcription and translation of the nucleotide sequence and synthesis of said stress protein or antigenic polypeptide in the cell. The nucleotide sequence(s) may be introduced as part of a vector, in accordance with conventional techniques.

It may be desirable to use regulatory control sequences that allow for inducible expression of the stress protein and/ or antigenic polypeptide. As mentioned above, it may be desirable to use regulatory control sequences that allow for constitutive expression of the stress protein.

A stress inducing stimulus is a stimulus that is capable of inducing a stress response in a cell that is subjected to the stimulus. Suitable stress-inducing stimuli are known in the art. A stress response may result in the induction of expression of a commensal Neisserial stress protein by the stimulated cell. Alternatively, a stress response may result in an increase in the expression of a commensal Neisseria! stress protein by the stimulated cell.

In certain embodiments, the stress-inducing stimulus comprises a (at least one) stimulus selected from the group consisting of, but not limited to: heat shock, osmotic shock (eg. by addition of high concentrations of an electrolyte such as NaCI to the cultivation medium), pH variation (eg. low pH), application of high pressure, cultivation under limited nutrient levels, exposure to heavy metals or oxidising agents, metabolic stress, respiratory stress and genetic modification.

It is an option to subject the cell to multiple stress-inducing stimuli (eg. selected from the group above).

In one embodiment, the stress-inducing stimulus comprises heat shock, also known as 'thermal stress'.

Suitable heat shock conditions are well-known in the art. In one embodiment, a 'heat shock' stimulus comprises subjecting (eg. incubating) a cell that is capable of expressing a commensal Neisserial stress protein at a temperature that is about 5- 20°C higher (preferably about 5, 6 or 7°C higher) than the normal, preferred growth temperature of the cell. By way of example, the cell may be incubated at a temperature in the region of 42-46°C, such as 43-45°C, such as about 44°C.

As mentioned above, in certain other embodiments, the induction of expression of a commensal Neisserial stress protein is achieved by genetic modification of a cell (eg. a commensal Neisseria cell, or a host cell recombinantly expressing a commensal Neisserial stress protein) to cause the constitutive expression of a commensal Neisserial stress protein gene. For example, the genetic modification may be the inactivation of repressor genes that suppress the expression of, or activation of inducer genes that stimulate the expression of, commensal Neisserial stress proteins. Suitable repressor genes include MisR/S and inducer genes include rpoH and the sigma factors. Other suitable genetic modifications are described in WO 02/020045 and citations referred to therein.

In one embodiment, the stress-inducing stimulus is applied to the ceil or induced in the cell at about the same time as induction of expression of the antigenic polypeptide, or shortly after. The optimum conditions for inducing stress proteins in cells of different species and different strains will vary slightly, and can readily be determined by simple trial and error, with the effect of a change of stress stimuli being assessed with regard to levels of stress protein production using conventional techniques. In this regard, the stress-inducing conditions {eg. length of exposure, incubation temperature and time) are not essential features of the invention, and may be varied so long as the stress-inducing stimulus results in the formation of the preparations (eg. mixtures or complexes) of the invention. Suitable stress-inducing stimuli are described in WO 01/013944 and citations referred to therein. The invention also provides a cell that expresses a commensal Neisserial stress protein and an antigenic polypeptide, wherein said cell has been subjected to a stress-inducing stimulus.

Suitable cells that express a commensal Neisserial stress protein and an antigenic polypeptide are discussed above, and suitable stress-inducing stimuli are discussed above.

In one embodiment, the cell is a commensal Neisseria, such as Neisseria lactamica.

In one embodiment, the cell is a recombinant host cell that has been genetically modified to express said commensal Neisseria! stress protein. In one embodiment, the ceil has been genetically modified to constitutively express the commensal Neisserial stress protein (eg. by inactivation of at least one repressor gene that inhibits the expression of said stress protein).

In one embodiment, the antigenic polypeptide is heterologous to said cell. In this embodiment, the cell is a recombinant host cell (eg. a recombinant commensal Neisserial cell or a recombinant host cell that has been genetically modified to express said commensal Neisserial stress protein), wherein said recombinant host cell has been genetically modified to express said antigenic polypeptide.

Once the cell has been subjected to the stress-inducing stimulus, and cultured for a suitable period to allow for protein expression (and optionally to allow complex formation) (such as for about a further 1-24 hours, such as about 2, 6, 12, 18 or 24 hours), the method comprises the step of recovering a preparation comprising said commensal Neisserial stress protein and said antigenic polypeptide from the stress-treated cell.

In one embodiment, the preparation recovered from the stress-treated cell comprises or consists of a mixture of polypeptides, wherein said polypeptide mixture comprises or consists of said commensal Neisserial stress protein and said antigenic polypeptide.

In one embodiment, the preparation recovered from the stress-treated cell comprises or consists of a polypeptide complex, wherein said polypeptide complex comprises or consists of said commensal Neisserial stress protein and said antigenic polypeptide.

Complexes of commensal Neisserial stress protein and said antigenic polypeptide may be found within the cell in which they are prepared. Alternatively, or in addition, complexes of commensal Neisserial stress protein and said antigenic polypeptide may have been secreted from the cell, for example into the cell medium.

Any conventional technique known in the art may be used to recover the stress protein and antigenic polypeptide from the cell.

In this regard, intracellular and/ or membrane complexes may be recovered using conventional techniques using standard cell lysis procedures. For example, the treated cell may be disrupted by homogenisation or ultrasonic fragmentation, followed by centrifugation and/ or filtration to clarify the homogenate and obtain a crude preparation of stress proteins and antigenic polypeptides (eg. in the form of complexes) in the supernatant.

Secreted stress proteins and antigenic polypeptides (eg. in the form of complexes) may also be recovered from the cell medium using standard procedures.

The invention therefore provides a cell lysate or cell homogenate; harvested cell culture fluid, cell culture supernatant or conditioned cell culture supernatant comprising a commensal Neisserial stress protein and an antigenic polypeptide. In one embodiment, the commensal Neisserial stress protein and an antigenic polypeptide are in the form of an immunogenic complex.

The invention also provides an isolated immunogenic preparation, such as an immunogenic mixture or complex prepared in accordance with (eg. obtained by) the above method.

In one embodiment, the method further comprises the step of enriching and/ or purifying the recovered commensal Neisserial stress protein and antigenic polypeptide (eg. enriching and/ or purifying recovered complexes comprising commensal Neisserial stress protein and antigenic polypeptide). For example, it may be desirable to purify the commensal Neisserial stress protein and antigenic polypeptide (eg. complexes thereof) from other host cell proteins, host ceil metabolites, un-complexed stress proteins and antigenic determinants, nucleic acids, endotoxins, chemical product related contaminants, lipids, media additives and media derivatives.

In one embodiment, the enrichment/ purification steps result in reduction of at least about 40 to 75% in cellular contaminants. Any conventional technique known in the art may be used to purify the stress protein and antigenic polypeptide - eg. as described in any of WO 97/10000, WO 97/10001 , WO 97/10002 or US Patent No 6,875,849. In one embodiment, the purification method includes affinity chromatography using ADP-matrix binding. In an alternative embodiment, the purification method includes free-solution isoelectric focusing (FF-IEF).

In a preferred embodiment, the purification method comprises the steps of (i) determining the isoelectric point (pi) of the stress protein and (ii) subjecting the source mixture (eg. cell lysate) comprising the stress protein and antigenic polypeptide to ion exchange, wherein the source mixture is buffered to a pH within 2 units of the pi of the stress protein, and wherein a salt gradient is used to elute the stress protein, as described in PCT/GB2009/051133 (published as WO 2010/026432, incorporated herein by reference) or as described in PCT/GB2010/051023 (incorporated herein by reference).

After ion exchange, in certain embodiments, the purified stress protein and antigenic polypeptide is present within at least one fraction, such as an eluate fraction. In one embodiment, the at least one fraction comprises a purified product or purification product, which fraction may be referred to as a "stress protein- antigenic polypeptide enriched preparation" (SEP). For example, the stress proteins and antigenic polypeptides may be present in the at least one fraction as a mixture. Alternatively, or in addition, the stress proteins and antigenic polypeptides may be present in the at least one fraction in the form of one or more stress protein/antigenic polypeptide complexes. The invention also provides a stress protein-antigenic polypeptide enriched preparation (SEP) prepared in accordance with (eg. obtained by) the above method.

The invention also provides an alternative method of preparing an immunogenic preparation (eg. mixture or complex) of the invention, wherein said method comprises combining a (at least one) commensal Neisserial stress protein and an (at least one) antigenic polypeptide to form a mixture of said commensal Neisserial stress protein and antigenic polypeptide. In one embodiment, said method comprises combining a (at least one) commensal Neisserial stress protein and an (at least one) antigenic polypeptide under conditions that promote formation of a complex between said commensal Neisserial stress protein and said antigenic polypeptide.

In accordance with this embodiment of the invention, antigenic polypeptides may be obtained by recombinant means, by chemical synthesis and/ or from natural sources such as purified or partially purified cell lysates.

In accordance with this embodiment of the invention, commensal Neisserial stress proteins may be obtained by recombinant means, by chemical synthesis and/ or from natural sources such as purified or partially purified cell lysates. The stress proteins may optionally be treated (prior to combining with the antigenic polypeptide) to remove any peptides that may be associated with them.

Suitable conventional treatments for removing unwanted peptides include treatment of the stress proteins with ATP or low pH. Excess ATP may be removed from the preparation by addition of apyranase and, if low pH is used, the pH may be adjusted to neutral pH.

The commensal Neisserial stress protein and antigenic polypeptide may then be combined by mixing and incubating in accordance with standard procedures. The mixing step may take from about 10 minutes to an hour or several hours. It is an option to use a ratio of greater than 1 part antigenic polypeptide to 1 part stress protein.

Optionally, the commensal Neisserial stress protein and antigenic polypeptide are combined until complexes form between the commensal Neisserial stress protein and antigenic polypeptide. The complexes of commensal Neisserial stress protein and antigenic polypeptide may then be recovered from the mixture. Optionally the mixture is enriched for the complexes of the invention. Uncomplexed stress proteins and/ or antigenic polypeptide may be removed by purification, in accordance with standard techniques.

The invention also provides an isolated immunogenic preparation prepared in accordance with (eg. obtained by) the above method. In one embodiment, the isolated immunogenic preparation comprises or consists of an immunogenic mixture or immunogenic complex.

The present invention also provides an immunogenic composition, wherein said composition comprises an immunogenic preparation comprising a commensal Neisserial stress protein and an antigenic protein as described above and a pharmaceutically acceptable carrier.

In one embodiment, the immunogenic preparation is obtained by a method as described above. In one embodiment, the immunogenic composition comprises a cell that expresses said immunogenic preparation. By way of example, the cell may be a cell as described above that expresses a commensal Neisseria! stress protein and an antigenic polypeptide, wherein said cell has been subjected to a stress-inducing stimulus. By way of example, said cell may genetically modified to express said commensal Neisserial stress protein constitutively, as described above. In one embodiment, said cell is a commensal Neisseria. In one embodiment, said cell is a host cell that recombinantly expresses said commensal Neisserial stress protein and/ or said antigenic polypeptide, as discussed above. In one embodiment, said cell is of a non-commensal species, in which case the cell may be attenuated or killed.

The invention also provides a method of preparing an immunogenic composition, comprising combining a pharmaceutically acceptable carrier with an immunogenic preparation comprising a commensal Neisseria stress protein and an antigenic polypeptide as defined above (or with a cell that expresses said immunogenic complex, as discussed above).

Optionally, the method comprises the step of combining the pharmaceutically acceptable carrier and the immunogenic preparation or cell with one or more of a salt, excipient, diluent, adjuvant, immunoregulatory agent and/ or antimicrobial compound.

In one embodiment, the immunogenic composition comprises more than one different immunogenic preparation (eg. complex) of the invention. For example, the immunogenic composition may comprise a mixture of different immunogenic preparations (eg. a mixture of different immunogenic complexes), which differ from each other because they comprise one or more different antigenic polypeptides. The different antigenic polypeptides may be derived from the same or different proteins, and/ or from the same or different strains or species. In one embodiment, the different antigenic polypeptides are different Neisserial polypeptides, such as different commensal and/ or pathogenic Neisserial polypeptides.

Alternatively, or in addition, the different immunogenic preparations in the immunogenic composition could differ from each other because they comprise one or more different commensal Neisserial stress proteins. The different commensal Neisserial stress proteins may be derived from the same or different commensal Neisserial species or strains. Alternatively, or in addition, the different commensal Neisserial stress proteins may be derived from the same or different families of stress proteins (eg. from the hsp60 family and/ or hsp70 family). In one embodiment, the different commensal Neisserial stress proteins are selected from GroEL (such as SEQ ID NO: 1 or fragments thereof as discussed above) and DnaK (such as SEQ ID NO: 2 or fragments thereof as discussed above).

By way of example, the immunogenic composition may comprise a cell (eg. a stress-treated and/ or genetically modified cell as discussed above) wherein said cell expresses multiple (eg. two or more) different immunogenic preparations (eg. complexes) of the invention. Alternatively, the immunogenic composition may comprise multiple (eg. two or more) cells, wherein at least two of said cells express different immunogenic preparations (eg. complexes) of the invention.

The immunogenicity of the immunogenic composition of the invention may be tested using a conventional in vitro assay, such as a mixed lymphocyte target culture assay (MLTA). In one embodiment, the immunogenic composition is a therapeutic or prophylactic formulation, or medicament, such as a vaccine.

As used herein, a "vaccine" is a formulation that, when administered to a subject stimulates a protective immune response against infection, or stimulates or desensitises the immune system in the treatment of a non-infectious medical condition such as an allergy or cancer. The immune response may be a humoral and/ or cell-mediated immune response. As described in more detail below, a vaccine of the invention can be used, for example, to protect a subject from the effects of infection by pathogenic Neisseria (eg. N. meningitidis or N. gonorrhoeae).

Immunogenic compositions, therapeutic formulations, medicaments and prophylactic formulations (eg. vaccines) of the invention comprise a pharmaceutically acceptable carrier, and optionally one or more of a salt, excipient, diluent and/ or adjuvant.

Administration of immunogenic compositions, therapeutic formulations, medicaments and prophylactic formulations (eg. vaccines) of the invention is generally by conventional routes e.g. intravenous, subcutaneous, intraperitoneal, or mucosal routes. The administration may be by parenteral injection, for example, a subcutaneous or intramuscular injection.

Accordingly, the immunogenic compositions, therapeutic formulations, medicaments and prophylactic formulations (eg. vaccines) of the invention are typically prepared as injectabies, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may alternatively be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules.

The active immunogenic ingredients (ie. immunogenic preparation comprising a commensal Neisseria stress protein and an antigenic polypeptide) are often mixed with excipients that pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.

Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. In some embodiments, however, the composition is in lyophilized form, in which case it may include a stabilizer, such as BSA. In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long term storage. The immunogenic composition (eg. vaccine) of the invention may further comprise an adjuvant. Examples of adjuvants which may be effective include but are not limited to: complete Freunds adjuvant (CFA), Incomplete Freunds adjuvant (IVA), Saporin, a purified extract fraction of Saporin such as Quil A, a derivative of Saporin such as QS-21 , lipid particles based on Saponin such as ISCOM/ISCOMATIX, E. coli heat labile toxin (LT) mutants such as LTK63 and/ or LTK72, aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetytmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 '-2'- dipalmitoyl-sn-glycero-3-hydroxyphosphoryl oxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/ Tween 80 emulsion.

Alternatively, the immunogenic composition (eg. vaccine) of the invention may be "substantially free of adjuvant". In this context, "substantially free of adjuvant" means that there is less than 0.05% adjuvant, such as less than 0.025% adjuvant, such as less than 0.001% adjuvant. In one embodiment, the immunogenic composition (eg. vaccine) may be completely free of adjuvant. Examples of buffering agents include, but are not limited to, sodium succinate (pH 6.5), HEPES (pH6.5) and phosphate buffered saline (PBS; pH 6.5 and 7.5). In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments and prophylactic formulations (eg. vaccines) of the invention are administered in a mouthwash or nasal spray.

Oral formulations such as mouthwashes may include conventional excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

Formulations for intranasal administration may in the form of nasal droplets or a nasal spray. An intranasal formulation may comprise droplets having approximate diameters in the range of 100-5000 μm, such as 500-4000 μm, 1000-3000 μιτι or 100-1000 μm. Alternatively, in terms of volume, the droplets may be in the range of about 0.001 -100 μΙ, such as 0.1 -50 μΙ or 1 .0-25 μΙ, or such as 0.001 -1 μΙ.

Alternatively, the immunogenic composition (eg. vaccine), therapeutic/ prophylactic formulation or medicament may be an aerosol formulation. The aerosol formulation may take the form of a powder, suspension or solution. Aerosol particles may be delivered using a nebulizer (eg. via the mouth) or nasal spray. An aerosol formulation may optionally contain a propellant and/ or surfactant. The size of aerosol particles is relevant to the delivery capability of an aerosol. Smaller particles may travel further down the respiratory airway towards the alveoli than would larger particles. In one embodiment, the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution may be selected to target a particular section of the respiratory airway, for example the alveoli. In the case of aerosol delivery of the medicament, the particles may have diameters in the approximate range of 0.1 -50 μm, preferably 1 -25 μm, more preferably 1 -5μm. The immunogenic composition (eg. vaccine) of the invention may contain 5% to 95% of active ingredient (eg. immunogenic complex), such as at least 10% or 25% of active ingredient, or at least 40% of active ingredient or at least 50, 55, 60, 70 or 75% active ingredient.

The immunogenic composition (eg. vaccine), therapeutic/ prophylactic formulation or medicament may be given in a single dose schedule, or in a multiple dose schedule. In a single dose schedule, the full dose is given at substantially one time. A multiple dose schedule is one in which a primary course of vaccination may be with 1 -10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or re-enforce the immune response, for example (for human subjects), at 1-4 months for a second dose, and if needed, a subsequent dose(s) after a further 1 -4 months. The dosage regimen will, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.

In one embodiment, the immunogenic composition (eg. vaccine) of the present invention may be administered as part of a 'prime-boost' vaccination regime.

Prime-boost vaccination regimes involve: Priming - ie. exposing a subject to one or more antigens or a vaccine; and subsequently: Boosting - ie. exposing the subject to one or more antigens or a vaccine. The 'boost' antigens/ vaccine may be different from the 'primer' antigens/ vaccine (known as "heterologous" prime- boost). In this regard, heterologous prime-boost immunization strategies have been shown to induce higher levels of effector T cell responses in subjects as compared with "homologous" boosting with the same vaccine.

Thus, in one embodiment of the invention, the subject's immune system is 'primed' by administration of a heterologous conventional vaccine and then 'boosted' by administration of the vaccine of the present invention. Alternatively, a subject's immune system may be 'primed' by administration of the vaccine of the present invention, and then 'boosted' by administration of a heterologous conventional vaccine. In a further embodiment of the invention, the subject's immune system is 'primed' by previous exposure to a Neisserial strain.

The 'priming' step may be carried out on the subject at any age - in the case of mammalian subjects (eg. human subjects), priming is conventionally carried out neonatally, or during infancy, adolescence or adulthood. The 'boosting' step may be carried out at any time after the 'priming' step. In the case of mammalian subjects (eg. human subjects), a boosting step may be carried out at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks after the priming step, or at least about 3, 6, 8 or 12 months after the priming step, or at least about 2, 5, 10, 15, 20, 25, 30, 35, or 40 or more years after the boosting step. In one embodiment, for a human subject, the priming step is carried out during infancy and the boosting step is carried out during adolescence.

The therapeutic formulation, medicament or prophylactic formulation (eg. a vaccine) is administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/ or therapeutically effective.

In this regard, as used herein, an "effective amount" is a dosage or amount that is sufficient to achieve a desired biological outcome. As used herein, a "therapeutically effective amount" is an amount which is effective, upon single or multiple dose administration to a subject for treating, preventing, curing, delaying, reducing the severity of, ameliorating at least one symptom of a disorder or recurring disorder, or prolonging the survival of the subject beyond that expected in the absence of such treatment.

Accordingly, the quantity of active ingredient to be administered, which is generally in the range of from 0.5 micrograms to 250 micrograms of antigenic polypeptide ml, such as 5-50 micrograms/ml, such as about 15 micrograms/ml, depends on the subject to be treated, capacity of the subject's immune system to generate a protective immune response, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be particular to each subject.

In one embodiment, the therapeutic formulation, medicament or prophylactic formulation (eg. a vaccine) of the invention may comprise one or more further agents. In this embodiment, the one or more further agents are combined with the therapeutic formulation, medicament or prophylactic formulation (eg. a vaccine) of the invention.

In alternative embodiments, said one or more other agents are administered to a subject (or are suitable for administration to a subject) separately from the therapeutic formulation, medicament or prophylactic formulation (eg. a vaccine) comprising the immunogenic preparation (or stress-treated cell) of the invention. Separate administration may be simultaneous or sequential, as discussed below.

Thus, in one embodiment, the immunogenic preparation (or stress-treated cell) of the invention and the one or more other agents are for simultaneous administration. In one embodiment, the methods and uses of the invention comprise simultaneous administration of the immunogenic preparation (or stress- treated cell) of the invention and the one or more other agents.

Simultaneous administration means administration at (substantially) the same time.

In one embodiment of 'simultaneous administration', the immunogenic preparation (or stress-treated cell) of the invention and said one or more other agents are combined into one composition (eg. a single or immunogenic composition of the invention as defined herein). This composition is administered to the subject thereby providing the immunogenic preparation (or stress-treated cell) of the invention and said one or more other agents to the subject simultaneously.

In an alternative embodiment of 'simultaneous administration', said one or more further agents are provided separately from the immunogenic preparation (or stress-treated cell) of the invention, but are administered to the subject at (substantially) the same time as the immunogenic preparation (or stress-treated cell) of the invention. This concurrent/ parallel administration regimen provides said immunogenic preparation (or stress-treated cell) of the invention and said one or more further agents to the subject at (substantially) the same time.

Alternatively, the immunogenic preparation (or stress-treated cell) of the invention and said one or more further agents are for administration to the subject sequentially - ie. (substantially) one after the other. Thus, in one embodiment, the methods and uses of the invention comprise sequential administration of the immunogenic preparation (or stress-treated cell) of the invention and the one or more other agents. In this embodiment, the immunogenic preparation (or stress- treated cell) of the invention and the one or more other agents are provided separately from each other (ie. in separate compositions), and are administered sequentially to the subject. The sequential administration of the immunogenic preparation (or stress-treated cell) of the invention and the one or more other agents provides the immunogenic preparation (or stress-treated cell) of the invention and the one or more other agents to the subject (substantially) one after the other. Thus, in one embodiment, the methods of the invention comprise administration of the immunogenic preparation (or stress-treated cell) of the invention and then administration of the one or more further agents. Alternatively, the one or more other agents may be administered and then the immunogenic preparation (or stress-treated cell) of the invention is administered.

The one or more other agents may comprise a (one or more) stress proteins other than commensal Neisserial stress proteins. For example, the one or more other agents may comprise one or more stress protein preparations (eg. complexes) comprising a stress protein other than a commensal Neisserial stress protein. The non-commensal Neisserial stress protein may be a stress protein of any organism - examples are known in the prior art (eg. WO 00/10597 and WO 01/13944, WO 02/20045 and PCT/GB2009/051133, published as WO 2010/026432, incorporated herein by reference).

Alternatively, or in addition, the one or more other agents may comprise one or more antigenic polypeptides. In one embodiment, the one or more other antigenic polypeptide is not bound (covalently or non-covalently) in a complex with any stress protein.

Alternatively, or in addition, the one or more other agents may be selected from immunoregulatory agents such as, for example, immunoglobulins, cytokines (eg. interleukins, interferons, TNFs and CSFs); and/ or one or more antimicrobial compounds, such as antibiotics or anti-viral compounds, for example one or more conventional anti-Neisserial drugs. Alternatively, or in addition, the one or more other agents may comprise a commensal Neisseria cell.

Alternatively, or in addition, the one or more other agents may be selected from immunogenic components or extracts of a Neisserial cell. The immunogenic components or extracts may be from a commensal Neisserial cell, a pathogenic Neisserial cell such as N. meningitidis or N. gonorrhoeae, or may comprise immunogenic components or extracts from both commensal and pathogenic Neisserial cells. Vaccines comprising a Neisserial immunogenic component or extract are described in WO 00/25811 and WO 00/50074.

Examples of suitable commensal Neisseria are discussed above, and include commensal Neisserial cells that have been genetically engineered to express a heterologous antigen. The commensal Neisseria (or immunogenic component or extract) may be from the same species and/ or strain as that of the commensal Neisserial stress protein. Alternatively, the commensal Neisseria (or immunogenic component or extract) may be from a different species and/ or different strain as that of the commensal Neisserial stress protein.

In one embodiment, the Neisserial immunogenic component or extract comprises a Neisserial outer membrane preparation ("OMP"), such as an outer membrane vesicle ("OMV") preparation.

Outer membrane vesicles (OMVs), also referred to as 'blebs', are discrete vesicles formed or derived from fragments of the outer membrane of a Gram negative bacterium such as Neisseria (eg. pathogenic or commensal Neisseria). OMVs typically comprise outer membrane proteins (OMPs), lipids, phospholipids, periplasms material and lipopolysaccharide (LPS). OMVs have a mean diameter of around 120nm and typically within the range of 80-200nm (such as 90-175nm, 100-150nm or 1 10-130nm). In one embodiment, the Neisserial immunogenic component or extract comprises a pathogenic Neisserial OMV. In one embodiment, the OMVs are enriched with a Neisserial (eg. pathogenic Neisserial) antigenic protein, as described in WO 00/2581 1. Alternatively, or in addition, a Neisserial immunogenic component or extract may comprise a protein fraction of said pathogenic or commensal Neisseria.

Techniques for obtaining an outer membrane preparation or protein fraction from Neisseria are known in the art (see WO 00/25811 or WO 00/50074).

A Neisserial outer membrane preparation (eg. an OMV preparation) or protein fraction can be obtained by culturing pathogenic or commensal Neisseria in the presence or absence of iron.

An antigenic component may be extracted from pathogenic or commensal Neisseria by a method comprising: (i) suspending the Neisseria cells in an aqueous solution of detergent; (ii) incubating the suspension so as to extract the antigenic component from the Neisseria; (iii) centrifuging the suspension to separate the suspension into a supernatant and a pellet; and (iv) fractionating the antigenic component from the supernatant.

This specific method can be modified according to the extraction protocol selected by the user. For example, instead of or as well as using detergent in the initial step (i), high salt concentration may be used. Alternative conventional techniques for obtaining OMVs from pathogenic or commensal Neisseria include the use of variation in salt concentration, treatment with detergents or chaotropic agents, variation in pH (high or low), enzymic digestion, mechanical disruption, deoxycholate extraction, Tris/HCI/EDTA extraction, and lithium acetate extraction. It will be appreciated by the skilled person that virtually any chemical and/ or physical technique is suitable that enables disruption of the bacterial cell outer membrane in order to release sufficient OMVs for purification and isolation.

The immunogenic preparation of the invention (eg. immunogenic mixture or complex of the invention), which comprises a commensal Neisserial stress protein and an antigenic polypeptide, stimulates an immune response in a subject.

Thus, in one embodiment, the invention provides the use of an immunogenic preparation (or stress-treated cell, or immunogenic composition) of the present invention, as described above, for the manufacture of a medicament for stimulating an immune response in a subject. The invention further provides an immunogenic preparation (or stress-treated cell, or immunogenic composition) of the present invention, as described above, for use in stimulating an immune response in a subject. The invention also provides a method of stimulating an immune response in a subject, comprising administering an immunogenic preparation (or stress-treated cell, or immunogenic composition) of the present invention, as described above.

Without being limited to any particular mode of action, it is contemplated that the immune response stimulated by the immunogenic complexes of the present invention may include both humoral and cell-mediated immune responses. A cell- mediated immune response may lead to a T cell cascade, such as a cytotoxic T cell cascade. In one embodiment, immune stimulation is a protective immune response, as measured by a protective effect in an in vivo survival assay. In one embodiment, immune stimulation is measured by an increased frequency in T lymphocytes specific for the antigenic polypeptide (ie. a T cell immune response). In one embodiment, the immune stimulation is a memory T cell immune response, such as a central memory T cell response (eg. a CCR7+ response). In one embodiment, immune stimulation is measured by an increase in antibody titer that is specific for the antigen in the vaccine.

In one embodiment, the immune response is against a pathogenic organism, such as a pathogenic bacterium, virus, fungus, yeast or protozoan.

In one embodiment, the invention provides the use of an immunogenic preparation (eg. immunogenic mixture or immunogenic complex) (or stress-treated cell, or immunogenic composition) as described above, for the manufacture of a medicament for treating or preventing an infection in a subject. The invention further provides an immunogenic preparation (eg. immunogenic mixture or immunogenic complex) (or stress-treated cell, or immunogenic composition), as described above, for use in treating or preventing an infection in a subject.

The invention also provides a method of treating or preventing an infection in a subject, comprising administering an immunogenic preparation (eg. immunogenic mixture or immunogenic complex) (or stress-treated cell, or immunogenic composition) of the present invention, as described above.

The term "infection" includes the proliferation of a pathogenic organism within and/ or on the tissues of a host organism.

The term "preventing an infection" includes preventing the initiation of an infection and/ or reducing the severity or intensity of an infection. The term "treating an infection" embraces therapeutic or preventative/ prophylactic measures, and includes post-infection therapy and amelioration of an infection.

Infections and diseases that can be treated or prevented using an immunogenic preparation (or immunogenic composition) of the invention include bacterial infections, viral infections, fungal or yeast infections, or protozoan infections.

In one embodiment, the infection is a bacterial infection, such as a bacterial infection caused by gram positive or gram negative bacteria, such as a bacterial infection caused by Acinetobacter, Actinobacillus, Actinomycetes, Aeromonas, Bacillus, Bordetella, Borrelia, Branhamella, Brucella, Calymmatobacterium, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterobacter, Erwinia, Erysipelothrix, Escherichia, Fanciselia, Haemophilus, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Pasturella, Pseudomonas, Proteus, Rickettsia, Salmonella, Shigella, Spirillum, Staphylococcus, Streptobacillus, Streptococcus, Streptomycetes, Treponema, Vibrio or Yersinia. In one embodiment, the infection is a Neisserial infection or Neisserial disease. In one embodiment, the infection is caused by Neisseria meningitidis. In one embodiment, the disease is meningitis. In one embodiment, the infection is caused by Neisseria gonorrhoeae. In one embodiment, the disease is gonorrhoea.

In one embodiment, the infection is a viral infection, such as an infection caused by rabies virus, herpes simplex virus, Epstein-Barr virus, vesicular stomatitis virus, vaccinia virus, Human immunodeficiency virus (HIV), Hepatitis A virus (HAV), hepatitis B (HBV), hepatitis C (HCV), human papillomavirus (HPV), Kaposi's Sarcoma-Associated Herpesvirus (KSHV), Respiratory Syncytial Virus, Ebola virus, Marburg virus, West Nile virus (WNV), St Louis Encephalitis virus (SLEV), Rift Valley Fever virus (RVFV), Influenza viruses, coronaviruses, rhinovirus, adenovirus, SIV, rotavirus, arbovirus, measles virus, polio virus, rubella virus, mumps virus, papova virus, varicella-zoster virus, varicella virus, huntavirus and cytomegalovirus.

In one embodiment, the infection is a yeast or fungal infection, such as an infection caused by Acremonium, Alternaria, Amylomyces, Arthoderma, Aspergillus, Aureobasidium, Blastochizomyces, Botrytis, Candida, Cladosporium, Crytococcus, Dictyostelium, Emmonsia, Fusarium, Geomyces, Geotrichum, Microsporum, Neurospora, Paecilomyces, Penicillium, Pilaira, Pityrosporum, Rhizopus, Rhodotorula, Saccharomyces, Stachybotrys, Trichophyton, Trichoporon or Yarrowia.

In one embodiment, the infection is a protozoan infection, such as an infection caused by Plasmodium falciparum, a leishmania sp., a trypanosome sp., or a Cryptosporidium sp.

In one embodiment, the immunogenic preparation or composition of the invention stimulates or desensitizes a subject's immune system to antigens associated with non-infectious medical conditions, such as allergies (eg. nut allergies) or cancers.

Thus, in one embodiment, the invention provides the use of an immunogenic preparation (eg. immunogenic mixture or immunogenic complex) (or stress-treated cell, or immunogenic composition) of the present invention, as described above, for the manufacture of a medicament for stimulating or desensitizing the immune system in a subject. The invention also provides an immunogenic preparation (eg. immunogenic mixture or immunogenic complex) (or stress-treated cell, or immunogenic composition) of the present invention, as described above, for use in stimulating or desensitizing the immune system in a subject.

The invention also provides a method of stimulating or desensitizing the immune system in a subject, comprising administering an immunogenic preparation (eg. immunogenic mixture or immunogenic complex) (or stress-treated cell, or immunogenic composition) of the present invention, as described above.

As used herein, the "efficacy" of a therapeutic/ prophylactic composition or medicament (eg. a vaccine) describes the ability of the therapeutic/ prophylactic composition or medicament to protect a subject (typically a mammalian subject eg. a human, bovine, porcine or equine subject) from challenge with a pathogen, or from a non-infectious medical condition. By way of example, "vaccine efficacy" may refer to the efficacy of a vaccine in preventing the initiation of an infection or non-infectious medical condition and/ or reducing the severity/ intensity of an infection or non-infectious medical condition.

A therapeutic/ prophylactic composition or medicament (eg. vaccine) of the invention may be administered to a subject already having an infection, or a condition or symptoms associated with an infection, to treat or prevent said infection. In one embodiment, the subject is suspected of having come in contact with an infectious organism, or has had known contact with an infectious organism, but is not yet showing symptoms of exposure.

A therapeutic/ prophylactic composition or medicament (eg. vaccine) of the invention may be administered to a subject already having a non-infectious medical condition, or a condition or symptoms associated with a non-infectious medical condition, to treat or prevent said non-infectious medical condition. In one embodiment, the subject is suspected of having come in contact with an allergen, or has had known contact with an allergen, but is not yet showing symptoms of exposure.

When administered to a subject that already has an infection or a non-infectious medical condition, or is showing symptoms associated with an infection or noninfectious medical condition, the therapeutic composition/ medicament (eg. vaccine) of the invention may cure, delay, reduce the severity of, or ameliorate one or more symptoms, and/ or prolong the survival of a subject beyond that expected in the absence of such treatment.

Alternatively, a therapeutic/ prophylactic composition or medicament (eg vaccine) of the invention may be administered to a subject who ultimately may acquire an infection or a non-infectious medical condition, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of said infection or non-infectious medical condition, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. In one embodiment, the subject has previously been exposed to an allergen or pathogen. For example, the subject may have had an infection in the past (but is optionally not currently infected). The subject may be latently infected with a pathogen. Alternatively, or in addition, the subject may have been vaccinated against the allergen or pathogen in the past. In one embodiment, the subject has been pre-exposed to a heterologous conventional vaccine against the allergen or pathogen in the past (eg. the subject's immune system has been 'primed'). In the context of the therapeutic uses and methods of the invention, a 'subject' is any animal subject that would benefit from stimulation of an immune response. Typical animal subjects are mammals, for example, human, bovine, porcine, ovine, caprine, equine, corvine, canine or feline subjects. In one embodiment, the subject is human, bovine, porcine or equine. Some pathogenic organisms such as pathogenic Neisseria are obligate human pathogens, Thus, in the context of these obligate human pathogens, the 'subject' is a human. The treatments and preventative therapies of the present invention are applicable to a variety of different subjects of different ages. In the context of humans, the therapies are applicable to children (eg. infants, children under 5 years old, older children or teenagers) and adults. In the context of other animal subjects, the therapies are applicable to immature subjects and mature/ adult subjects. The treatments and preventative therapies of the present invention are applicable to subjects who are immunocompromised or immunosuppressed (eg. human patients who have HIV or AIDS, or other animal patients with comparable immunodeficiency diseases), subjects who have undergone an organ transplant, bone marrow transplant, or who have genetic immunodeficiencies.

A 'neutralisation test' examines the capability of antisera raised to a specific antigen to neutralise (ie. inhibit or prevent) a particular biological process associated with the functionality of the antigen. In general terms, antisera are raised to the antigen in an appropriate animal model (for example, mice, guinea pigs, rabbits, goats, sheep, horse) using the immunisation protocol that is appropriate to the host. For example, a standard immunisation protocol for guinea pigs might be immunisation with 0.5 nmoles antigen on day 1 , followed by two further 0.5 nmole inoculums over an eight-week period. Two weeks after the final dose, the sera would be obtained from the animals and used for neutralisation test. The neutralisation test is performed by mixing the sera with the active antigen, or a protein containing the antigen, in vitro prior to assessment of the functionality of the antigen in an appropriate test.

A 'challenge test' examines the ability of the antigen to raise a sufficient host response in order to neutralise (ie. inhibit or prevent) the pathogenesis of the agent from which the antigen is derived. In general terms, susceptible animals are inoculated with appropriate doses of the test antigen over an appropriate time period. For example, a standard immunisation protocol for botulinum neurotoxin in mice would be initial immunisation with 5μg antigen on day 1 , followed by 5μg antigen on day 14 and 5μg antigen on day 28.

On completion of the immunisation schedule, the animals would be challenged with a test dose of the test agent and observed for susceptibility to the agent. An antigen that demonstrated potential as a vaccine candidate would protect the animals from succumbing to the effects of the agent.

The challenge test therefore differs conceptually from the neutralisation test. The neutralisation test assesses the ability of anti-antigen sera to inactivate an agent in vitro. The challenge test assesses the ability of an antigen to raise a host response to the test agent - ie. the challenge test assesses in vivo efficacy.

As used herein, the terms "nucleic acid sequence", "nucleotide sequence" and "polynucleotide" are used interchangeably and do not imply any length restriction. As used herein, the terms "nucleic acid" and "nucleotide" are used interchangeably.

The term "recombinant" as used herein intends a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation: (1 ) is not associated with all or a portion of a polynucleotide with which it is associated in nature; or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) does not occur in nature. This artificial combination is often accomplished by via conventional chemical synthesis techniques, or by the artificial manipulation of isolated segments of nucleic acids - eg. by conventional genetic engineering techniques.

When applied to a nucleic acid sequence, the term "isolated" denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment. Methods for isolating nucleic acid sequences are known in the art.

Polynucleotide sequences include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.

Polynucleotides may be prepared by any means known in the art. For example, large amounts of polynucleotides may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines. Polynucleotides may also be produced by chemical synthesis, eg. by the phosphoramidite method or the triester method, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

A nucleic acid sequence can be obtained by conventional cloning procedures, such as PCR, or can be synthesized using nucleic acid synthesis machines. An alternative conventional way to prepare a full-length polynucleotide is to synthesize a specified set of overlapping oligonucleotides (eg. 40 to 100 nucleotides). Other sequences may be added that contain signals for proper initiation and termination of transcription and translation.

In view of the degeneracy of the genetic code, considerable sequence variation is possible among polynucleotides. One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, some polynucleotides encompassed by degenerate sequences may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the present invention. A nucleic acid sequence is substantially homologous (or substantially similar) to another (eg. a reference nucleic acid sequence) if, when optimally aligned (with appropriate nucleotide insertions, deletions, and/ or substitutions) with the reference nucleic acid sequence (or its complementary strand) using conventional techniques, there is nucleotide sequence identity in at least 80, 82, 84, 86, 88, 92, 94, 96, 98 or 99% of the nucleotide bases. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences may be compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Alternatively, a nucleic acid sequence is substantially homologous (or substantially similar) to a corresponding naturally-occurring sequence when the two molecules are capable of hybridizing under selective hybridization conditions. Selectivity of hybridization exists when hybridization occurs which is substantially more selective than total lack of specificity. Typically, selective hybridization will occur when, over a stretch of at least about 50 nucleotides, there is at least 80, 82, 84, 86, 88, 92, 94, 96, 98, 99 or 100% nucleotide sequence identity. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about 100 nucleotides, usually at least about 200, 500 or 1000 nucleotides. Nucleic acid hybridization will be affected by such conditions as salt concentration (eg. NaCI), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30°C, typically in excess of 37°C and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. However, the combination of parameters is much more important than the measure of any single parameter.

One of ordinary skill in the art appreciates that different species exhibit "preferential codon usage". As used herein, the term "preferential codon usage" refers to codons that are most frequently used in cells of a certain species, thus favouring one or a few representatives of the possible codons encoding each amino acid. For example, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells ACC is the most commonly used codon; in other species, different Thr codons may be preferential. Preferential codons for a particular host cell species can be -introduced into the polynucleotides of the present invention by a variety of methods known in the art, Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species.

Thus, in one embodiment of the invention, a nucleic acid sequence is codon optimized for expression in a host cell.

A "fragment" of a polynucleotide of interest comprises a series of consecutive amino acid residues from the sequence of said full-length polynucleotide. By way of example, a "fragment" of a polynucleotide of interest may comprise (or consist of) at least 150 consecutive nucleic acid residues from the sequence of said polynucleotide (eg. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 consecutive nucleic acid residues of said polynucleotide). A fragment may include at least one antigenic determinant and/ or may encode at least one antigenic epitope of the corresponding polypeptide of interest.

A polynucleotide of interest, or variant or fragment thereof, may encode a polypeptide that has the ability to induce a "recall response" of a T-lymphocyte (eg. CD4+, CD8+, effector T cell or memory T cell such as a TEM or TCM), which has been previously exposed to an antigenic component of a pathogen or allergen.

Alternatively, or in addition, an antibody capable of binding to a polypeptide encoded by the polynucleotide of interest, or fragment or variant, may be also capable of binding to the pathogen or allergen.

The term "polypeptide" throughout this specification is synonymous with the terms "oligopeptide", "peptide" and "protein". These terms are used interchangeably and do not refer to a specific length of the product. These terms embrace post- translational modifications such as glycosylation, acetylation and phosphorylation.

As used herein, an 'isolated' polypeptide is substantially free from other proteins with which it is co-produced as well as from other contaminants. For instance, an isolated polypeptide is substantially free of material or other proteins from the cell, bacterial, or tissue source from which it was derived.

As used herein, a "purified" molecule is substantially free of its original environment and is sufficiently pure for use in pharmaceutical compositions. A substantially pure polypeptide, as used herein, refers to a polypeptide that is at least about 50% (w/w) pure; or at least about 60%, 70%, 80%, 85%, 90% or 95% (w/w) pure; or at least about 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure. Polypeptides of the present invention (and mixtures or complexes thereof) may be purified from commensal Neisseria, or may be purified from other cell-types that express these polypeptides or complexes (eg. because they are transformed with recombinant nucleic acids encoding these polypeptides). An expressed polypeptide may be purified by, for instance, a combination of hydrophobic interaction chromatography, ion exchange chromatography and ceramic hydroxyl apatite chromatography. Other chromatographic techniques well known to the art of protein purification, such size exclusion chromatography, may be used. Polypeptide purity or homogeneity may be indicated by, for example, polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel, or using HPLC. Polypeptides may be soluble or predominantly soluble (for instance, at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or even 99% soluble).

A polypeptide is substantially homologous to a reference polypeptide (such as any one of the reference SEQ ID NOs identified in this application, including fragments thereof) if it comprises or consists of an amino acid sequence having at least 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% amino acid sequence identity with the amino acid sequence of said reference polypeptide. As used herein, the terms "sequence identity" and "sequence homology" are considered synonymous.

There are many established algorithms available to align two amino acid sequences. Typically, one sequence acts as a reference sequence, to which test sequences may be compared. The sequence comparison algorithm calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alignment of amino acid sequences for comparison may be conducted, for example, by computer implemented algorithms (eg. GAP, BESTFIT, FASTA or TFASTA), or BLAST and BLAST 2.0 algorithms. The percent identity may be calculated as:

In a homology comparison, the identity may exist over a region of the sequences that is at least 50 amino acid residues in length (eg. at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 amino acid residues in length - eg. up to the entire length of the reference sequence). Substantially homologous polypeptides have one or more amino acid substitutions, deletions, or additions. In many embodiments, those changes are of a minor nature, for example, involving only conservative amino acid substitutions. Conservative substitutions are those made by replacing one amino acid with another amino acid within the following groups: Basic: arginine, lysine, histidine; Acidic: glutamic acid, aspartic acid; Polar: glutamine, asparagine; Hydrophobic: leucine, isoleucine, valine; Aromatic: phenylalanine, tryptophan, tyrosine; Small: glycine, alanine, serine, threonine, methionine. Substantially homologous polypeptides also encompass those comprising other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide, or an affinity tag.

Polypeptides described herein may comprise non-naturally occurring amino acid residues. In this regard, in addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-W-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine) may be substituted for amino acid residues of the polypeptides described herein. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues of the polypeptides described herein. Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro- glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2- azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4- fluorophenylalanine. Essential amino acids, such as those in the polypeptides described herein, can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. The identities of essential amino acids can also be inferred from analysis of homologies with related family members of the polypeptide of interest.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening. Methods are known for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display. Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a polypeptide of the invention. As an illustration, DNA molecules can be digested with Bal31 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for the desired activity. An alternative to exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions, or stop codons to specify production of a desired fragment. Alternatively, particular polynucleotide fragments can be synthesized using the polymerase chain reaction. A polypeptide sequence may contain one or more analogs of an amino acid {eg. an unnatural amino acid), or a substituted linkage, as compared with the sequence of a reference polypeptide. In a further embodiment, a polypeptide may be a mimic of the reference polypeptide, which mimic reproduces at least one epitope of the reference polypeptide. Mutants of the polypeptide sequences described herein can be generated through DNA shuffling. Briefly, mutant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.

Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned mutant polypeptides. Mutagenized nucleic acid molecules that encode polypeptides of the invention, or fragments thereof, can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

A "fragment" of a polypeptide of interest comprises a series of consecutive amino acid residues from the sequence of said polypeptide. By way of example, a "fragment" of a polypeptide of interest may comprise (or consist of) at least 50 consecutive amino acid residues from the sequence of said polypeptide (eg. at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 500 to 600 consecutive amino acid residues of said polypeptide). A fragment may include at least one epitope of the polypeptide of interest. A polypeptide or fragment thereof may possess the active site of a reference polypeptide.

A polypeptide or fragment thereof may have a common antigenic cross-reactivity and/or substantially the same in vivo biological activity as a reference peptide. For example, the polypeptides, or polypeptide fragments, and reference polypeptides share a common ability to induce a "recall response" of a T-lymphocyte (eg. CD4+, CD8+, effector T cell or memory T cell such as a TEM or TCM), which has been previously exposed to an antigenic component of a pathogen.

Alternatively, or in addition, an antibody capable of binding to a polypeptide of interest, or fragment, may be also capable of binding to the reference peptide.

SEQ ID NOs described herein:

SEQ ID NO: 1 = GroEL Protein sequence (N. lactamica Y92-1009)

MAAKDVQFGNEVRQKMVNGVNILANAVRVTLGPKGRNVVVDRAFGGPHITKDGVTVAKEIE LKDKFENMGAQMVKEVASKTNDVAGDGTTTATVLAQSIVAEGMKYVTAGMNPTDLKRGIDK AVAALVEELKNIAKPCDTSKEIAQVGSISANSDEQVGAIIAEAMEKVGKEGVITVEDGKSL ENELDWEGMQFDRGYLSPYFINDAEKQIAALDNPFVLLFDKKISNIRDLLPVLEQVAKAS RPLLIIAEDVEGEALATLVVNNIRGILKTVAVKAPGFGDRRKAMLQDIAILTGGTVIAEEV GLSLEKATLDDLGQAKRIEIGKENTTIIDGFGDAAQIEARVAEIRQQIETATSDYDKEKLQ ERVAKLAGGVAVIKVGAATEVEMKEKKDRVEDALHATRAAVEEGWAGGGVALLRARAALE NLHTGNADQDAGVQIVLRAVESPLRQIVANAGGEPSVVNKVLEGKGNYGYNAGSGEYGDM IEMGVLDPAKVTRSALQHAASIAGLMLTTDCMIAEIPEEKPAMPDMGGMGGMGGMM

SEQ ID NO: 2 = DnaK Protein sequence (N. lactamica Y92-1009)

MAKVIGIDLGTTNSCLAISENGQTKVIENAEGARTTPSVIAYLDGGEILVGAPAKRQAVTN AKNTIYAAKRLIGHKFEDKEVQRDIESMPFEIIKANNGDAWVKAQGKELSPPQISAEVLRK MKEAAEAYLGEKVTEAVITVPAYFNDSQRQATKDAGRIAGLDVKRIINEPTAAALAFGMDK GDNKDRKVAVYDLGGGTFDISIIEIANLDGDKQFEVLATNGDTFLGGEDFDQRLIDYIIDE FKKEQGIDLKQDVMALQRLKEAAEKAKIELSSGQQTEINLPYITMDATGPKHLAMKITRAK FESLVEDLIARSIEPCRTALKDAGLSTGDIDDVILVGGQSRMPKVQEAVRDFFGKEPRKDV NPDEAVAVGAAIQGEVLSGGRSDVLLLDVTPLSLGIETMGGVMTKLIQKNTTIPTKASQVF STAEDNQSAVTIHVLQGERERASANKSLGQFNLGDIAPAPRGVPQIEVTFDIDANGILHVS AKDKGTGKAANITIQGSSGLSEEEIERMVKDAEANAEEDKKLTELVASRNQAEALIHSVKK SLADYGDKLDAAEKEKIEAALKEAEEAVKGDDKAAIDAKAEALGAASQKLGEIVYAQAQAE AQAGEGAQADAS SQKDDDWDADFTEVKDDKK

Discussion of the Figures

Aspects and embodiments of the invention are illustrated by the Examples provided below, and with reference to the following Figures, in which:

Figure 1 illustrates Western blots of stress protein-enriched vaccines from N. meningitidis (A) and N. lactamica (B) prepared in Example 1. The preparations have been probed with antisera specific for Hsp60 or Hsp70. The presence of Hsp60 and Hsp70 in both preparations is confirmed.

Figure 2 illustrates the protective immunogenicity of the stress protein-enriched vaccines of the invention comprising commensal Neisserial stress proteins (annotated as NL HspC), as compared with a stress protein-enriched vaccine comprising N. meningitidis stress proteins (annotated as MC58 HspC), N. meningitidis OMVs (annotated as MC58 OMVs) and buffer only (as a negative control) in a mouse lethal challenge model.

Panel A of Figure 2 demonstrates that mice immunized with a stress protein- enriched preparation from N. lactamica (NL HspC) provide complete protection from meningococcal challenge, with all the mice surviving (10 out of 10 mice surviving). This is in contrast to the HspC vaccine prepared from N. meningitidis (MC58 HspC), where only half the mice survive (5 out of 10). The protection is also compared to an OMV vaccine prepared from N. meningitidis strain MC58 which has a homologous PorA protein to the challenge strain (see MC58 OMVs), and would thus be expected to provide strong protection. However, only 6 out of the 10 mice survived following immunisation with the MC58 OMV vaccine. Three out of 10 mice survived when immunized with buffer alone. Hence, panel A of Figure 2 confirms that a N. lactamica HspC vaccine of the invention unexpectedly provided greater protection than both (i) a N. meningitidis MC58 HspC vaccine and (ii) a N. meningitidis MC58 OMV vaccine.

Panel B of Figure 2 illustrates the health of the mice during the experiment. Each healthy mouse started with a health score of 5, and points were then deducted for stages of meningococcal disease until death, which is given a score of 0. It can be seen that the mice immunized with the N. lactamica HspC vaccine (NL HspC) remain in considerably better health than those mice given either the meningococcal HspC vaccine (MC58 HspC) or the meningococcal OMV vaccine (MC58 OMVs). All mice given the N. lactamica HspC vaccine are completely healthy at 60 hours post challenge.

Thus the results in panels A and B of Figure 2 clearly illustrate the advantages of the N. lactamica HspC vaccine. Figure 3 shows the results of an immunogenicity study, which measured the ability of antibodies raised in mice against buffer only, meningococcal OMVs (MC58 OMVs), meningococcal HspC vaccine (MC58 HspC), N. lactamica HspC vaccine or a homologous strain control meningococcal OMV vaccine prepared from each test organism to mediate opsonophagocytosis against a panel of 7 diverse meningococcal strains.

It can be seen from Figure 3 that the N. lactamica HspC vaccine mediated greater opsonophagocytosis than the MC58 HspC vaccine for all 7 strains - see panels (A)-(G). For several of the strains, the N. lactamica HspC vaccine induced opsonophagocytosis at a level approaching or equivalent to the homologous strain OMV vaccine, which is expected to provide a strong response. As opsonophagocytosis is believed to be involved in protection and recovery from meningococcal disease, these results indicate that the N. lactamica HspC vaccine unexpectedly elicits a greater cross-strain protective response than that of either the N, meningitidis MC58 HspC vaccine or N. meningitidis MC58 OMV vaccine. Figure 4 shows the results of an immunogenicity study, in which sera raised against the vaccines described in Figure 3 were assessed for their ability to elicit antibody responses that mediate the binding of complement components C3b or iC3b onto the surface of a panel of 7 meningococcal strains. The deposition of these complement components is important for opsonophagocytosis and thus the data presented in panels (A)-(G) of Figure 4 provide additional evidence of the cross-strain opsonophagocytosis mediated by the N. lactamica HspC vaccine, which is greater than that mediated by the MC58 HspC vaccine or OMV vaccine. Figure 5 shows the results of an immunogenicity study, in which sera raised against the vaccines described in Figure 3 were assessed for their ability to elicit antibody responses that mediate the binding of complement components C5b-9 or membrane attack complex (MAC) onto the surface of a panel of 7 meningococcal strains. Antibody mediated MAC deposition is the cause of serum bactericidal activity, which is acknowledged as a correlate of protection for meningococcal disease vaccines. As illustrated in panels (A)-(G) of Figure 5, the N. lactamica HspC vaccine response was more effective at C5b-9 deposition than the N. meningitidis HspC vaccine, for all strains. For strains M01 240101 and NZ98/254, the C5b-9 deposition in response to the N. lactamica HspC vaccine was greater than in response to the homologous strain OMV vaccine.

These results again demonstrate the greater vaccine potential of the HspC vaccine of the present invention, which comprises commensal Neisserial stress proteins.

Figure 6 compares the results obtained in the C5b-9 deposition study with the result obtained in a Serum Bactericidal Assay using meningococcal strain 44/76- SL. Antibody mediated C5b-9/ MAC deposition is the cause of serum bactericidal activity, which is acknowledged as a correlate of protection for meningococcal disease vaccines. Figure 7 shows the results of a Serum Bactericidal Assay, in which sera raised against the vaccines described in Figure 3 were assessed for their bactericidal activity against a panel of 2 meningococcal strains. The values shown in the "44/76-SL" column were obtained in duplicate assays. As illustrated in Figure 7, for both strains, the sera raised against the N. lactamica HspC vaccine demonstrated more effective bactericidal activity than the sera raised against the N. meningitidis HspC vaccine.

EXAMPLES

Example 1 : Preparation of Neisserial stress protein preparations

Cultures of an acapsulate variant of Neisseria meningitidis strain MC58 (Mol Microbiol. 1995, 18(4): 741-54) or Neisseria lactamica (Infect. Imm. 2002, 70(7): 3621-26) were heat shocked at 44°C.

The cells were then killed by heat treatment at 56°C for 30 mins, and processed to recover stress protein-enriched preparations, as described in PCT/GB2009/051133 (published as WO 2010/026432, incorporated herein by reference) and as described in PCT/GB2010/051023 (incorporated herein by reference).

In brief, cells were resuspended in 50mM HEPES, pH6.8 and lysed by cycles of sonication and disruption in an Emulsiflex C5 homogeniser (Avestin Inc.). The cellular homogenate was clarified by centrifugation for 45min at 13,000g and the clarified extract filtered sequentially through 0.8, 0.45 and 0.2pm membrane filters before loaded onto a 5ml HiTrap Capto Q column (GE Healthcare). After extensive washing of the column with 50mM HEPES buffer, pH 6.8, protein fractions were batch eluted using increasing concentrations of NaCI (150mM, 350mM, 500mM). Eluted fractions containing Hsp70 and Hsp60 were analysed by gel electrophoresis and Western blotting and the stress protein complex fractions were eluted by 350 mM NaCi, buffer exchanged into saline and characterised using SDS-PAGE and Western blotting.

Typical results obtained for the stress protein-enriched vaccines from N. meningitidis (A) and N. lactamica (B) are shown in Figure 1.

Example 2: In vitro reconstitution of stress protein-polypeptide preparations

Purified, ATP-treated commensal Neisserial GroEL and/or DnaK (9mg) is mixed with the antigenic polypeptide of interest (1 mg) and incubated for 3 hours at room temperature in a binding buffer containing 20mM sodium phosphate, pH 7.2, 350mM NaCI, 3mM MgCI₂, 1 mM PMSF. The resulting preparation is centrifuged through Centricon 10 assembly (Millipore) to remove unbound polypeptide.

Example 3: Animal vaccination and challenge method

Groups of 30 NIH mice (six to eight weeks old, Harlan, UK) were immunized with either:

> 10 μg of Neisseria lactamica HspC {in 50mM HEPES 150mM NaCI pH 6.8);

> 10 μg of Neisseria meningitidis MC58 Cap-HspC (in 50mM HEPES 150mM NaCI pH 6.8);

> 10 μg of N. meningitidis MC58 OMVs (in 50mM HEPES 150mM NaCI pH 6.8); or

> 50mM HEPES 150mM NaCI pH 6.8 only.

This took place on days 0, 21 and 28 by subcutaneous injection (0.2 ml). On Day 35, terminal sera were collected from 10 mice.

10 mice per group were challenged with 1x10⁷ CFU or 1x10⁸ CFU clinical meningococcal strains, including N. meningitidis strain 44/76-SL.

The challenge dose was made as follows. A frozen stock of N. meningitidis stored at -70°C in Frantz medium containing 30% (v/v) glycerol was thawed and spread onto blood agar to produce a lawn of growth. Plates were incubated overnight at 37°C with 5% C0₂, used to inoculate 10ml Frantz media with EDDHA for iron restriction, which were incubated at 37°C shaking for 4hr. After four hours, a 1 :10 dilution of the culture was prepared and the absorbance measured at 600nm. Using this absorbance measurement, the amount of culture needed to give the required challenge dose was calculated.

Total inoculum required (ml) x 0.1 = 2 x 10⁸ cfu/ ml of neat culture,

Α600nm Each mouse received 0.5ml of the final volume of challenge inoculum at the appropriate dilution. A serial dilution was plated out on blood agar in duplicate and incubated overnight. The following day, colonies were counted to calculate the exact dose given to the mice. Human transferrin was then added to a final 10mg per dose. The challenge dose was injected interperitoneally (IP) into each mouse. After 24 hours a further 10 mg transferrin dose was given IP to each surviving mouse.

Following challenge, animals were monitored every four hours over a four day period for sickness. The symptoms recorded for each mouse were translated into a health score. A healthy mouse was given a score of 5, ruffled fur or eyes shut was given 4, ruffled and eyes shut was given 2, immobile mice were given a score of 1 , and any dead mice were given a score of 0.

The results are illustrated in Figure 2.

Example 4: Opsonophagocytosis Assay (OPA)

The granulocytic cell line HL60 was used as a reproducible source of phagocytic cells. Azide-killed meningococci labelled internally with the fluorescent dye BCECF were used as the target bacteria; IgG-depleted human plasma was used as the complement source and a single-point determination of OP activity was made at a serum dilution of 1 :20; all assays were performed in duplicate. Flow cytometry was used to determine the percentage of HL60 cells taking up the labelled target meningococci and the intensity of fluorescence uptake; the data were expressed as the signal of test antibody minus the signal from the no antibody, complement-only control (Fl-C)-

The results are illustrated in Figure 3. Example 5: C3c and C5b-9 Deposition Assay

Azide-killed meningococci were incubated with test antibody and IgG-depleted human plasma for 30mins at room temperature, washed twice, and sheep-anti- human C3c (FITC) and mouse-anti-human C5b-9 (AlexaFluor 647) used to measure deposition by incubation for 20min at 40C. Flow cytometry was used to detect the percentage of meningococci showing fluorescence (complement binding) and the intensity of that fluorescence (level of complement). The data were expressed as the signal of test antibody minus the no-antibody, complement- only control (Fl-C').

The results are illustrated in Figures 4 and 5. Antibody-mediated complement C3c deposition has been shown to correlate with opsonophagocytosis, and it is also now accepted that opsonophagocytosis is important for protection from meningococcal disease (see Granoff DM. "Relative importance of complement-mediated bactericidal and opsonic activity for protection against meningococcal disease"; Vaccine, 2009, Jun 24; 27 Suppl. 2: B117-25).

Complement C5-9b is the membrane attack complex that is responsible to antibody and complement-mediated killing of N. meningitidis. We have shown that C5b-9 deposition correlates well with serum bactericidal activity measured in a conventional killing and viable count assay (see "Correlation of high throughput flow cytometry opsonophagocytosis and antibody-mediated membrane attack complex assays with killing opsonophagocytosis and bactericidal antibody assays"; Stephen Taylor, Charlotte Brookes, Rachel Kenneil, Chuk Tsang, Michael Hudson and Andrew Gorringe; Poster presentation, European Meningococcal Disease Society Conference, Manchester, June 2009).

Example 6: Serum Bactericidal Activity

Serum bactericidal activity is an accepted correlate of protection for meningococcal disease vaccines, as evidenced by: Borrow R, et al., "Neisseria meningitidis group B correlates of protection and assay standardization-international meeting report Emory University, Atlanta, Georgia, United States, 16-17 March 2005"; Vaccine, 2006 Jun 12; 24(24): 5093-107; and Frasch CE, et al., "Bactericidal antibody is the immunologic surrogate of protection against meningococcal disease" Vaccine, 2009 Jun 24; 27 Suppl 2: B112-6.

Typical results obtained in the serum bactericidal assay are shown in Figures 6 and 7.

All documents referred to in this specification are herein incorporated by reference. Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

1. An immunogenic preparation, comprising a commensal Neisserial stress protein and an antigenic polypeptide.

2. An immunogenic preparation according to Claim 1 , wherein said commensal Neisserial stress protein is a member of the Hsp60 family or a member of the Hsp70 family.

3. An immunogenic preparation according to Claim 1 or Claim 2, wherein said commensal Neisserial stress protein is an N. lactamica stress protein.

4. An immunogenic preparation according to any preceding claim, wherein the stress protein is induced in response to a stress-inducing stimulus and is not a constitutively expressed stress protein.

5. An immunogenic preparation according to any preceding claim, wherein said antigenic polypeptide comprises a Neisserial antigen.

6. An immunogenic preparation according to any preceding claim, wherein said antigenic polypeptide is heterologous to commensal Neisseria.

7. A vaccine composition comprising the immunogenic preparation as claimed in any one of the preceding claims as the immunogenic determinant.

8. A vaccine composition according to Claim 7, for use in treating or preventing meningitis.

9. Use of a vaccine composition according to Claim 7, in the preparation of a medicament for the treatment of meningitis.

10. A method for the treatment or prophylaxis of meningitis, the method comprising the steps of:

providing an immunogenic preparation according to any of Claims 1 - 6 or a vaccine composition according to Claim 7; and

- administering a prophylactically effective or therapeutically effective amount of said immunogenic preparation or vaccine composition to a subject.

11. An immunogenic preparation according to any of Claims 1-6, for use as a booster vaccine to a first vaccine, for example as a booster vaccine to a vaccine comprising N. lactamica stress protein.

12. A method of preparing an immunogenic preparation according to any of Claims 1 -6, comprising the steps of:

13. A method according to Claim 12, wherein said recovered commensal Neisserial stress proteins are induced stress proteins and not constitutively- expressed stressed proteins.

14. A method according to Claim 12 or Claim 13, wherein said cell is a commensal Neisseria, such as Neisseria lactamica.

15. A method according to any of Claims 12-14, wherein said cell is a recombinant host cell that has been genetically modified to express said commensal Neisserial stress protein.

16. A method according to any of Claims 12-15, wherein said antigenic polypeptide comprises a Neisserial antigen.

17. A method according to any of Claims 12-16, wherein said antigenic polypeptide is heterologous to said cell.

18. A method according to any of Claims 12-17, wherein said stress-inducing stimulus comprises heat shock.

19. A method of preparing an immunogenic preparation according to any of Claims 1 -6, comprising combining said commensal Neisserial stress protein and said antigenic polypeptide under conditions that promote formation of a complex between said commensal Neisserial stress protein and said antigenic polypeptide.

20. A cell that expresses a commensal Neisserial stress protein and an antigenic polypeptide, wherein said cell has been subjected to a stress-inducing stimulus.

21 . A cell according to Claim 20, wherein said cell is a commensal Neisseria, such as Neisseria lactamica, or wherein said cell is a recombinant host cell that has been genetically modified to express said commensal Neisserial stress protein.

22. A cell according to Claim 20 or 21 , wherein said antigenic polypeptide is heterologous to said cell.

23. A method of preparing an immunogenic composition, such as a vaccine, comprising combining an immunogenic preparation according to any of Claims 1 -6 or a cell according to any of Claims 20-22 with a pharmaceutically acceptable carrier.

24. An immunogenic composition, such as a vaccine, which comprises an immunogenic preparation according to any of Claims 1 -6 or a cell according to any of Claims 20-22, and a pharmaceutically acceptable carrier.

25. An immunogenic preparation according to any of Claims 1 -6, a cell according to any of Claims 20-22, or an immunogenic composition according to

Claim 24, for use in stimulating an immune response in a subject.

26. A method of stimulating an immune response in a subject, comprising administering to the subject an immunogenic preparation according to any of Claims 1-6, a cell according to any of Claims 20-22, or an immunogenic composition according to Claim 24.

27. A method of treating or preventing an infection in a subject, comprising administering to the subject an immunogenic preparation according to any of Claims 1 -6, a cell according to any of Claims 20-22, or an immunogenic composition according to Claim 24.

28. An immunogenic preparation, cell, immunogenic composition, or method, as hereinbefore described and/ or as illustrated in any of the Examples and/ or Figures.