EP2274320A1

EP2274320A1 - Methods for preparing immunogenic compositions

Info

Publication number: EP2274320A1
Application number: EP09733672A
Authority: EP
Inventors: Ricahrd L. Broughton; Corey Mallett; Frahad Namdaran; Krikor Torossian; Joseph Zimmerman
Original assignee: ID Biomedical Corp of Quebec; GlaxoSmithKline LLC
Current assignee: ID Biomedical Corp of Quebec; GlaxoSmithKline LLC
Priority date: 2008-04-25
Filing date: 2009-04-24
Publication date: 2011-01-19
Also published as: CA2722558A1; WO2009132244A1

Abstract

The present invention relates methods of preparing a composition comprising the steps of: releasing from at least one cell a composition comprising at least one outer membrane protein from said at least one cell comprising contacting said at least one said cell with at least one agent capable of solubilizing at least one lipid and optionally an osmalalic agent, forming a mixture comprising said at least one outer membrane protein, at least one endogenous liposaccharide and cell debris; adding an agent capable of separating said cell debris from said at least one outer membrane protein; separating said separated cell debris from said mixture; removing at least one agent capable of solubilizing at least one lipid and said optional osmolalic agent from said at least one outer membrane protein wherein said at least one outer membrane protein remains soluble. Also, included within this invention are compositions comprising at least one outer membrane protein and an endogenous liposacharide wherein said endogenous liposaccharide is in the range of about 0.03 grams to about 0.99 grams endogenous liposaccharide to 1.0 gram of at least one outer membrane protein weight.

Description

METHODS FOR PREPARING IMMUNOGENIC COMPOSITIONS

FIELD OF THE INVENTION The present invention provides methods for making compositions, including immunostimulatory compositions, methods for making immunogenic compositions comprising said immunostimulatory compositions, methods for making vaccines with said immunostimulatory compositions, immunogenic compositions and vaccines made by said methods.

BACKGROUND OF THE INVENTION

Nasal vaccines are a growing segment of the research and development efforts of industry and academic laboratories. The goal of nasal vaccines is to provide better protection to individuals against mucosal-borne pathogens and to reduce side effects associated with the traditional intramuscular route of administration. Protection against such pathogens would be improved by generating greater immunity at mucosal sites in the nose, mouth, and lungs which is generally observed by an increase in secretory IgA antibody production at these mucosal sites. Nasal vaccines have been shown to be especially effective for generating these secretory antibodies. The proteins translated from OMP (porin) genes of Neisseria meningitidis have been shown to have adjuvant properties and are known mitogens of B cells. These proteins alone or in combination with other adjuvants can stimulate a strong IgA response in humans and other mammals to several antigens that are co-administered at mucosal sites of delivery. Nasal delivery of these OMP -based vaccines is complicated, however, by the nature of these proteins and many of the antigens that are formulated with them. . Outer membrane proteins

(OMPs) are multi-transmembrane proteins comprised of hydrophobic domains that render them insoluble in aqueous solutions in the absence of a surfactant.

OMP -based immunostimulatory compositions and vaccines may be prepared as described in the art (see, e.g., U.S. Pat. No. 5,726,292 or U.S. Pat. No. 5,985,284, incorporated herein by reference). However, the methods described in U.S. Patent No. 5,726,292 and 5,985,284 may take several days to complete and require a high amount of detergent for the OMPs to remain soluble. The methods also use high concentration ethanol and ammonium sulfate precipitation steps that require special handling for safety. Furthermore the precipitated product forms a floating pellet after ammonium sulfate precipitation which diminishes recovery. Thus, currently described methods are limited in scalability. There is, therefore, a need for new methods of producing OMP -based compositions, and immunostimulatory compositions and immunogenic compositions and vaccines comprising OMP -based compositions.

SUMMARY OF THE INVENTION

The present invention provides methods of preparing compositions comprising the steps of:

releasing from at least one cell a composition comprising at least one outer membrane protein from said at least one cell comprising contacting said at least one said cell with at least one agent capable of solubilizing at least one lipid and optionally an osmolalic agent, forming a mixture comprising said at least one outer membrane protein, at least one endogenous liposaccharide and cell debris; adding an agent capable of separating said cell debris from said at least one outer membrane protein; separating said separated cell debris from said mixture; removing at least one agent capable of solubilizing at least one lipid and said osmolalic agent from said at least one outer membrane protein wherein said at least one outer membrane protein remains soluble.

Also, provided herein are compositions comprising at least one outer membrane protein and an endogenous liposacharide wherein said endogenous liposaccharide is in the range of about 0.03 grams to about 0.99 grams endogenous liposaccharide to 1.0 gram of at least one outer membrane protein weight.

Brief Description of the Drawings

Figure 1 : Flow Diagram of V2 method. Figure 2: Flow Diagram of Vl and V2 method. Figure 3: Anti-H3N2 (detergent-split A/New York) Serum IgG Responses in

Mice Administered Intranasal Influenza Antigen Formulated with Vl Proteosome, V2 Proteosome, and Protollin. Figure 4: Anti-H3N2 (detergent-split A/New York) Lung IgA Responses in Mice

Administered Intranasal Influenza Antigen Formulated with Vl Proteosome, V2

Proteosome, and Protollin.

Figure 5: Anti-H3N2 (detergent-split A/New York) HAI Responses in Mice Administered Intranasal Influenza Antigen Formulated with Vl Proteosome, V2

Proteosome, and Protollin.

Figure 6: Lung viral titers from immunized and challenged mice.

Figure 7: RSV-specific serum neutralization titers following V2 and FG immunization (from pooled serum samples). Figure 8: RSV-specific serum IgG and BAL IgA titers following V2 and FG immunization.

Figure 9: RSV-specific serum IgG and BAL IgA titers following V2 and FG immunization.

Figure 10: Toll-Like Receptor Cell Based Assays. NF-κB activation indicated that V2 Proteosome act through TLRl and TLR2 as well as a the TLR4 receptor in a concentration dependent manner.

Figure 11: TLR-2 Competition Assay. Specific inhibition of the TLR2 signaling by anti-TLR2 antibodies.

Figure 12: Assessment of TLR-1/2 Activity in a dose response manner of V2P after Storage for Six Months at Different Temperatures.

Figure 13: Immunogenicity of a subunit Proteosome-influenza vaccine in TLR4^7",

MyD88^"A and wild-type C57BL/6 mice.

DETAILED DESCRIPTION OF THE INVENTION Glossary

As used herein "blebs" refers to membrane vesicles that form and are released from Gram negative pathogens during growth. Blebs from Neisseria meningitidis can be isolated from cultures of N. meningitidis by differential centrifugation.

As used herein "diafiltration" or "DF" refers to a method that enables one to change the small molecular weight components of a solution, i.e. salts, detergents, peptides, by continuously filtering the solution through a membrane of selected pore size while adding a buffer containing the desired components to the solution. Typically, the volume of the solution being treated by diafiltration is kept constant by adding the desired buffer at the filtration rate.

As used herein "diafiltration volume" or "DV" refers to the volume of a solution being treated by diafiltration. As used herein "Trans-Membrane Pressure" or "TMP" refers to the pressure that occurs during filtration of a liquid solution achieved by forcing the liquid through a filter of selected pore size using pressure. During diafiltration this pressure can be controlled by adjusting the outlet valve of the diafiltration device. Filtration performance is often related to TMP. As used herein "release" or "releasing" means separating one component from another. Thus, releasing can include but is not limited to isolating, separating, and extracting. In releasing one component, such as an OMP, from a cell, the OMP may contain other cellular elements.

"Liposaccharide," as used herein, refers to native (isolated or prepared synthetically with a native structure) or modified lipopolysaccharide or lipooligosaccharide. Liposaccharides may be endogenous to a first bacterium or may be derived from a second Gram-negative bacteria, such as Shigella flexneri or Plesiomonas shigelloides, or other Gram-negative bacteria (including Alcaligenes, Bacteroides, Bordetella, Borrellia, Brucella, Campylobacter, Chlamydia, Citrobacter, Edwardsiella, Ehrlicha, Enterobacter, Escherichia, Francisella, Fusobacterium, Gardnerella,

Hemophilus, Helicobacter, Klebsiella, Legionella, Leptospira (including Leptospira interrogans), Moraxella, Morganella, Neiserria, Pasteurella, Proteus, Providencia, other Plesiomonas, Porphyromonas (including Porphyromonas gingivalis), Prevotella, Pseudomonas, Rickettsia, Salmonella, Serratia, other Shigella, Spirllum, Veillonella, Vibrio, or Yersinia species). Included within the definition of liposaccharide is both lipooligosaccharide (LOS), which is understood in the art to mean a liposaccharide having a glycan chain consisting of 10 or fewer monosaccharide subunits, and lipopolysaccharide (LPS), which is understood in the art to mean a liposaccharide having a glycan chain comprising more than 10 monosaccharide subunits. Thus, LOS and LPS may be endogenous or exogenous. A liposaccharide may be in a detoxified form (i.e., having the Lipid A core removed) or may be in a form that has not been detoxified. For example, an LPS that contains multiple lipid A species such as P. gingivalis LPS may be used in the compositions described herein (see, e.g., Darveau, et ah, Infect. Immun. 72:5041-51 (2004)). The liposaccharide may be prepared, for example, as described in U.S. Patent

Application Publication No. 2003/0044425.

"Bacteria(um)(l)" means a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enter obacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, and (ii) an archaeon, including but not limited to Archaebacter.

"OMP -based composition" as used herein, refers to preparations of outer membrane proteins (OMPs, including some porins) from Gram-negative bacteria, such as, but not limited to, Neisseria species (see, e.g., Lowell et al, J. Exp. Med. 167:658, 1988; Lowell et al., Science 240:800, 1988; Lynch et al., Biophys. J. 45: 104, 1984; Lowell, in "New Generation Vaccines" 2nd ed., Marcel Dekker, Inc., New York, Basil, Hong Kong, page 193, 1997; U.S. Pat. No. 5,726,292; U.S. Pat. No. 4,707,543), which are useful as a carrier or in compositions for immunogens, such as bacterial or viral antigens. Some OMP -based compositions are immunostimulatory. Some OMP -based compositions may be referred to as "Proteosome," which are hydrophobic and safe for human use. Proteosome have the capability to auto-assemble into vesicle or vesicle-like OMP clusters of about 20 nm to about 800 nm, and to noncovalently incorporate, coordinate, associate (e.g., electrostatically or hydrophobically), or otherwise cooperate with protein antigens (Ags), particularly antigens that have a hydrophobic moiety. Any preparation method that results in the outer membrane protein component in vesicular or vesicle-like form, including multi-molecular membranous structures or molten globular-like OMP compositions of one or more OMPs, is included within the definition of Proteosome. Proteosome may be prepared, for example, as described in the art (see, e.g., U.S. Pat. No. 5,726,292 or U.S. Pat. No. 5,985,284). Proteosome prepared according to procedures set forth herein may also contain an endogenous liposaccharide (LPS or LOS) originating from the bacteria used to produce the OMP porins (e.g., Neisseria species). LOS is about 20-30% of the total OMP composition by weight in OMP -based composition described herein and made by the methods described herein.

Proteosome are composed primarily of chemically extracted outer membrane proteins (OMPs) from Neisseria menigitidis (mostly porins A and B as well as class 4 OMP), maintained in solution by detergent (Lowell GH. Proteosome for Improved Nasal, Oral, or Injectable Vaccines. In: Levine MM, Woodrow GC, Kaper JB, Cobon GS, eds, New Generation Vaccines. New York: Marcel Dekker, Inc. 1997; 193-206). Although a narrow portrait of the lipid content is available for Proteosome, recent unpublished observations indicate that phosphatidylethanolamine (PE) and phosphatidylglycol (PG) are co-purified with OMPs. Proteosome can be formulated with a variety of antigens such as purified or recombinant proteins derived from viral or bacterial sources (Plant, et al, Vaccine 2001; 20(1-2): 218-25; Chabot, e? α/. Vaccine 2005; 23(11): 1374-83; Jones, et al Vaccine 2005; Jones, et al Vaccine 2003; 21(25-26): 3706-12), Cyr, et al Vaccine 2007 or even LPS (Jones, et al Vaccine 2004; 22(27-28): 3691-7 and Orr, et al Infect. Immun. 1993 61: 2390-2395) by diafiltration or traditional dialysis methods. The gradual removal of detergent allows the formation of particulate hydrophobic complexes of approximately 100-200nm in diameter (Lowell GH. Proteosome for Improved Nasal, Oral, or Injectable Vaccines. In: Levine MM, Woodrow GC, Kaper JB, Cobon GS, eds, New Generation Vaccines. New York: Marcel Dekker, Inc. 1997; 193-206). "Proteosome: LPS or Protollin" as used herein refers to preparations of

Proteosome admixed (e.g., by the exogenous addition) with at least one kind of lipo- polysaccharide to provide an OMP-LPS composition (which can function as an immunostimulatory composition). Thus, the OMP-LPS composition can be comprised of two of the basic components of Protollin, which include (1) an outer membrane protein preparation of Proteosome prepared from Gram-negative bacteria, such as Neisseria meningitidis, and (2) a preparation of one or more liposaccharides. Liposaccharides may be endogenous (i.e., naturally contained with the OMP Proteosome preparation), may be admixed or combined with an OMP preparation from an exogenously prepared liposaccharides (i.e., prepared from a different culture or microorganism than the OMP preparation), or may be a combination thereof. Such exogenously added LPS may be from the same Gram-negative bacterium from which the OMP preparation was made or from a different Gram-negative bacterium. Protollin should also be understood to optionally include lipids, glycolipids, glycoproteins, small molecules, or the like, and combinations thereof. The Protollin may be prepared, for example, as described in U.S. Patent Application Publication No. 2003/0044425, incorporated herein by reference. ProtQosomQ-Shigella-flexneri 2a LPS complexes, known as Protollin, have been administered in Phase I and II clinical trials as a vaccine against dysentery, to more than 100 volunteers and were found to be safe and non-toxic (Fries, et al. Infect Immun 2001; 69(7): 4545-53.). Protollin was delivered at doses of up to 1.5 mg of LPS intranasally, without adverse events (Jones, et al. Vaccine 2004; 22(27-28): 3691-7)

"Excipient" as used herein refers to any substance added to a composition that is not responsible for the principle activity of the composition. Excipients may be used to increase the stability, consistency or deliverability of the active ingredient. The immunogenic compositions of the present invention may be administered in the form of a pharmaceutical composition comprising purified polypeptide in conjunction with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to patients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the vaccinating agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrans, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents.

"Mammal" as used herein includes any and all mammals including humans. An "immunogenic composition" as used herein refers to any one or more compounds or agents or immunogens capable of priming, potentiating, activating, eliciting, stimulating, augmenting, boosting, amplifying, or enhancing an adaptive (specific) immune response, which may be cellular (T cell) or humoral (B cell), or a combination thereof. Preferably, the adaptive immune response is protective, which may include neutralization of a virus (decreasing or eliminating virus infectivity). A representative example of an immunogen is a microbial antigen (such as one or more RSV antigens or one or more influenza antigens).

An "immunostimulatory composition" as used herein refers to any composition that enhances an immune response when administered to a mammal, including a human. An immunostimulatory composition may enhance mucosal and/or adaptive immune response and/or cellular and/or humoral immune response and/or innate immune response of the mammal. For instance, an immunostimulatory composition may enhance a mammals immuno response to a specific antigen when co-administered with the antigen. Adjuvants may act as immunostimulatory compositions.

An "antigen" refers to any agent or substance that stimulates an immune response, either cellular and/or humoral, either alone or in combination or linked or fused to another substance. Antigens are often foreign microorganisms such as bacteria or viruses, or the substances they produce, including but not limited to peptides, proteins, lipids, carbohydrates, glycoproteins, glycosaminoglycans and complexes of two or more of the above. An antigen can be a peptide, polypeptide or protein or fragment thereof of at least about 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids in length or greater. Antigens can be produced artificially by chemical synthesis or molecular biology techniques. Often, artificial antigens are designed to elicit immune responses upon exposure or multiple exposures without the potential consequence of acquiring the disease against which the immune response has been stimulated to protect. The antigen can comprise a "carrier" polypeptide and a hapten, e.g., a fusion protein or a carrier polypeptide fused or linked (chemically or otherwise) to another composition. The antigen can be recombinantly expressed in an immunization vector, which can be simply naked DNA comprising the antigen's coding sequence operably linked to a promoter, e.g., a simple expression cassette.

As used herein "conjugate" or "conjugated" refers to two molecules that are bound to each other. For example, a first polypeptide may be covalently or non- covalently bound to a second polypeptide. The first polypeptide may be covalently bound by a chemical linker or may be genetically fused to the second polypeptide, wherein the first and second polypeptide share a common polypeptide backbone.

As used herein "fusion polypeptide" means any polypeptide wherein the first and second polypeptide share a common polypeptide backbone. As used herein "hybrid antigen" refers to any antigen wherein part or all of an antigen is conjugated to another molecule. By way of example, a hybrid antigen could comprise both an immunogenic domain of an antigen as well as a second polypeptide or fusion partner. The fusion partner may be both an immunological fusion partner and or an expression enhancing partner. As understood in the art the immunogenic domain of an antigen may be a fragment and/or variant of the whole antigen capable of eliciting a similar immunogenic response as the whole antigen. Also, by way of example, an immunogenic domain may be conjugated to a fusion partner derived from protein D, a surface protein of the gram-negative bacterium, Haemophilus influenza B (WO91/18926). Other fusion partners include, but are not limited to, the non-structural protein from influenzae virus, NSl (hemagglutinin). Typically the N terminal 81 amino acids are utilized, although different fragments may be used provided they include T- helper epitopes.

"Variant" as the term is used herein, is a or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Variants may also include, but are not limited to, polypeptides or fragments thereof having chemical modification of one or more of its amino acid side groups. A chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at amino acid side groups include, without limitation, acylation of lysine-ε-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the terminal amino group include, without limitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications.

Furthermore, one or more side groups, or terminal groups, may be protected by protective groups known to the ordinarily-skilled protein chemist.

As used herein "fragment," when used in reference to a polypeptide, is a polypeptide having an amino acid sequence that is the same as part but not all of the amino acid sequence of the entire naturally occurring polypeptide. Fragments may be "free-standing" or comprised within a larger polypeptide of which they form a part or region as a single continuous region in a single larger polypeptide. By way of example, a fragment of whole antigen would include 10, 15, 20, 25, 30 or more contiguous amino acids of a polypeptide antigen of naturally occurring antigen. Furthermore, fragments of a polypeptide may also be variants of the naturally occurring partial sequence. For instance, a fragment of a naturally occurring polypeptide antigen may also be a variant having amino acid substitutions within its partial sequence.

As used herein "lipid" refers to any of a group of organic compounds, including the fats, oils, waxes, sterols, and triglycerides, that are insoluble in water but soluble in nonpolar organic solvents, are oily to the touch, and together with carbohydrates and proteins constitute the principal structural material of living cells. Lipids of the present invention include, but are not limited to, phosphatidylethanolamine (PE), phosphatidylglycol (PG), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), and cardiolipin (CL).

As used herein "synergistic" or "synergistic effect" refers to an outcome that is greater than the sum of the two parts. For example, an immunogenic composition having a synergistic effect on a first immune pathway together with a second immune pathway would elicit an improved immune response of either or both pathways compared to each other, individually.

As used herein "microorganism" includes but is not limited to any bacteria, virus, protozoa, fungi, or prion found in nature. Antigens can be derived from either part or all of a microorganism. For example, an antigen derived from a microorganism may include, but is not limited to, a polypeptide present on the surface of the microorganism. The polypeptide from said microorganism may be genetically or chemically fused to a second polypeptide, which may be endogenous or exogenous to said microorganism.

"Microorganism(s)" may include (1) prokaryote, including but not limited to, (a) Bacteria(l)(um), meaning a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (b) an archaeon, including but not limited to Archaebacter, and (2) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans.

As used herein "allergen" means any immunogenic compound or organism or derivative, variant or fragment thereof capable of eliciting an allergic response in a mammal, including a human. Examples of allergens include, but are not limited to, antigens derived from house dust mites, Grass pollen, Ragweed pollen, cats, trees, molds and foods.

As used herein "innate immune response" refers to an response wherein a host produces immune cells and/or mechanism that defend a host from infection by other organisms, in a non-specific manner. During innate immune response, the cells of the innate system recognize, and respond to, pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. Innate immune responses provide immediate defense against infection, and are found in all classes of plant and animal life.

As used herein "weight/volume" refers to a percentage of a component of a composition over a given volume of the composition.

"Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," including but not limited to when such polynucleotide or polypeptide is introduced back into a cell.

"Transformed" as known in the art, is the directed modification of an organism's genome or episome via the introduction of external DNA or RNA, or to any other stable introduction of external DNA or RNA.

"Transfected" as known in the art, is the introduction of external DNA or RNA into a microorganism, including but not limited to recombinant DNA or RNA. Compositions of the present invention can be made using a number of different microorganisms. Microorganisms may be virulent or avirulent. Virulence of a microorganism can be measured by several methods known in the art including, but not limited to, an organism's ability to invade epithelial cells. See Maurelli, et al. Infection and Immunity , Jan 1984 p. 397-401. OMPs extracted from a microorganism will typically have native or endogenous lipopolysacharrides associated with them. When virulent strains are used, the cells are killed by heat or by chemical treatment with phenol subsequent to fermentation to ensures that the bacteria are no longer infectious. In the methods provided herein outer membrane proteins and LOS are isolated. The LOS structure is not modified nor it endotoxin potency intentionally diminished, although hypothetically may be shielded by the OMPs. However, when administered intranasally the compositions of the present invention have been shown to be safe.

The present invention provides methods for making compositions, including immunostimulatory compositions, methods for making immunogenic compositions comprising said immunostimulatory compositions, methods for making vaccines with said immunostimulatory compositions, immunogenic compositions and vaccines made by said methods. Also included within the present invention are uses for such compositions and vaccines. Immunostimulatory compositions of the present invention, immunogenic compositions comprising the immunostimulatory compositions of the present invention and vaccines of the present invention may be used as treatment for an existing disease or prophylactic ally to prevent the occurrence or worsening of a disease.

In particular, the present invention provides methods of preparing an composition comprising the steps of:

releasing from at least one cell a composition comprising at least one outer membrane protein from said at least one cell comprising contacting said at least one said cell with at least one agent capable of solubilizing at least one lipid and optionally an osmalalic agent, forming a mixture comprising said at least one outer membrane protein, at least one endogenous liposaccharides and cell debris; adding an agent capable of separating said cell debris from said at least one outer membrane protein; separating said separated cell debris from said mixture; removing at least one agent capable of solubilizing at least one lipid and said optional osmolalic agent from said at least one outer membrane protein wherein said at least one outer membrane protein remains soluble. In some aspects the agent capable of separating cell debris from outer membrane protein is not ammonium sulfate. In some aspects, the agent capable of separating cell debris is low concentration (e.g., <500 g/1 of protein solution, <200 g/1 of protein solution, <100 g/1 of protein solution) ammonium sulfate. In another aspect the agent is 20% ethanol. In some aspects, said at least one cell is a Gram negative bacterium. The gram negative bacterium may be a Meningococcus and the Meningococcus may be from Group B type 2b. Said at least one cell may be from the genus Neisseria including Neisseria meningitides. Bacteria can be selected from the group of wild type Neisseria meningitidis group B, Haemophilus influenzae type b, Neisseria gonorrhoeae, Escherichia coli, Pseudomonas aeruginosa. Bordetella pertussis, Haemophilus aegyptius, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenza, Moraxella catarrhalis, Neisseria meningitides, Shigella flexneri Plesiomonas shigelloides, Alcaligenes, Bacteroides, Bordetella, Borrellia, Brucella, Campylobacter, Chlamydia, Citrobacter, Edwardsiella, Ehrlicha, Enterobacter, Escherichia, Francisella, Fusobacterium, Gardnerella, Hemophilus, Helicobacter, Klebsiella, Legionella, Leptospira (including Leptospira interrogans), Moraxella, Morganella, Neiserria, Pasteurella, Proteus,

Providencia, other Plesiomonas, Porphyromonas (including Porphyromonas gingivalis), Prevotella, Pseudomonas, Rickettsia, Salmonella, Serratia, other Shigella, Spirllum, Veillonella, Vibrio, or Yersinia species.

In another aspect of the present invention, said Gram negative bacteria are engineered bacteria. Bacteria can be engineered to overexpress certain porins such as, but not limited to, PorA and PorB and/or engineered to underexpress at least one other protein. Bacteria can be engineered by various methods known in the art including site directed mutagenisis, or disruption or deletion of part or all of an encoding gene. Bacteria can also be engineered to over or under express certain polypeptides such as outer membrane proteins when cultured under selective growth conditions. Furthermore, and as understood in the art, bacteria can be engineered by the addition of certain encoding sequences into the genome of the bacteria or by expression vector. By way of example, DNA encoding wild type and/or modified PorA can be inserted into the genome of bacteria by transformation or can be inserted in a cell by transfection. When said transformed cell is cultured the cell may express more PorA than a bacterium that has not been engineered.

In a further aspect of the present invention, the agent capable of solubilizing at least one lipid is a detergent. Detergent mixtures capable of solubilization may be comprised of but not limited to one or more of: lauryl dimethylbetaine (LDB), sodium deoxycholate (DOC), Lauryldimethylamine-oxide (LDAO), Cetyltrimethylammoniumbromide (CTAB), Sodium dodecylsulfate (SDS), N- Lauroylsarcosine, Sodium octyl sulfate, Triton, Trizma® dodecyl sulfate, Nonidet™ P 40, Pentaethylene glycol and derivatives, Polyoxyethylene (20) sorbitan monolaurate, Polyoxyethylene 40 stearate, Saponin, T WEEN® 20 and related substances, Decanoyl-N- methylglucamide (Mega-10), 3-[(3-Cholamidopropyl)dimethylammonio]-2 -hydroxy- 1- propanesulfonate (CHAPSO), Sorbitan monolaurate (Span-20), Polyoxyethylene[23]lauryl ether (BRIJ) and Dimethyloctylphosphine oxide (APO). In some aspects of the present invention, only about 200 ppm of detergent is necessary to keep the OMP -based immunostimulant soluble, which has been shown to be a safe concentration in non-clinical and clinical studies. Previous methods of making Proteosome required at least 2000 - 10,000 ppm of detergent for solubility which was then stored in 10,000 -15,000 ppm and required removal by dialysis when formulated with the antigen. The methods described here provide for a stable and soluble immunostimulant preparation containing safe levels of detergent, less than 500 ppm. Furthermore, a detergent reduction step is not required during formulation of the OMP base immunostimulant with the desired antigen to make an immunostimulatory composition.

Optional osmolalic agent may comprise sugar and/or salt. In some instances the optional osmolalic agent is calcium chloride. In another aspect, the agent capable of separating said cell debris from said at least one outer membrane protein comprises at least one alcohol. Said at least one alcohol may be ethanol. Ethanol may be added to said mixture comprising at least one outer membrane protein, at least one endogenous liposaccharides and cell debris to a total volume by weight of up to about 50%. Cell debris may be separated from said mixture by precipitation. Cell debris may be removed from said mixture by a method selected from: centrifugation, depth filtration, and filtration. In another aspect of the method, removing at least one agent capable of solubilizing lipids and optional osmolalic agent from said at least one outer membrane protein and wherein said at least one outer membrane protein remains soluble is done by diafiltration. In another aspect, the methods of the present invention further comprise sterilizing said composition by micro filitration. Additionally, the methods of the present invention comprise adding a composition comprising phosphate buffered saline during diafiltration. The methods of the present invention also provide for concentration and ultrafiltration of the compositions described herein. In another aspect, low molecular weight proteins are removed from the mixture. Low molecular weight proteins include, but are not limited to, proteins having a molecular weight of less than or equal to about 100 kDa, or less than about 50 kDa or less than about 25 kDa, or less than about 10 kDa. Said at least one outer membrane may be released with detergent at a concentration of about 40,000 ppm to about 70,000 ppm. In another aspect, concentration of said detergent after said removing step is less than 20,000 ppm. In another aspect of the present invention the composition is homogenized. Homogenization of the composition will produce uniform size particles of outer membrane proteins in solution. In another aspect the compositions of the present invention act as immunostimulatory compositions. Thus, the methods provided for preparing compositions include methods for preparing immunostimulatory compositions.

Another aspect of the present invention comprises admixing said composition with at least one antigen to form an immunogenic composition. One of the surprising benefits of the present invention is that compositions prepared by the methods described are not precipitated and resolubilized in detergent as previous methods for the preparation of Proteosome. The OMPs remain soluble in aqueous solution at low concentration of detergent meaning levels below 500 ppm or below 200 ppm. Toxicology studies have been carried out to demonstrate the safety of detergents at this level. Furthermore, LPS from a second organism does not need to be added to the OMPs to maintain soluble OMPs. Therefore, compositions, immunostimulatory compositions, immunogenic compositions and vaccines made by the presently described methods can be mixed with antigen in solution free of detergent. Thus, these mixtures do not need to undergo diafiltration for the production of an immunogenic composition or vaccine. The elimination of the precipitation step and the detergent extraction step from previous methods as described herein reduces the preparation time of the compositions herein and provides for large scale methods for the preparation of compositions, immunostimulatory compositions, immunogenic compositions and vaccines. The ability to prepare immunogenic compositions and vaccines by admixing the compositions and antigen eliminates losses of antigens observed with previous uses of Proteosome.

The antigens used in the presently described methods may be at least one whole or disrupted microorganism. The microorganism may be selected from virus, bacteria, fungi and protozoa. The antigen may be from influenza virus. The antigen may comprise at least one hemagglutinin; it may be monovalent or trivalent. Additionally, antigens may comprise a fragment and/or variant and/or hybrid antigen from the group of: cancer antigen, influenza virus, malarial protozoa, HIV, birch pollen, DerPl, grass pollen, RSV, non-typeable H. influenzae and Morexella. Antigens may be recombinant antigen and/or a hybrid antigen.

In another aspect of the present invention, administering said composition or immunogenic composition comprising said immunostimulatory composition to a mammal induces an enhanced immune response in said mammal. The compositions and/or immunogenic compositions of the present invention can be administered by a route selected from: intranasal, sublingual, rectal, intramuscular, intravenous, intraperitoneal, mucosal, enteral, parenteral and inhalation. The mucosal route is via the nasal, oropharyngeal, ocular or genitourinary mucosa. In another aspect, administering said composition or immunogenic composition comprising said composition to a mammal induces an innate immune response against infections, including viral and bacterial infections. The compositions of the present invention may be administered to a mammal in need thereof for the treatment of a neurological disease. The neurological disease may be, but is not limited to, an amyloidal disease such as Alzheimer disease. As used herein a "neurological disease or disorder" refers to any condition involving a neuronal abnormality, including but not limited to, a neurodegenerative disease or disorder. For instance, neurodegenerative diseases and disorders are neurological disease characterized by destruction or deterioration of selective neuronal and/or myelin populations. Exemplary neurodegenerative diseases include, but are not limited to, acute diseases such as stroke (ischemic or haemorrhagic), traumatic brain injury and spinal cord injury as well as chronic diseases including Alzheimer's disease, fronto-temporal dementias (tauopathies), peripheral neuropathy, Parkinsonian syndromes such as Parkinson's disease (PD), Creutzfeldt- Jakob disease (CJD), Prion diseases, Schizophrenia, amyotrophic lateral sclerosis (ALS), multiple sclerosis, cerebral amyloid angiopathy (CAA), Huntington's disease, inclusion body myositis, and mild cognitive impairment (MCI). Neurodegenerative disease is associated with progressive nervous system dysfunction, and often leads to atrophy of affected central or peripheral nervous system structures.

Alzheimer's disease (AD) is a beta amyloid (b-amyloid) associated disease which is a progressive neurodegenerative disorder that is the predominant cause of dementia in people over 65 years of age. AD is characterized by massive neuronal cell loss in certain brain areas, and by the deposition of proteinaceous material in the brains of AD patients. These deposits contain neurofibrillary tangles and b-amyloid plaques. The major protein component of the b-amyloid plaque is A beta (Ab). Increased accumulation of Ab has been postulated to significantly contribute to the pathogenesis of AD, and is also associated with various other amyloidoses and neurological disorders also referred to herein as "b-amyloid associated disease," such as Parkinson's disease, Down syndrome, diffuse Lewy body disease, progressive supranuclear palsy, and Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch Type (HCHWA-D), cerebral amyloid angiopathy (CAA), and mild cognitive impairment (MCI).

The administration of proteosome-based compositions in connection with neurological disease is disclosed in United States Patent Publication 2006/0229233, the entirety of which is incorporated herein by reference.

In another embodiment of the present invention, compositions are provided comprising at least one outer membrane protein and endogenous liposacharide wherein said endogenous liposaccharide is in the range of about 0.03 grams to about 0.99 grams endogenous liposaccharide to 1.0 gram of at least one outer membrane protein weight. The composition may comprises endogenous liposaccharide is in the range of about 15% to about 300% of the composition by weight. The ratio of liposaccharide to OMP may be about 0.6: 1.0 by weight. The composition may further comprises lipid wherein said lipid comprises about 1.5 to about 1.6 gram lipid per 1.0 gram at least one outer membrane protein by weight. The composition may further comprise DNA, wherein said DNA comprises <1.0% of the total composition by weight. In some instances, the liposaccharide is LOS. LOS content of a composition can be measured using a chemical assay such as, but mot limited to, measuring 3-deoxy-d-mα««o-2-octulosonic acid (Kdo) content, which forms part of the LOS oligosaccharide chain. The total protein content of the composition can be measured by such assays as Lowry or bicinchoninic acid (BCA) protein assay kit (Peirce).

In another aspect of the invention, the composition further comprises an antigen. The antigen may be at least one whole or disrupted microorganism. The microorganism may be selected from virus, bacteria, fungi and protozoa. The antigen may be from influenza virus. The antigen may comprise at least one hemagglutinin; it may be monovalent or trivalent. Influenza vaccines, of all kinds, are usually trivalent vaccines. They generally contain antigens derived from two influenza A virus strains and one influenza B strain. A standard 0.5 ml injectable dose in most cases contains 15 μg of haemagglutinin antigen component from each strain, as measured by single radial immunodiffusion (SRD) (J. M. Wood et al.: An improved single radial immunodiffusion technique for the assay of influenza haemagglutinin antigen: adaptation for potency determination of inactivated whole virus and subunit vaccines. J. Biol. Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborative study of single radial diffusion and Immunoelectrophoresis techniques for the assay of haemagglutinin antigen of influenza virus. J. Biol. Stand. 9 (1981) 317-330). The influenza virus strains to be incorporated into influenza vaccine each season are determined by the World Health Organisation in collaboration with national health authorities and vaccine manufacturers.

In some aspects of the present invention compositions are provided comprising about 75 μg/mL of flu antigen, which may be a trivalent flu antigen. Compositions comprising flu antigen can comprise about lOμg, 25 μg, 65 μg, and 160μg of total protein from at least one cell comprising an outer membrane protein in about a 0.2 mL sample. Total protein from a cell comprising at least one outer membrane protein is typically about 60% to about 70% outer membrane protein by mass (including Class I, II and IV outer membrane proteins). Compositions comprising about 75 μg/mL of flu antigen and about lOμg, 25μg, 65μg, and 160μg of total protein in a 0.2 mL sample from at least one cell comprising an outer membrane protein can further comprise PBS buffer. The compositions can be prepared by the methods described herein. Thus, the present invention also provides for the use of the composition described herein as a immunogenic composition and/or a vaccine for the treatment or prevention of influenza as well as the use of the composition and methods described herein to manufacture a immunogenic composition and/or vaccine for the treatment or prevention of influenza. Influenza viral strains and influenza antigens

The influenza virus antigen or antigenic preparation thereof may be produced by any of a number of commercially applicable processes. Said influenza virus or antigenic preparation thereof may be derived from the conventional embryonated egg method, by growing influenza virus in eggs and purifying the harvested allantoic fluid egg-derived. Alternatively the influenza virus or antigen preparation thereof may be cell-culture derived using cell or cell culture to grow the virus or express recombinant influenza virus surface antigens. Suitable cell substrates for growing the virus include for example dog kidney cells such as MDCK or cells from a clone of MDCK, MDCK-like cells, monkey kidney cells such as AGMK cells including Vero cells, suitable pig cell lines, or any other mammalian cell type suitable for the production of influenza virus for vaccine purposes. Suitable cell substrates also include human cells e.g. MRC-5 cells or the Per.Cό cell line. Suitable cell substrates are not limited to cell lines; for example primary cells such as chicken embryo fibroblasts and avian cell lines such as chicken or duck cell lines (e.g. EBx cell line such as EB 14 or EB24 derived from chicken or duck embryonic stem cells respectively) are also included. Suitable insect cells are Sf9, Sf2 or Hi5. Alternative cells are yeast cells (such as Saccharomyces cerevisiae or Pichia pastoris) for recombinant Influenza A antigens for example, or plants.

In one embodiment, an influenza virus or antigenic preparation thereof for use according to the present invention may be a split influenza virus or split virus antigenic preparation thereof. In an alternative embodiment the influenza preparation may contain another type of inactivated influenza antigen, such as inactivated whole virus or purified HA and NA (subunit vaccine), or an influenza virosome. In a still further embodiment, the influenza virus may be a live attenuated influenza preparation. A split influenza virus or split virus antigenic preparation thereof for use according to the present invention is suitably an inactivated virus preparation where virus particles are disrupted with detergents or other reagents to solubilize the lipid envelope. Split virus or split virus antigenic preparations thereof are suitably prepared by fragmentation of whole influenza virus, either infectious or inactivated, with solubilizing concentrations of organic solvents or detergents and subsequent removal of all or the majority of the solubilizing agent and some or most of the viral lipid material. By split virus antigenic preparation thereof is meant a split virus preparation which may have undergone some degree of purification compared to the split virus whilst retaining most of the antigenic properties of the split virus components. For example, when produced in eggs, the split virus may be depleted from egg-contaminating proteins, or when produced in cell culture, the split virus may be depleted from host cell contaminants. A split virus antigenic preparation may comprise split virus antigenic components of more than one viral strain. Vaccines containing split virus (called 'influenza split vaccine') or split virus antigenic preparations generally contain residual matrix protein and nucleoprotein and sometimes lipid, as well as the membrane envelope proteins. Such split virus vaccines will usually contain most or all of the virus structural proteins although not necessarily in the same proportions as they occur in the whole virus. Examples of commercially available split vaccines are for example FLUARIX™, FLUSHIELD™, or FLUZONE™. Split flu may be produced using a solvent/detergent treatment, such as tri-w-butyl phosphate, or diethylether in combination with Tween™ (known as "Tween-ether" splitting) of by using other splitting agents including detergents or proteolytic enzymes or bile salts, for example sodium deoxycholate. Detergents that can be used as splitting agents include cationic detergents e.g. cetyl trimethyl ammonium bromide (CTAB), other ionic detergents e.g. laurylsulfate, taurodeoxycholate, or non-ionic detergents such as the ones described above including Triton X-IOO (for example in a process described in Lina et al, 2000, Biologicals 28, 95-103) and Triton N-101, or combinations of any two or more detergents. The preparation process for a split vaccine may include a number of different filtration and/or other separation steps such as ultracentrifugation, ultrafiltration, zonal centrifugation and chromatography (e.g. ion exchange) steps in a variety of combinations, and optionally an inactivation step eg with heat, formaldehyde or β- propiolactone or U. V. or any combination thereof which may be carried out before or after splitting. The splitting process may be carried out as a batch, continuous or semi- continuous process. A preferred splitting and purification process for a split immunogenic composition is described in WO 02/097072. Preferred split flu vaccine antigen preparations according to the invention comprise a residual amount of Tween 80 and/or Triton X-100 remaining from the production process, although these may be added or their concentrations adjusted after preparation of the split antigen. In one embodiment of both Tween 80 and Triton X-100 are present. The preferred ranges for the final concentrations of these non-ionic surfactants in the vaccine dose, arising from the antigenic preparation, are:

- Tween 80: 0.01 to 1%, or about 0.1% (v/v)

- Triton X-100: 0.001 to 0.1 (% w/v), or 0.005 to 0.02% (w/v). Alternatively, the influenza virus may be in the form of a whole virus vaccine.

This form may prove to be an advantage over a split virus vaccine for a pandemic situation as it avoids the uncertainty over whether a split virus vaccine can be successfully produced for a new strain of influenza virus. For some strains the conventional detergents used for producing the split virus can damage the virus and render it unusable. Although there is always the possibility to use different detergents and/or to develop a different process for producing a split vaccine, this would take time, which may not be available in a pandemic situation. In addition to the greater degree of certainty with a whole virus approach, there is also a greater vaccine production capacity than for split virus since considerable amounts of antigen are lost during additional purification steps necessary for preparing a suitable split vaccine.

In another embodiment, the influenza virus preparation is in the form of a purified sub-unit influenza vaccine. Sub-unit influenza vaccines generally contain the two major envelope proteins, HA and NA, and may have an additional advantage over whole virion vaccines as they are generally less reactogenic, particularly in young vaccinees. Sub-unit vaccines can be produced either recombinantly or purified from disrupted viral particles. Examples of commercially available sub-unit vaccines are for example AGRIPPAL™, or FLUVIRTN™. In a specific embodiment, sub-unit vaccines are prepared from at least one major envelope component such as from haemagglutinin (HA), neuraminidase (NA), or M2, suitably from HA. Suitably they comprise combinations of two antigens or more, such as a combination of at least two of the influenza structural proteins HA, NA, Matrix 1 (Ml) and M2, suitably a combination of both HA and NA, optionally comprising Ml. Suitably, the influenza components are produced by recombinant DNA technology, i.e. results from, or is expressed from, a nucleic acid resulting from recombinant DNA manipulations, including live recombinant vector (vaccinia) or recombinant subunit protein (baculovirus/insect cells, mammalian cells, avian cells, yeast, plants or bacteria). Suitable insect cells are Spodoptera frugiperda (Sf9) insect cells or High Five (Hi5) insect cells developed from Trichoplusia ni (Invitrogen) and suitable baculovirus are Autographa californica nuclear polyhedrosis virus (AcNPV) (Baculogold, Becton

Dickinson, PharMingen) or the so-called Bacmid system.

In one embodiment, the influenza virus preparation is in the form of a virosome.

Virosomes are spherical, unilamellar vesicles which retain the functional viral envelope glycoproteins HA and NA in authentic conformation, intercalated in the virosomes' phospholipids bilayer membrane. Examples of commercially available virosomal vaccines are for example INFLEXAL V™, or INVA VAC™.

In another embodiment, the sub-unit influenza components are expressed in the form of virus-like-particles (VLP) or capsomers, suitably plant-made or insect cells-made VLPs. VLPs present the antigens in their native form. The VLP sub-unit technology may be based entirely on influenza proteins, or may rely on other virus such as the murine leukaemia virus (MLV) and may therefore comprise a non-influenza antigen such as MLV gag protein. A suitable VLP comprises at least one, suitably at least two influenza proteins, optionally with other influenza or non-influenza proteins, such as Ml and HA, HA and NA, HA, NA and Ml or HA, NA and MLV gag. It may be produced either in plant cells or insect cells. VLPs can also carry antigens from more than one influenza strain, such as VLPs made from two seasonal strains (e.g. HlNl and H3N2) or from one seasonal and one pandemic strain (e.g. H3N2 and H5N1) for example.

Accordingly, in one embodiment the immunogenic compositions and uses thereof according to the invention comprise an influenza virus antigen or antigenic preparation thereof from influenza virus grown on eggs or on cell culture. In another embodiment, said influenza virus antigen or antigenic preparation thereof comprises a whole virus, a split virus, a virosome or one or more purified antigen chosen from: HA, NA, Ml, M2. In another embodiment, said purified antigen(s) are prepared from influenza virus grown in mammalian, avian or insect cells. Specifically, said purified antigen(s) are recombinantly produced. They can be in the form of a Virus-like-particle.

The influenza virus strain may be a reassortant strain, produced by classical reassortant techniques or by reverse genetics techniques, with the reassortant virus being rescued in the presence of in the absence of a helper virus. These techniques are well known in the art. When influenza virus is cell-derived, the amount of residual host cell DNA is reduced to low levels, in order to minimize the tumourigenic potential of the vaccine. Host cell DNA will normally not exceed 10 ng per dose of vaccine, and suitably be less than 1 ng, less than 100 pg, less than 50 pg or less than 25 pg per dose. Validated methods used to assess residual DNA levels are for example: blotting techniques or quantitative PCR, e.g. Southern blot, slot blots, the Threshold™ system from Molecular devices.

In one embodiment, the influenza preparation is prepared in the presence of low level of thiomersal, or in the absence of thiomersal. In another embodiment, the resulting influenza preparation is stable in the absence of organomercurial preservatives, in particular the preparation contains no residual thiomersal. In particular the influenza virus preparation comprises a haemagglutinin antigen stabilized in the absence of thiomersal, or at low levels of thiomersal (generally 5 μg/ml or less). Specifically the stabilization of B influenza strain is performed by a derivative of alpha tocopherol, such as alpha tocopherol succinate (also known as vitamin E succinate, i.e. VES). Such preparations and methods to prepare them are disclosed in WO 02/097072.

The invention can be operated with vaccine including inter-pandemic, pandemic or pre-pandemic influenza strains. The vaccines may include influenza virus strains which are not strictly matching the then circulating strain, and are effective for example against "influenza drift variant", i.e. new strains that have changed enough to cause an epidemic again among the general population; through a process termed "antigenic drift."

Suitably the influenza virus strain or strains to be included in the immunogenic or vaccine composition is/are interpandemic (seasonal) strain(s), i.e. circulating influenza viruses that are related to those from the preceding epidemic, or strain(s) being associated with a pandemic outbreak or having the potential to be associated with a pandemic outbreak (herein a "pre-pandemic strain"). Different strains may be included in a multivalent composition, such as a mixture of interpandemic strains, a mixture of pandemic strains or a mixture of both.

Interpandemic strains are for example strains which circulate globally during interpandemic periods such as but not limited to: HlNl, H1N2, H3N2 or B. Commercially available influenza vaccines are a trivalent combination including one influenza B strain and two influenza A strains (HlNl, H3N2). A suitable composition therefore contains antigens prepared from the three WHO recommended strains of the appropriate influenza season, usually two influenza A virus strains and one influenza B strain. A standard 0.5 ml injectable dose in most cases contains (at least) 15 μg of haemagglutinin antigen component from each strain, as measured by single radial immunodiffusion (SRD) (J.M. Wood et al.: An improved single radial immunodiffusion technique for the assay of influenza haemagglutinin antigen: adaptation for potency determination of inactivated whole virus and subunit vaccines. J. Biol. Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborative study of single radial diffusion and Immunoelectrophoresis techniques for the assay of haemagglutinin antigen of influenza virus. J. Biol. Stand. 9 (1981) 317-330). Another suitable composition contains four influenza strains such the three classical strains, and an additional B strain (Commun Dis Intell 2006, 30, 350-357) or an additional H3N2 strain (Vaccine 1992, 10, 506-511).

Another suitable composition for use in the present invention comprises a pandemic influenza strain, or an influenza strain susceptible to be associated with a pandemic, in the form of a monovalent pandemic or pre-pandemic composition, alone, in combination or in addition to one or more seasonal (i.e. interpandemic) strains. Pandemic or pre-pandemic strains are for example from an avian or pig origin.

The features of an influenza virus strain that give it the potential to cause a pandemic or an outbreak of influenza disease associated with pandemic influenza strains are: (i) the influenza virus must undergo a major change that results in a completely new virus (eg a new haemagglutinin as compared to haemagglutinin of currently circulating strains); (ii) the new virus is pathogenic for humans and (iii) the new virus must be transmissible from human to human. A new haemagglutinin may emerge at unpredictable levels with a totally different subtype from strains circulating the season before, with resulting antigens varying from 20% to 50% from the corresponding protein of strains that were previously circulating in humans, through a phenomenon called "antigenic shift" which results in virus escaping 'herd immunity' and establishing pandemics. Therefore the new HA has not been evident in the human population for an extended period of time, probably a number of decades, such as H2. Or it may be a haemagglutinin that has not been circulating in the human population before, for example H5, H9, H7 or H6 which are found in avian species (birds). In either case the majority, or at least a large proportion of, or even the entire population has not previously encountered the antigen and is immunologically naϊve to it. At present, the influenza A virus that has been identified by the WHO as one that potentially could cause a pandemic in humans is the highly pathogenic H5N1 avian influenza virus. Therefore, the pandemic vaccine according to the invention will suitably comprise H5N1 virus. Two other suitable strains for inclusion into the claimed composition are H9N2 or H7N1.

Suitably the vaccines for use in the invention will include any one of the following 16 HA subtypes (Hl-H 16) and/or any one of the nine NA subtypes (N1-N9) that have been identified for influenza A viruses. Suitably the vaccine will include a HA and a NA moiety, but the vaccine can also include an antigen from a recombinant origin, in this case it may be HA only, or it may be any combination of one or more of HA, NA, Ml, and M2. Suitably three seasonal (e.g. HlNl, H3N2, B) strains are present. Suitably four strains are present that are from the group of: four seasonal strains (e.g. HlNl, H3N2, two B strains; or HlNl, B, two H3N2 strains) or the group of one pandemic (e.g. avian) strain plus three seasonal strains (e.g. HlNl, H3N2, B). Suitable A strains are, but not limited to: interpandemic strains such as: HlNl,

H3N2, and pandemic strains or strains susceptible to be associated with a pandemics for example strains having at least one of the H5, H2, H7, H9 or HlO subtype, specifically H2N2, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2 and H10N7. Within a given subtype, different variant are possible, such as within the H5 subtype the following variants are: clade 1, clade 2, clade 3 etc.

Suitably the HA is from at least three, at least four influenza strains. One or two B strains from two different lineage (such as B/yamagata or B/Victoria) may be included.

In specific embodiments, the immunogenic composition contains (i) an haemagglutinin (HA) from a single influenza strain, referred to as a "monovalent" influenza composition; or (ii) a HA from more than one influenza strain, referred to as a "multivalent" influenza composition.

A suitable multivalent composition for use according to the invention is a bivalent composition comprising haemagglutinin (HA) from two influenza virus strains such as but not exclusively two strains associated to a pandemic or susceptible to be associated with a pandemic, e.g. H5 or H2, a trivalent composition comprising HA from three influenza virus strains, optionally from two A strains, and one B strain such as but not limited to B/Yamagata or B/Victoria, a quadrivalent composition comprising haemagglutinin (HA) from four influenza virus strains or a pentavalent composition comprising haemagglutinin (HA) from five influenza virus strains. A suitable quadrivalent composition comprises haemagglutinin from two A strains and two B strains from different lineage (such as B/Yamagata or B/Victoria). Alternatively a quadrivalent composition comprises haemagglutinin from three A strains (optionally HlNl, H3N2, and one A strain associated to a pandemic or susceptible to be associated to a pandemic) and one B strain (such as B/Yamagata or B/Victoria). Another alternative quadrivalent composition comprises haemagglutinin from four interpandemic A strain, or from four A strains at least one of which being associated to a pandemic or susceptible to be associated to a pandemic, such as avian strains such as H5 + H2 + H7 + H9. Specifically a multivalent adjuvanted pandemic composition such as a pandemic bi-valent (e.g. H5+H2) or trivalent or quadrivalent (e.g. H5 + H2 + H7 + H9) offers the advantage of a pre-emptive immunisation against pandemic influenza A threats subtypes and durable priming against threat subtypes. Optionally, such a pandemic vaccine may be combined with a seasonal vaccine. A multivalent composition can also comprise more than 5 influenza strains such as 6, 7, 8, 9 or 10 influenza strains. When two B strains are used in a multivalent seasonal composition, they can suitably be from two different lineages (optionally from B/Victoria and B/Yamagata). At least one of said B strain, suitably both B strains, will be from a circulating lineage. Such a composition is particularly suitable for children.

Suitably the HA per strain is at the usual 15 μg HA per strain, as determined by SRID. The HA per strain may alternatively be a low amount of HA (optionally 10 μg HA per strain or below). Suitably the HA per strain is at about or below 5 μg, at about 2.5 μg or below. Said low amount of HA may be as low as practically feasible provided that it allows to formulate a vaccine which fulfils the requirements of the binding Pharmacopeia such as the international e.g. EMEA or FDA criteria for efficacy, as detailed below (see Table s 1 and 2 and the specific parameters as set forth). The amount of HA per strain can be as little as 3.8 μg per human vaccine dose, or even as little as 1.9 μg per dose. A vaccine dose of 0.5 ml is suitably used. Advantageously, a vaccine dose according to the invention, in particular but not exclusively a low HA amount vaccine, may be provided in a smaller volume than the conventional injected split flu vaccines, which are generally about 0.5, 0.7 or 1 ml per dose. The low volume doses according to the invention are suitably below 500 μl, typically below 300 μl and suitably not more than about 200 μl or less per dose. A dose volume of 0.2 ml is suitable for intranasal administration and may be administered in two fractions of 0.1 ml per nostril. Slight adaptation of the dose volume will be made routinely depending on the HA concentration in the original bulk sample, or depending on the delivery route with smaller doses being given by the intranasal or intradermal route, or depending on the target population (for example infants may receive half of an adult human dose).

The influenza medicament of the invention suitably meets certain international criteria for vaccines. Standards are applied internationally to measure the efficacy of influenza vaccines. Serological variables are assessed according to criteria of the European Agency for the Evaluation of Medicinal Products for human use (CHMP/BWP/214/96, Committee for Proprietary Medicinal Products (CPMP). Note for harmonization of requirements for influenza vaccines, 1997 ^'. CHMP/BWP/214/96 circular N°96-0666: 1 -22) for clinical trials related to annual licensing procedures of influenza vaccines (Table 3). The requirements are different for adult populations (18-60 years) and elderly populations (>60 years) (Table 3). For the annual re-registration of interpandemic influenza vaccines, at least one of the assessments (seroconversion factor, seroconversion rate, seroprotection rate) should meet the European requirements, for all strains of influenza included in the vaccine. The proportion of HI titres equal or greater than 1:40 is regarded most relevant because these titres are expected to be the best correlate of protection [Beyer W et al. 1998. Clin Drug Invest.;15: l-12].

As specified in the "Guideline on dossier structure and content for pandemic influenza vaccine marketing authorization application. (CHMP/VEG/4717/03, April 5th 2004, or more recently EMEA/CHMP/VWP/263499/2006 of 24 Jan 2007 entitled

'Guidelines on flu vaccines prepared from viruses with a potential to cause a pandemic', available on wvw,_craea.,.ci3.j.I?.t)" issued by the European Medicines Agency's Committee, in the absence of specific criteria for influenza vaccines derived from non circulating strains, it is anticipated that a pandemic candidate vaccine should (at least) be able to elicit sufficient immunological responses to meet suitably all three of the current standards set for existing vaccines in unprimed adults or elderly subjects, after two doses of vaccine. The EMEA Guideline describes the situation that in case of a pandemic the population will be immunologically naive and therefore it is anticipated that all three

CHMP criteria for seasonal vaccines should be fulfilled by pandemic candidate vaccines. No explicit requirement to prove it in pre-vaccination seronegative subjects is required. However, Guidance for pre-pandemic vaccine expects that for vaccines used for primary immunisation of a previously immunologically naive population, influenza vaccines used for pandemic preparedness should induce high seroprotection rates, preferably after one or at most two doses. All three criteria (seroprotection rate, GMT increase and response rate) as defined in guideline CPMP/BWP/214/96 should be fulfilled.

The compositions for use in the present invention suitably meet at least one such criteria for the strain included in the composition (one criteria is enough to obtain approval), suitably at least two, or typically at least all three criteria for protection as set forth in Table 1.

Table 1 (CHMP criteria)

A 70% seroprotection rate is defined by the European Medicines Agency's Committee for Medicinal Products for Human Use (CHMP) as one of three criteria normally required to be met for an annual seasonal influenza vaccine and which CHMP is also expecting a pandemic candidate vaccine to meet. However, mathematical modeling has indicated that a vaccine that is, at the population level, only 30% efficient against certain drifted strains may also be of benefit in helping to reduce the magnitude of a pandemic and that a pandemic vaccination campaign using a (pre-pandemic) vaccine with

30% efficacy against infection (30% reduction in susceptibility) against the pandemic strain (cross-protection of 30%) could effectively reduce the clinical attack rate by 75% and consequently morbidity/mortality within the population (Ferguson et al, Nature 2006).

The U.S. FDA has published a draft guidance (CBER draft criteria) (available from the Office of Communication, Training and Manufacturers Assistance (HFM-40), 1401 Rockville Pike, Suite 200N, Rockville, MD 20852-1448, or by calling 1-800-835- 4709 or 301-827-1800, or from the Internet at hUp://vvΛvw.fda.gυv/cber/auideliiies.hlm) on Clinical Data Needed to Support the Licensure of Pandemic Influenza Vaccines, and the proposed criteria are also based on the CHMP criteria. FDA uses slightly different age cut-off points. Appropriate endpoints similarly include: 1) the percent of subjects achieving an HI antibody titer > 1 :40, and 2) rates of seroconversion, defined as a fourfold rise in HI antibody titer post-vaccination. The geometric mean titer (GMT) should be included in the results, but the data should include not only the point estimate, but also the lower bound of the 95% confidence interval of the incidence rate of seroconversion, and the day 42 incidence rate of HI titers > 1 :40 must exceed the target value. These data and the 95% confidence intervals (CI) of the point estimates of these evaluations should therefore be provided. FDA draft guidance requires that both targets be met. These FDA- issued criteria are summarized in Table 2.

Table 2 (CBER draft criteria)

Accordingly, in one aspect of the invention, it is provided the vaccine composition will be able to induce an immune response against influenza virus which meets at least one criteria, suitably two, suitably all three criteria for protection set out above. Specifically at least one of the following criteria is met for the or all strains present in the vaccine after one single dose. Accordingly there is also provided a one dose intranasal influenza vaccine, optionally a low amount influenza vaccine, wherein the adjuvant is herein defined.

Populations to vaccinate are children, adults and elderly. The target population to vaccinate is the entire population, e.g. healthy young adults (e.g. aged 18-50 or 18-60), elderly (typically aged above 60) or infants/children/adolescents. The target population may in particular be naϊve, or immuno-compromised or immuno-suppressed. Immunocompromised or immuno-suppressed humans generally are less well able to respond to an antigen, in particular to an influenza antigen, in comparison to healthy adults. In a specific aspect, the vaccine is administered intranasally. Typically, the vaccine is administered locally to the nasopharyngeal area, suitably without being inhaled into the lungs. It is desirable to use an intranasal delivery device which delivers the vaccine formulation to the nasopharyngeal area, without or substantially without it entering the lungs. Suitable devices for intranasal administration of the vaccines according to the invention are spray devices. Suitable commercially available nasal spray devices include Accuspray™ (Becton Dickinson). Nebulisers produce a very fine spray which can be easily inhaled into the lungs and therefore does not efficiently reach the nasal mucosa. Nebulisers are therefore not preferred. Suitable spray devices for intranasal use are devices for which the performance of the device is not dependent upon the pressure applied by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is applied. These devices make it easier to achieve a spray with a regular droplet size. Pressure threshold devices suitable for use with the present invention are known in the art and are described for example in WO91/13281 and EP311863B and EP516636, incorporated herein by reference. Such devices are commercially available from Pfeiffer GmbH and are also described in Bommer, R. Pharmaceutical Technology Europe, Sept 1999. Suitable intranasal devices produce droplets (measured using water as the liquid) in the range 1 to 200 μm, suitably 10 to 120 μm. Below 10 μm there is a risk of inhalation, therefore it is desirable to have no more than about 5% of droplets below 10 μm. Droplets above 120 μm do not spread as well as smaller droplets, so it is desirable to have no more than about 5% of droplets exceeding 120 μm. Bi-dose delivery is a further suitable feature of an intranasal delivery system for use with the vaccines according to the invention. Bi-dose devices contain two sub-doses of a single vaccine dose, one sub-dose for administration to each nostril. Generally, the two sub-doses are present in a single chamber and the construction of the device allows the efficient delivery of a single sub-dose at a time. Alternatively, a monodose device may be used for administering the vaccines according to the invention.

Thus, the invention provides in one aspect the use of a non-live influenza virus antigen preparation and an adjuvanted as herein defined in the manufacture of a vaccine formulation for a one-dose nasal vaccination against influenza. The vaccine may be administered in a mono-dose format or a bi-dose format (generally one sub-dose, optionally of 0.1 ml each, for each nostril).

In another aspect, the invention provides in another aspect the use of a low dose of non-live influenza virus antigen and an adjuvant as herein defined in the manufacture of a mucosal vaccine for immunisation against influenza. Although the vaccine of the invention may be administered as a single dose, components thereof may also be co-administered together at the same time or at different times (for instance influenza antigens could be administered separately, suitably at the same time as the administration of the adjuvant). In addition to a single route of administration, 2 different routes of administration may be used when two injections are administered. For example, the first administration (e.g. priming dose) of adjuvanted influenza antigens may be administered IM (or ID) and the second administration (e.g. booster dose) may be administered IN (or ID). In addition, the vaccines of the invention may be administered IM for priming doses and IN for booster doses, or vice versa.

In a further aspect, the invention provides a pharmaceutical kit comprising an intranasal spray device and a one-dose influenza virus vaccine. Suitably the one-dose influenza vaccine is non-live, optionally a split virus vaccine. Suitably the device is a bi- dose delivery device for two sub-doses of vaccine, optionally for two sub-doses of 0.1 ml each of vaccine.

Additionally, antigens may comprise a fragment and/or variant and/or hybrid antigen from the group of: cancer antigen, influenza virus, malarial protozoa, HIV, birch pollen, DerPl, grass pollen, RSV, non-typeable H. influenzae and Morexella. Antigens may be recombinant antigen and/or a hybrid antigen. The immunostimulatory compositions of the present invention can act as TLRl, TLR2, and TLR4 agonists. The compositions of the present invention are immunostimulatory compositions.

In another embodiment, methods are provided for preparing a composition comprising the steps of: culturing at least one cell comprising an outer membrane protein; killing said at least one cell with heat to form a cell paste; releasing at least one outer membrane protein from said cell; contacting said cell paste with at least one agent capable of solubilizing at least one lipid and optionally an osmolalic agent, forming a mixture comprising said at least one outer membrane protein, at least one endogenous liposaccharide and cell debris; adding an agent capable of separating said cell debris from said at least one outer membrane protein; separating said separated cell debris from said mixture; removing at least one agent capable of solubilizing at least one lipid and said optional osmolalic agent from said at least one outer membrane protein wherein said at least one outer membrane protein remains soluble.

Compositions of the present invention may also comprise excipients. Cells cultures of the present invention may be grown in animal-free media and/or serum free media.

Cells may be cultured in spinner flasks, roller bottles or hollow fibre systems but it is preferred for large scale production that stirred tank reactors are used particularly for suspension cultures. The stirred tanks are adapted for aeration using e.g. spargers, baffles or low shear impellers. For bubble columns and airlift reactors direct aeration with air or oxygen bubbles maybe used. Where the cells are cultured in a serum free culture media the media may be supplemented with a cell protective agent such as pluronic F-68 to help prevent cell damage as a result of the aeration method. Depending on the cell characteristics, either microcarriers maybe used as growth substrates for anchorage dependent cell lines or the cells maybe adapted to suspension culture (which is typical). The culturing of cells, particularly invertebrate cells may utilize a variety of operational modes such as fed-batch, repeated batch processing (see Drapeau et al (1994) cytotechnology 15: 103-109), extended batch method or perfusion culture. Although recombinantly transformed mammalian cells may be cultured in serum-containing media such as fetal calf serum (FCS), such cells may be cultured in synthetic serum-free media such as disclosed in Keen et al (1995) Cytotechnology 17: 153-163, or commercially available media such as ProCHO-CDM or UltraCHO™ (Cambrex NJ, USA), supplemented where necessary with an energy source such as glucose and synthetic growth factors such as recombinant insulin. The serum- free culturing of cells may require that those cells are adapted to grow in serum free conditions. One adaptation approach is to culture such cells in serum containing media and repeatedly exchange 80% of the culture medium for the serum- free media so that the cells learn to adapt in serum free conditions (see e.g. Scharfenberg K et al (1995) in Animal Cell technology: Developments towards the 21st century (Beuvery E.C. et al eds), pp619-623, Kluwer Academic publishers).

The following examples illustrate various aspects of this invention. These examples do not limit the scope of this invention which is defined by the appended claims.

Examples

Example 1: Preparation of OMP-based Composition

An OMP-based composition was made from a soluble mixture of purified integral outer membrane and lipooligosaccharides in solution from Heat-killed Neisseria meningitidis, solubilized by a zwitterionic detergent, as schematized in Figure 1 and described below.

OMPs extraction from the whole cells with a zwitterionic detergent Two hundred and fifty (250) grams of Neisseria meningitidis Strain 8047 cell paste was thawed for 12-24 hours at 2-8°C and suspended in IM sodium acetate buffer pH 5.0. The diluted cell paste was then mechanically homogenized using an IKA Ultra- Turrax homogenizer on ice for 20-30 minutes. The homogenized solution was then further diluted with 1.5 vol Milli-Q water and homogenized for 20-30 minutes at room temperature. Subsequently, one suspension volume of IM CaCl₂/6% lauryl dimethylbetaine (LDB) was added and the suspension was homogenized for an additional 60 minutes at room temperature. OMPs were extracted from the cell paste and the mixture is well solubilized. 20% Ethanol Precipitation

After the initial cell paste solubilization, ethanol at 4°C was slowly added to a final concentration of 20% v/v ethanol while homogenizing. For this step, a slow flow rate of ethanol addition, combined with efficient mixing was used so as to not create local high ethanol concentration in the suspension that might precipitate the proteins of interest. After ethanol addition, the suspension was homogenized for an additional 14-16 minutes at room temperature. The suspension was then clarified by pumping the mixture at a flux rate of 100 LMH on two 0.1m² disposable and scalable POD depth filters (Millipore Cat. MCOHC01FS1). This filtration step retains cellular debris (high molecular weight proteins) and nucleic acid that were both precipitated with the ethanol. Filters were then immediately chased using an equal volume of In-House Chase buffer (0.08M sodium acetate, 0.4M CaCl₂, 20% EtOH and 0.1%LDB).

1OX Concentration The OMP- filtrate was concentrated 1OX on a Pellicon Mini 0.1 m², 3OkDa ultrafiltration cassette (Millipore Cat. P2C030C01) at a flow rate of 33Oml/min and with a TMP manually adjusted at 10-11 psi. Afterwards, a micro-BCA assay (MTDV-0036, Rev.2) was performed on the 1OX concentrate and the suspension is incubated at 2-8°C overnight.

Diaflltration until [LDB] < 200ppm

After 1OX concentration, the solution was highly concentrated in LDB detergent (6.3 ± 0.1%) and contained undesirable impurities such as ethanol, sodium acetate and calcium chloride. A diaflltration was performed on the 1OX retentate 1) to reduce the detergent concentration, 2) to lower undesirable impurities, 3) to remove the lower molecular weight proteins and finally, 4) to have the OMPs and LOS in solution and in the final and human injectable PBS buffer.

The diaflltration was performed on another 3OkDa ultrafiltration cassette (Millipore Cat. P2C030C01) and two different buffers were used to diafilter the bulk at a constant volume. The first buffer was the TEN IX buffer pH 8.0 (5OmM TrisBase,

1OmM EDTA and 15OmM NaCl) against which the suspension was diafiltered for 20 DV. Product was further diafiltered against the PBS Buffer at pH 7.4 (Gibco, Cat. 70011-044) until the LDB concentration was below 200 ppm, as determined by an on-line HPLC method (MTDV-0042). For this diafiltration step, it was important to diafilter first against the TEN buffer to remove calcium from the retentate before it comes in contact with PBS. This step avoided possible precipitate formation between the phosphate in the PBS buffer and the calcium from the calcium chloride buffer.

Concentration to 4.5mg/ml and if necessary, Diafiltration until [LDB] < 300ppm

The suspension, which was below 200ppm LDB, was concentrated using the same ultrafiltration cassette and set-up to 4.5mg/ml using the micro-BCA assay result obtained on the Retentate after 1OX concentration and considering a loss of 50%. After concentration, LDB concentration was measured. If LDB concentration was >300ppm, the suspension was diafiltered against an additional volume of PBS buffer. I f LDB concentration was lower than 300ppm, no additional volume of PBS buffer was passed.

Sterile Filtration The final product was sterile filtered in the BioSafety Cabinet at a constant pressure of 50psi and through two 0.22 sterilizing Grade and disposable Millipk-60 filter units (Millipore, Cat.MPGL066H2)

Example 2; Composition made by V2 or Vl Method

OMP -based composition was prepared by the method described in Example 1 and Figures 1 and 2 (referred to as V2 below) as well as by the methods described in US Patent No. 5,726,292 and 5,985,284 (referred to as Vl below). Composition of each OMP -based composition is summarized in Table 3 herein.

Table 3

Example 3. Immunogenicity and potency of intranasal monovalent influenza immunogenic compositions

The immunogenicity and potency of OMP -based composition prepared by the methods described in Example 1 (referred to herein as V2 Proteosome) was compared with the immunogenicity and potency if OMP -based composition prepared by the methods described in US patent No. 5,726,292 and 5,985,284 (Vl Proteosome) and monovalent detergent-split influenza immunogenic compositions comprising OMP -based compositions described in PCT7US02/07108 referred to herein as Protollin. The protocol used for testing is described below:

Studv Design

Table 4

Group Λ Split Flu Antigen (meg HA)

IN.T Adjuvant

No. [A/New York (H3N2)]

1 10 0.3 V2 Proteosome (1 meg)

2 10 1 V2 Proteosome (1 meg)

3 10 3 V2 Proteosome (1 meg)

4 10 10 V2 Proteosome (1 meg)

5 10 0.3 Protollin (3 meg)

6 10 1 Protollin (3 meg)

7 10 3 Protollin (3 meg)

8 10 10 Protollin (3 meg)

9 10 0.3 Vl Proteosome (est. 0.6 meg)

10 10 1 Vl Proteosome (est. 2 meg)

11 10 3 Vl Proteosome (est. 6 meg)

12 10 10 Vl Proteosome (est. 20 meg)

13 10 0.3 PBS

14 10 1 PBS

15 10 3 PBS

16 10 10 PBS

Anesthetized six- to eight-week-old female BALB/c mice were immunized intranasally on study days 0 and 14 with indicated amounts of hemagglutinin (HA) antigen formulated with V2 Proteosome (Groups 1 through 4), Protollin (Groups 5 through 8), or Vl Proteosome (Groups 9 through 12). Control animals received the same doses of HA antigen without adjuvant (Groups 13 through 16). Whole blood and lung lavage was collected at sacrifice on study day 21, and the amplitude of the antibody response to the immunizing detergent-split flu antigen was determined by enzyme-linked immunosorbent assay (ELISA - measurement of serum IgG and lung IgA binding antibody) and hemagglutination inhibition using rooster erythrocytes (HAI - measurement of total functional serum antibody).

Results

The mouse serum IgG responses to different doses of H3N2 detergent-split influenza antigen (range of 0.3 to 10 meg) admixed with Vl Proteosome, Protollin, or V2 Proteosome are shown in Figure 3. At the doses used, potent anti-A/New York antibody responses were observed in the serum IgG ELISA when the antigen was administered with OMP -based compositions, but not when the same doses of antigen were administered with PBS buffer alone, indicating a strong immunostimulatory effect. A dose dependent increase in serum IgG was observed in each group regardless of the

OMP -based composition. In this immunological compartment, 1 meg of V2 Proteosome compares favorably with 3 meg of Protollin, and both these compositions are superior to Vl Proteosome at antigen doses of 0.3, 1, and 3 meg, in increasing serum IgG. Additional data from this study show similar immune responses for V2 Proteosome used at 3 and 10 meg to those observed with 1 meg.

The mouse lung IgA responses to different doses of H3N2 detergent-split influenza antigen (range of 0.3 to 10 meg) combined with Vl Proteosome, Protollin, or V2 Proteosome are shown in Figure 4. At the doses used, potent anti-A/New York antibody responses were observed in the lung IgA ELISA when the antigen was administered with Proteosome or Protollin, but not when the same doses of antigen were administered with PBS buffer alone, indicating a strong immunostimulatory effect for the OMP -based compositions. A dose-dependent increase in lung IgA was observed in each group regardless of OMP -based composition. In this immunological compartment, 1 meg of V2 Proteosome elicits lower levels of antibody than 3 meg of Protollin, but both compositions are superior to Vl Proteosome at antigen doses of 0.3, 1, and 3 meg in eliciting lung IgA.

The mouse serum HAI responses to different doses of H3N2 detergent-split influenza antigen (range of 0.3 to 10 meg) combined with Vl Proteosome, Protollin, or V2 Proteosome are shown in Figure 5. At the doses used, potent anti-A/New York antibody responses were observed in serum HAI when the antigen was administered with

Proteosome or Protollin. The serum HAI responses were not performed on the group that received antigen alone because previous studies demonstrated that no antibody was elicited under these conditions. A dose dependent increase in total antibody was observed in each group regardless of the OMP -based composition. In this immunological compartment, all three compositions elicit comparable antibody levels at all antigen doses.

Example 4; Allergen with V2 Proteosome

V2 proteosome can be evaluated as an immunostimulatory composition for use in therapeutic allergy vaccines using a murine allergy / asthma model in which mice made allergic to Der p 1 (a major house dust mite allergen) by intraperitoneal injection of the allergen adsorbed to alum are administered vaccines containing recombinant proDer p 1 plus V2 proteosome via the intranasal / intrapulmonary route.

Administration of a vaccine containing recombinant proDer p 1 plus V2 proteosome should drive Der p 1 -specific Th2-suppressive cellular immune responses, as measured by suppression of Th2 cytokine (IL-5, IL 13) responses and induction of the ThI cytokine interferon (IFN)-gamma upon stimulation of spleen cells from vaccinated mice with natural Derpl.

The finding that V2 proteosome alone prevented eosinophil influx, but did not induce Der p 1 -specific humoral or cellular responses, suggests that the protective effect of V2 proteosome for eosinophil influx is due to non-specific immunomodulatory activity associated with this composition. The fact that the protective effect was still present three weeks following the final vaccination indicates the non-specific effect is fairly long-lived. These findings suggest that, aside from its usefulness as a immunostimulatory composition and vaccine adjuvant, V2 proteosome may also have a potential use as a monotherapy for the treatment for allergy.

Example 5; RSV with V2 Immunostimulatory Composition

8 BALB/c mice were anaesthetized by isoflurane inhalation and immunized IN with a volume of 25μl (12.5 μl/nare) twice on days 0 and 14 with 9, 3 and lμg of V2 formulated with 2μg of V2. Mice also received under the same conditions, the same dose of FG with 3μg of Protollin. Finally, the protection efficacy of IN immunizations with V2 was compared to IM injections with 2 doses of ASOlB (2μg) formulated with 2μg of FG. Two weeks post-boost, 5 mice in each group were anesthetised and challenged IN with 107 TCID50 of Long strain RSV. Serums and bronchoalveolar lavages (BAL) were collected from the remaining mice at this time. Challenged mice were euthanized 4 days post- challenge when lungs were collected for virus titration.

Lungs from immunized animals were washed, weighed and homogenized individually in RSV media (D- MEM with 50% 199-H media, 0.5% fetal bovine serum, 2mM glutamine and 50μg/mL gentamicin (all Invitrogen, Burlington, ON)) with an automated Potter homogenizer then centrifuged at 2655 x g for 2 minutes at 4°C. The supernatants were titered and RSV was detected by indirect immunofluorescence. A well was considered positive when >1 fluorescent syncytium was detected and titers were normalized per gram of lung tissue. Titrations were performed on Vero cells and detected by immunofluorescence (reported as log of number of infectious virions per gram of lung homogenate). The bars represent the geometric means of each group. These results are shown in Figure 6.

Pooled sera from immunized animals were serially diluted from a starting dilution of 1:8 in RSV media in 96-well plates (20 μl/well). Control wells contained RSV media only or goat polyclonal anti-RSV antibody at 1:50 (Biodesign international). 500-1000 infectious doses of RSV Long strain were added, the plates were incubated for 20 minutes at 33°C and the mixture was transferred to 96-well flat-bottomed plates previously seeded with 1x105 cells/mL Vero cells. After 5-6 days at the same temperature, supernatants were removed; plates were washed with PBS and adhering cells fixed with 1% paraformaldehyde in PBS for 1 hour, followed by indirect immunofluorescence (IFA) as described for lung titration. RSV-specific serum neutralization titers following V2 and FG immunization are shown from pooled serum samples. These results are shown in Figure 7.

Antigen-specific IgG and IgA titers were determined on individual serum samples by ELISA. Briefly, 96-well plates were coated with 7.5μg/ml of UV-inactivated RSV A or B or homologous protein (FG) and incubated overnight at 4°C. Serum or BAL samples were serially diluted starting at 1 :50 (IgG) or 1:2 (IgA), respectively. Bound antibody was detected with HRP-conjugated anti-mouse IgG, (Sigma). RSV-specific serum IgG, and BAL IgA titers were quantified by ELISA. RSV-specific serum IgG and BAL IgA titers following V2 and FG immunization. RSV-specific serum IgG, and BAL IgA titers were quantified by ELISA. Results are shown as geometric mean of 3 animals per group with upper and lower 95% confidence intervals. Antigen-specific IgG and IgA titers are shown in Figures 8 and 9, respectively. V2 Proteosome with FG (chimeric recombinant RSV antigen) elicited complete protection against challenge in mice. Intranasal (IN) immunization with V2 Proteosome and FG elicited good systemic (IgG) and mucosal (IgA) neutralization titers, while intramuscular (IM) immunization with the same antigen did not elicit mucosal antibodies. Importantly, IN immunization with V2 Proteosome and FG elicit both systemic and mucosal RSV A and B cross-reactive antibodies. Again, IM immunization with the same antigen does not elicit mucosal responses.

Example 6; Trivalent Flu adjuvanted with V2 Adjuvant An immunogenic composition against H. influenzae is prepared by admixing an

OMP -based composition of Example 1 with trivalent flu.

Preparation of trivalent flu antigen Antigen comprising equal portion of the following three antigens are prepared:

Η1N1, Η3N2 and B strain. The strains for each antigen are designated seasonally by the World Health Organization (WHO).

Briefly, preparation of each component of the trivalent composition involves harvesting allantoic fluid from virus inoculated eggs followed by clarification, inactivation of the virus, concentration by diafiltration/ultrafiltration, banding the virus on sucrose gradient density centrifugation, pelleting, extracting the re-suspended pellet with Triton X-IOO, or NP-40 or other suitable detergent, and centrifuging and collecting the supernatant. This method is repeated as required, pooled and stored at 2-8 ⁰C.

Split flu trivalent antigen can be combined with OMP -based immunostimulatory composition at a concentration of about 30 mg/mL for each antigen. Dosing of the trivalent flu vaccine may be at a dose of 15 μg/0.5 mL for an intramuscular injection. Dosing for an intranasal injection may include 15 μg/0.2 mL for each antigen for a total antigen dose of 75 μg/mL, which can be administered by splitting into two equal doses per nostril. V2 Proteosomes can be diluted from an initial concentration of about 4.5 mg of total protein/mL to about 3.0 mg total protein/mL to a dilution of about 800μg/mL, 325μg/mL, 125μg/mL or 50 μg/mL by mixing with appropriate volumes of influenza antigens and PBS diluent, to give antigen concentrations listed in this example. An intranasal injection of 0.2 mL would therefore deliver 160μg, 65μg, 25μg or 10 μg of total protein. About 60% to about 70% of the total protein of the V2 proteosome comprise Class I, II and IV outer membrane proteins. It is expected that the minimally effective V2 proteosome dose to adjuvant influenza antigens will be found within this dose range.

Example 7; V2 Proteosome act as TLR agonist

OMP -based compositions are derived from the outer membrane proteins (OMP) of Neisseria meningitides and are potent inducers of both mucosal and systemic immunity in humans and animals. V2 proteosome has been shown capable of interacting with antigens to form immunological compositions which, when instilled intranasally (Ln.), elicit enhanced mucosal and systemic immune responses in different models of disease such as influenza. To investigate whether Proteosomes act through Toll-like receptor (TLR) signalling, TLR experimental models such as in vitro cell-based assays and TLR knockout mice were used.

NF-κB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents, leading to cell activation, differentiation, or apoptosis. Innate immunity pathway components such as Toll-like receptors agonists are well known to activate NF -kB tranlocation and thereby activating promoter containing NF -KB binding elements (Janeway, CA, P Travers, M Walport, MJ Shlomchik. Immunobiology , Book. New York: Garland Publishing, 2005. Akira, 2006. TLR signaling. Curr Top Microbiol Immunol. 2006;311: 1-16. Janeway CA Jr, Medzhitov R. 2002. Innate immune recognition. Amut Rev Immunol. 20: 197-216). Reporter constructs utilizing the NF-κB promoter element driving the expression of the secreted alkaline phosphatise (SEAP) are used to measure such activity. HEK293 cells stably transfected with the gene coding for human TLR 1 and 2 or TLR4/MD2/CD14 (Invivogen, San Diego, CA) were cultured in 24-well plates in 500 μl/well DMEM supplemented with 10% heat-inactivated FBS in a 5% CO₂ incubator (DMEM media). At 80% confluence, cultures were transiently transfected with 500 ng/ml SEAP (secreted form of human embryonic alkaline phosphatase) reporter plasmid (pNifty2-Seap) (Invivogen) in the presence of lipofectamine 2000 (Invitrogen,

Carlsbad, CA) in culture medium. Plasmid DNA and lipofectamine were diluted separately in serum-free medium and incubated at room temperature for 5 minutes. After incubation, the diluted DNA in lipofectamine-DMEM solution were mixed and the mixtures were incubated at room temperature for 20 min. Aliquots of 100 μl of the

DNA/lipofectamine mixture containing 500 ng of plasmid DNA and lipofectamine were added on top of 400 μl of DMEM media of to each well of the cell culture plate, and the cultures were continued for 16 hours. After transfection, medium was replaced with fresh DMEM culture medium without serum, adjuvants were added to the cultures, and the cultures were continued for 5 hours. At the end of the treatment with agonists, 50 μl of culture supernatant was collected from each culture wells and used for SEAP assay following manufacturer's protocol (Invivogen). Briefly, culture supernatants were incubated with QUANTI-Blue phosphate substrate (Invivogen) and the purple color generated was measured by a plate reader at 650 nm. The data are shown as the relative optic density measure at 650nm which reflect the TLR-dependent NF -KB activity.

Transfected cells were exposed for 5 hours to V2 Proteosome at different concentration or PBS as a negative control. A dose per well of Oμg, 0.01 μg, 0.1 μg, and 1.0 μg for TLR1/2 assay was used. For TLR4 assay, a dose of 0, 0.1 μg, and 1.0 μg per well was used. The results of this study are shown in Figures 1OA and 1OB. NF-κB activation indicated that V2 Proteosome acted through TLR1-TLR2 (see Figure 10A) as well as the TLR4 receptor in a concentration dependent manner (see Figure 10B). These results demonstrate the involvement of TLRs in vitro by V2 Proteosome and suggest that V2 Proteosome likely activates the innate arm of the immune system. Data also indicated that pre- incubation of TLR-2 transfected HEK-293 cells with excess of neutralizing anti-TLR2 antibodies abolished NF -kB activation potential of V2 Proteosomes (see Figure 11). Moreover, control cells (not transfected with TLR2) confirmed that TLR2 is a receptor targeted by V2 Proteosome components.

The stability of V2 proteosomes after 6 months storage at -80⁰C, 2-8°C and 37°C was assessed using the TLR1/2 cell-based assay (see Figure 12). A similar activation and stability was demonstrated using the TLR4 cell based assay (data not shown). The immunogenicity of a mucosally delivered subunit Proteosome-influenza vaccine was assessed in TLR2, TLR4 and MyD88 knockout mice. A/New Caledonia split flu antigens were instilled by intranasal route with or without V2 Proteosomes to TLR4^7", MyDδδ^7" mice and to their wild type counterpart, i.e. C57BL/6. Immunization schedule was performed as follow: First intranasal immunization was performed at day 0, and a boost performed at day 14 with the same formulation for both immunizations (either with split flu alone or either split flu combined with V2 Proteosomes). At day 28, mice were euthanized and exsanguinated by cardiac puncture for antibody determination by ELISA using split influenza A/New Caledonia as solid-phase antigen. Co-instillation of SFV with V2 Proteosomes increased the immunogenicity of the subunit influenza vaccine in C57BL/6 mice (Figure 13). In addition, the immunogenicity of the subunit influenza vaccine tested decreased significantly in TLR4^{" "} and MyD88^{" "} confirming the involvement of the TLR4 and the adaptor molecule MyD88 in the elicitation of influenza specific immune response. In conclusion, these data provide evidence that V2 proteosomes are able to induce specific TLR1/2 and TLR4 signalling and suggest that V2 proteosome formulated vaccines with antigens such as influenza could be highly effective and excellent candidates for mucosal immunization using the intranasal route.

Claims

CLAIMS:

1. A method of preparing a composition comprising the steps of:

releasing from at least one cell a composition comprising at least one outer membrane protein from said at least one cell comprising contacting said at least one said cell with at least one agent capable of solubilizing at least one lipid and optionally an osmalalic agent, forming a mixture comprising said at least one outer membrane protein, at least one endogenous liposaccharide and cell debris; adding an agent capable of separating said cell debris from said at least one outer membrane protein; separating said separated cell debris from said mixture; removing at least one agent capable of solubilizing at least one lipid and said optional osmolalic agent from said at least one outer membrane protein wherein said at least one outer membrane protein remains soluble.

2. The method of claim 1, wherein said at least one cell is Gram negative bacteria.

3. The method of claim 2, wherein said Gram negative bacteria is a Meningococcus.

4. The method of claim 3, wherein said Meningococcus is from Group B type 2b.

5. The method of claim 2, wherein said at least one cell is from the genus Neisseria.

6. The method of claim 2, wherein the bacteria is selected from the group of wild type Neisseria meningitidis group B, Haemophilus influenzae type b, Neisseria gonorrhoeae, Escherichia coli, Pseudomonas aeruginosa. Bordetella pertussis, Haemophilus aegyptius, Haemophilus ducreyi, Haemophilus parainfluenza, Moraxella catarrhalis, and Neisseria meningitides, Shigella flexneri or Plesiomonas shigelloides, Alcaligenes, Bacteroides, Bordetella, Borrellia, Brucella, Campylobacter, Chlamydia, Citrobacter, Edwardsiella, Ehrlicha, Enter obacter, Escherichia, Francisella, Fusobacterium, Gardnerella,

Hemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Moraxella, Morganella, Neiserria, Pasteurella, Proteus, Providencia, Plesiomonas, Porphyromonas, Prevotella, Pseudomonas, Rickettsia, Salmonella, Serratia, other Shigella, Spirllum, Veillonella, Vibrio, and Yersinia species.

7. The method of claim 6, wherein the bacteria is Neisseria meningitidis.

8. The method of claim 2, wherein the said gram negative bacteria is engineered bacteria.

9. The method of claim 1, further comprising admixing said composition with at least one antigen to form an immunogenic composition.

10. The method of claim 1, wherein the agent capable of solubilizing at least one lipid is a detergent.

11. The method of claim 10, wherein the detergent is at a low concentration.

12. The method of claim 1, wherein the optional osmolalic agent comprises sugar and/or salt.

13. The method of claim 1, wherein the optional osmolalic agent is calcium chloride.

14. The method of claim 1, wherein the agent capable of separating said cell debris from said at least one outer membrane protein comprises at least one alcohol.

15. The method of claim 14, wherein said at least one alcohol is ethanol.

16. The method of claim 15, wherein said ethanol is added to said mixture comprising at least one outer membrane protein, at least one endogenous liposaccharide and cell debris to a total volume by weight of up to about 50%.

17. The method of claim 1, wherein said cell debris is separated from said mixture by precipitation.

18. The method of claim 1, wherein said cell debris is removed from said mixture by a at least one method selected from: centrifugation, depth filtration, and filtration.

19. The method of claim 1, wherein said removing at least one agent capable of solubilizing lipids and osmolalic agent from said at least one outer membrane protein wherein said at least one outer membrane protein remains soluble is by diafiltration.

20. The method of claim 1, further comprising sterilizing said composition by microfilitration.

21. The method of claim 19, further comprising adding a composition comprising phosphate buffered saline during diafiltration.

22. The method of claim 1, wherein low molecular weight proteins are removed from the mixture.

23. The method of claim 10, wherein said at least one outer membrane is released with detergent at a concentration of about 40,000 ppm to about 70,000 ppm.

24. The method of step 23, wherein the concentration of said detergent after said removing step is less than 20,000 ppm.

25. The method of claim 9, wherein said at least one antigen is from a whole or disrupted microorganism.

26. The method of claim 25, wherein said microorganism is selected from virus, bacteria, fungi, and protozoa.

27. The method of claim 26, wherein said at least one antigen is from an influenza virus.

28. The method of claim 27, wherein said at least one antigen comprises at least one hemagglutinin.

The method of claim 9, wherein said at least one antigen comprises a fragment and/or variant and/or hybrid antigen from the group of: cancer antigen, influenza virus, malarial protozoa, HIV, birch pollen, DerPl, grass pollen, RSV, non- typeable H. influenzae and Morexella.

29. The method of claim 9, wherein said at least one antigen is a recombinant antigen and/or a hybrid antigen.

30. The method of claim 9, further comprising administering said immunogenic composition to a mammal and inducing an enhanced immune response in said mammal.

31. The methods of claim 31, wherein said mammal is a human.

32. The method of claim 31 , wherein said immunogenic composition is administered by a route selected from: rectal, intramuscular, subcutaneous, intravenous, intraperitoneal, mucosal, enteral, parenteral and inhalation.

33. The method of claim 31 , wherein said immunogenic composition is administered by a mucosal route.

34. The method of claim 34, wherein the mucosal route is via the nasal, oropharyngeal, ocular or genitourinary mucosa.

35. The method of claim 1, further comprising administering said composition to a mammal and inducing an innate immune response against a viral infection.

36. The method of claim 1, further comprising administering said composition to a mammal in need thereof for the treatment of a neurological disease.

37. The method of claim 37, wherein said mammal is a human.

38. The method of claim 38, wherein the disease is an amyloidal disease.

39. The method of claim 39, wherein the disease is Alzheimer disease.

40. The method of claim 1, further comprising homogenizing said composition.

41. The method of claim 41, wherein homogenizing said composition creates uniform sized particles of outer membrane proteins.

42. The method of claim 1, wherein said composition is an immunostimulatory composition.

43. An composition comprising at least one outer membrane protein and an endogenous liposacharide wherein said endogenous liposaccharide is in the range of about 0.03 grams to about 0.99 grams endogenous liposaccharide to 1.0 gram of at least one outer membrane protein weight.

44. The composition of claim 44, wherein the endogenous liposaccharide is in the range of about 15% to about 300% of the composition by weight.

45. The composition of claim 44, further comprising at least one lipid wherein said lipid comprises about 1.5 to about 1.6 grams of lipid per 1.0 gram at least one outer membrane protein by weight.

46. The composition of claim 44, further comprising DNA, wherein said DNA comprises <1.0% of the total composition by weight.

47. The composition of claim 44, further comprising at least one antigen.

48. The composition of claim 48, wherein said at least one antigen is a whole or disrupted microorganism.

49. The composition of claim 49, wherein said microorganism is selected from virus, bacteria, fungi and protozoa.

50. The composition of claim 50, wherein said at least one antigen is from an influenza virus.

51. The composition of claim 51, wherein said at least one antigen comprises at least one hemagglutinin.

52. The composition of claim 48, wherein at least one antigen comprises a fragment and/or variant and/or hybrid antigen from the group of: influenza virus, malarial protozoa, HIV, birch pollen, DerPl, grass pollen, RSV, non-typeable H. influenzae and Morexella.

53. The composition of claim 48, wherein said at least one antigen is a recombinant antigen and/or a hybrid antigen.

54. The composition of claim 44, wherein the composition is capable of acting as a TLRl, TLR2, and TLR4 agonist.

55. The composition of claim 48 wherein said composition comprises more than one antigen.

56. The composition of claim 56 wherein said antigen is a trivalent flu antigen.

57. The composition of claim 57 comprising about 75 μg/mL of flu antigen.

58. The composition of claim 57 wherein about a 0.2 mL sample of said composition comprises total protein from at least one cell comprising at least one outer membrane protein selected from the group of: lOμg, 25μg, 65μg, and

160μg.

59. The composition of claim 58 further comprising PBS buffer.

60. The composition of claim 44, wherein the composition is an immunostimulatory composition.

61. The composition of claim 44, wherein said liposaccharide is an LOS.

62. A vaccine comprising the composition of claim 44.

63. A method of preparing an composition comprising the steps of:

culturing at least one cell comprising an outer membrane protein; killing said at least one cell with heat to form a cell paste; releasing at least one outer membrane protein from said cell; contacting said cell paste with at least one agent capable of solubilizing at least one lipid and an osmolalic agent, forming a mixture comprising said at least one outer membrane protein, at least one endogenous liposaccharide and cell debris; adding an agent capable of separating said cell debris from said at least one outer membrane protein; separating said separated cell debris from said mixture; removing at least one agent capable of solubilizing at least one lipid and said osmolalic agent from said at least one outer membrane protein wherein said at least one outer membrane protein remains soluble.

64. The method of claim 63, further comprising adding excipients to said composition.

65. The method of claim 63, wherein said culture is grown in animal free media.

66. The method of claim 63, further comprising adding at least one antigen to said composition.

67. The method of claim 63, wherein said composition is an immunostimulatory composition.