CA2362204C - Immunogenic complexes and methods relating thereto - Google Patents

Abstract

The present invention relates generally to an immunogenic complex comprising a charged organic carrier and a charged antigen and, more particularly, a negatively charged organic carrier and a positively charged antigen. The complexes of the present invention are useful, inter alia, as therapeutic and/or prophylactic agents for facilitating the induction of a cytotoxic T-lymphocyte response to an antigen.

Description

IMMUNOGENIC COMPLEXES AND METHODS RELATING THERETO
FIELD OF THE INVENTION

The present invention relates generally to an immunogenic complex comprising a charged organic carrier and a charged antigen and, more particularly, a negatively charged organic carrier and a positively charged antigen. The complexes of the present invention are useful, inter alia, as therapeutic and/or prophylactic agents for facilitating the induction of a cytotoxic T-lymphocyte response to an antigen.
BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

There is an increasing belief that co-delivery of antigen and adjuvant to the same antigen-presenting-cell (APC) is preferable and sometimes essential for induction of appropriate immune responses. For example, the ability of saponin-based adjuvants to induce CD8 CTL responses is attributed to their ability to cause endosomal escape of antigen, a mechanism which requires co-delivery. Particle formation which comprises a stable complex of adjuvant and antigen is the simplest way to achieve co-delivery.
The usefulness of ISCOMTM technology derives partly from the immunomodulatory activity of saponins and partly from their ability to form complexes with hydrophobic or amphipathic immunogens. However, many molecules lack hydrophobic regions and in fact such molecules are preferred as recombinant proteins because of their easier expression and purification.

Accordingly, there is a need to develop immunogenic complexes which facilitate the co-delivery of antigens and carriers which otherwise do not usually form sufficiently stable complexes. For example, complexes comprising antigens which lack hydrophobic regions together with adjuvant.

In work leading up to the present invention, the inventors have developed an immunogenic complex based on the electrostatic association of an antigen and an organic carrier, such as an adjuvant. This electrostatic association permits co-delivery of the antigen and the organic carrier to the immune system. Accordingly, by establishing an electrostatic association, antigens of interest. (irrespective of their hydrophobicity) can be co-delivered with an; organic carrier , for the purpose, for example, of inducing a cytotoxic T-lymphocyte response to the antigen.

SUMMARY OF THE ICON

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The subject specification contains amino acid sequence information prepared using the programme Patentln Version 2.0, presented herein after the bibliography. Each amino acid sequence is identified in the sequence listing by the numeric indicator <
210 >
followed by the sequence identifier (e. g. < 210 > 1, < 210 > 2, etc.). The length, type of - 20 sequence (protein (PRT), etc) and source organism for each amino acid sequence are indicated by information provided in the numeric indicator fields < 211 >, <
212 > and < 213 > , respectively. Amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field < 400 > followed by the sequence identifier (e. g. < 400 > 1, < 400 > 2, etc).

One aspect of the present invention relates to an immunogenic complex comprising a charged organic complex and a charged antigen which organic complex and antigen are electrostatically associated.

Another aspect of the present invention more particularly provides an immunogenic complex comprising a negatively charged organic carrier and a positively charged antigen which organic carrier and antigen are electrostatically associated.

Still another aspect of the present invention provides an immunogenic complex comprising a negatively charged organic carrier and a positively charged protein which organic carrier and protein are electrostatically associated.

Yet another aspect of the present invention provides an immunogenic complex comprising a negatively charged adjuvant and a positively charged protein which adjuvant and protein are electrostatically associated.

Yet still another aspect of the present invention provides an immunogenic complex comprising a negatively charged adjuvant and a positively charged protein, wherein said negatively charged adjuvant is a naturally negatively charged adjuvant which has been modified to increase the degree of its negative charge, which adjuvant and protein are electrostatically associated.

Still another aspect of the present invention provides an immunogenic complex comprising a negatively charged adjuvant and a positively charged protein, wherein said positively charged protein is a naturally positively charged protein which has been modified to increase the degree of its positive charge, which adjuvant and protein are electrostatically associated.

Still yet another aspect of the present invention provides an immunogenic complex comprising a negatively charged adjuvant and a positively charged protein, wherein said negatively charged adjuvant is a naturally negatively charged adjuvant which has been modified to increase the degree of its negative charge and said positively charged protein is a naturally positively charged protein which has been modified to increase the degree of its positive charge, which adjuvant and protein are electrostatically associated.

In accordance with an aspect of the present invention, there is provided an electrostatically-associated immunogenic complex, comprising: (A) a negatively-charged organic complex that comprises a saponin and a sterol, and (B) a positively-charged antigen, where said organic complex and antigen are associated only by electrostatic interaction.

Another further aspect of the present invention relates to a method of eliciting, inducing or otherwise facilitating, in a mammal, an immune response to an antigen said method comprising administering to said mammal an effective amount of an immunogenic complex or a vaccine composition as hereinbefore described.

Yet another further aspect of the present invention relates to a method of treating a disease condition in a mammal said method comprising administering to said mammal an effective amount of an immunogenic complex or a vaccine composition as hereinbefore described wherein administering said composition elicits, induces or otherwise facilitates an immune response which inhibits, halts, delays or prevents the onset or progression of the disease condition.

Still another further aspect the present invention relates to the use an immunogenic complex or vaccine composition as hereinbefore defined in the manufacture of a medicament for inhibiting, halting, delaying or preventing the onset or progression of a disease condition.

Still yet another further aspect of the present invention relates to an agent for use in inhibiting, halting, delaying or preventing the onset or progression of a disease condition.
Said agent comprising an immunogenic complex or vaccine composition as hereinbefore defined.

Single and three letter abbreviations used throughout the specification are defined in Table 1.

Single and three letter amino acid abbreviations Amino Acid Three-letter One-letter Abbreviation Symbol Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamine Gln Q
Glutamic acid Glu E
Glycine Gly G
Histidine His H
Isoleucine Ile I

Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P

Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V

Any residue Xaa x BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation of the sucrose gradient analysis of ISCOMATRIXTM
formulated with DPPC (Figure 1 A), CDL (Figure 1 B), DPL (Figure 1 C), MPL
(Figure 1 D), DPA (Figure 1 E) and DPPG (Figure IF). In each case it can be seen that lipid and 3H
overlap indicating incorporation of each lipid into the ISCOMATRIXTM
structure.

Figure 2 is a graphical representation of the sucrose gradient analysis of four of the ISCOMATRIXTM formulations of Example 1 after mixing with HpE. It can be seen that most of the HpE is with CDL and DPL ISCOMATRIXTM but only part is associated with DPPC and DPPG ISCOMATRIXTM.

Figure 3 is a graphical representation of the sucrose gradient analysis of two of the ISCOMATRIXTM formulations of Example 1 after mixing with ESO. It can be seen that most of the ESO is associated with DPL ISCOMATRIXTM but only part is associated with DPPC ISCOMATRIXTM

Figure 4 is a graphical representation of antibody responses to ESO
formulations. It can be seen that ESO associated ISCOMATRIXTM incudes higher antibody responses than ESO
alone especially in the Thl subtype IgG2a.

Figure 5 is a graphical representation of CTL analysis of mice immunised with ESO
(Figures 5A, 5C) and ESO associated ISCOMATRIXTM (Figures 513, 5D) using SLLMWITQCFL (<400>1)(Figure 5A, 513) and SLLMWITQC (<400>2) (Figure 5C, 5D) peptides for stimulation and targets. It can be seen that ESO associated ISCOMATRIXTM
induces a CTL response but ESO alone does not.

Figure 6 is a graphical representation of the sucrose gradient analysis of six of the ISCOMATRIXTM formulations of Example 1 after mixing with E6E7. It can be seen that most of the E6E7 is associated with CDL, DPL and DPA ISCOMATRIX', less associated with the MPL and DPPG ISCOMATRIXTM and even less again with the DPPC
ISCOMATRIXTM.

Figure 7 is a graphical representation of CTL analysis in mice immunised with ISCOMATRIXTM (Figure 7A) and E6E7 DPPC ISCOMATRIXTM (Figure 7B). It can be seen that E6E7 DPL ISCOMATRIXTM induces CTL responses but E6E7 DPPC
ISCOMATRIXTM does not.

Figure 8 is a graphical representation of the sucrose gradient analysis of two of the ISCOMATRIXTM formulations from Example 1 after mixing with HpC. It can be seen that more HpC is associated with DPL ISCOMATRIXTM than with DPPC ISCOMATRIXTM
where there is very little association.

Figure 9 is a graphical representation of the sucrose gradient analysis of two DPPC

ISCOMATRIXTM formulations after mixing with E6E7 at pH6 (Figure 6A) and pH7.2 (Figure 6B). It can be seen that more E6E7 associates with DPPC ISCOMATRIXTM
at pH6 than at pH7.2.

Figure 10 is a graphical representation of the sucrose gradient analysis of ISCOMATRIXTM
formulations after mixing with modified HpC from Example 11. It can be seen that addition of 6K to HpC increases the association with DPPC ISCOMATRIXTM to a level comparable to that with the 6H and CHL ISCOMATRIXTM formulation.

Figure 11 is a graphical representation of the sucrose gradient analysis of four polytope ISCOMTM and ISCOMATRIXTM formulations from Example 13. It can be seen that there is some association of the 6K polytope with ISCOMATRIXTM but there is no association if the 6K are not present. The 6K polytope association with ISCOMATRIXTM was comparable to hydrophobic incorporation of the PAL polytope into ISCOMsTM but less than association between 6H polytope and CHL ISCOMATRIXTM

Figure 12 is a graphical representation of the CTL analysis of four synthetic polytope ISCOMATRIXTM formulations from Example 13. It can be seen that 6K polytope ISCOMATRIXTM induced CTL responses against all 4 epitopes in the polytope (Figure 12C) but the polytope ISCOMATRIXTM formulation without a tag only induced a low CTL

response to one of the epitopes (Figure 12D). The CTL responses for the 6K
polytope ISCOMATRIXTM were comparable to those induced with the PAL polytope ISCOMTM
(Figure 12A) and the 6H polytope CHL ISCOMATRIXTM (Figure 12B).

Figure 13 is a graphical representation of the sucrose gradient analysis of ten recombinant ISCOMATRIXTM formulations from Example 16. It can be seen that the combination of adding a 6K tag with CDL or DPL ISCOMATRIXTM gives increased association over with DPPC ISCOMATRIXTM and to then combine these with low pH increase the capacity to associate even further. The association achieved with the combination of 6K, CDL
ISCOMATRIXTM and low pH gave almost complete association of the polytope with ISCOMATRIXTM and the association was greater than could be achieved with 6H
polytope CHL ISCOMATRIXTM.

Figure 14 is a graphical representation of the CTL analysis of the 6K polytope CDL
ISCOMATRIXTM pH4.3 (Figure 14A) and 6H polytope CHL ISCOMATRIXTM (Figure 14B) formulations. It can be seen that CTL responses were induced to all 4 epitopes in the polytope for both the formulations but the responses were very low to the TYQ
epitope.
Figure 15 is a graphical representation of the liposomes mixed with E6E7 from Example 18. It can be seen that most of the E6E7 was associated with the DPL liposomes but very little E6E7 was associated with the DPPC liposomes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated, in part, on the development of an immunogenic complex formulation which utilises electrostatic interactions to associate an antigen and a carrier thereby facilitating, inter alia, the co-delivery of these molecules to the immune system. The immunogenic complexes of the present invention are particularly suitable for use in facilitating the stimulation of cytotoxic T-lymphocyte responses to immunogens which do not comprise hydrophobic regions.

Accordingly, one aspect of the present invention relates to an immunogenic complex comprising a charged organic carrier and a charged antigen which organic carrier and antigen are electrostatically associated.

Reference to a "complex" should be understood as describing an entity of two or more different interacting chemical components.

Reference to a "charged" organic carrier or antigen should be understood as a reference to an organic carrier or antigen which exhibits an overall positive electrical charge or an overall negative electrical charge. By "overall" is meant the summation of the individual positive and negative charges which a given molecule comprises. Where the summation of the individual positive and negative charges results in overall electrical neutrality, the molecule is not regarded as "charged" within the context of the present invention.
Preferably, the antigen comprises an overall positive charge and the organic carrier comprises an overall negative charge.

Accordingly, the present invention more particularly provides an immunogenic complex comprising a negatively charged organic carrier and a positively charged antigen which organic carrier and antigen are electrostatically associated.

Reference to "electrostatically associated" is a reference to the organic carrier and the antigen being linked, bound or otherwise associated by means which include electrostatic interaction. Accordingly, it should be understood that in some instances the electrostatic interaction will be the only attractive force which results in complexing of the antigen and the organic carrier. However, in other instances the formation of the electrostatic interaction may also lead to, or be associated with, the formation of other interactive forces.

Reference to "antigen" should be understood as a reference to any molecule against which it is sought to induce an immune response, and in particular, a cytotoxic T-lymphocyte response. The antigen may be either a proteinaceous or a non-proteinaceous molecule, which molecule may or may not be immunogenic if it were administered in isolation. The antigen of the present invention may be naturally derived or it may be recombinantly or synthetically produced. Following its isolation or synthesis the antigen may require further modification (for example, structural or sequence modification to improve its antigenicity) prior to use in the present invention. Antigens suitable for use in the present invention include, but are not limited to, core proteins or nucleoproteins isolated from viruses, non-core viral proteins such as virus-like particles (VLPs), antigens of malignant and non-malignant cells, bacterial antigens, parasite antigens and synthetic and recombinant polytopes.

Preferably, the antigen is a protein. The term "protein" should be understood to encompass reference to proteins, polypeptides and peptides and derivatives and equivalents thereof. The protein may be glycosylated or unglycosylated, phosphorylated or dephosphorylated to various degrees and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins. Reference hereinafter to a "protein" includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.

As hereinbefore defined, the antigen of the present invention may also be a polytope. The subject polytope may be produced by synthetic or recombinant means (for example refer International Patent Publication No. WO 96/03144).

According to this preferred embodiment, there is provided an immunogenic complex comprising a negatively charged organic carrier and a positively charged protein which organic carrier and protein are electrostatically associated.

In this regard, the antigen which is included in the immunogenic complex of the present invention may be, in its initial or natural form, positively charged, negatively charged or of neutral charge. Where an antigen is positively charged, it may nevertheless be weakly positively charged and may therefore require modification to increase its degree of positive charge such that complex formation with the negatively charged organic carrier is better facilitated. For example, wherein an antigen is weakly positively charged, increasing the degree of its positive charge may be achieved by any one of a number of methods known to those skilled in the art including, but not limited to, chemically adding further positive charge to the antigen or recombinantly adding positive charge such as by adding polylysine to the antigen. This is of particular use where the antigen is a protein.
Other methods which may be utilised to increase the degree of an antigen's positive charge include, but are not limited to, pH modification, chemical modifications or neutralisation of an antigen's negative charges with positively charged molecules such as arginine.
Similarly, where an antigen is neutral or negatively charged, its overall charge can be converted to an overall positive charge by utilising such methodology. Conversion of a negatively charged antigen to express an overall positive charge may be of particular importance where the antigen is a protein, since most proteins are naturally negatively charged.

Once the charge of the antigen of interest is sufficiently positive, it becomes necessary to ensure that precipitation of the positively charged antigen does not occur prior to complex formation with the organic carrier. In this regard, any suitable method for preventing antigen precipitation may be utilised. For example, antigen solubility may be maintained by disrupting the forces that cause antigen aggregation. Disruption of these forces can be achieved, for example, by incorporating into the antigen solution chaotrophic agents such as urea and guanidine, solvents such as DMSO (dimethyl sulfoxide) and acetonitrile, intermediates such as zwitterions, detergents such as Triton X-100 and CHAPS
(3-[(3-cholamidopropyl)-dimethylammonioj-2-hydroxy-l-propanesulfonate), reducing agents such as DTT (dithiothreitol) and cysteine and chelating agents such as EDTA
(ethylene diaminetetraacetic acid). Solubility can also be maintained by altering the pH
of the antigen solution or by chemical modification of the antigen to introduce polar or ionic molecules such as by alkylation or acetylation. A gradual or phased removal of these solubilising agents when the antigen has been brought into contact with the "organic carrier" or mild denaturation of the antigen can lead to a controlled precipitation of antigen with concomitant increased association with the organic carrier.

Reference to "organic carrier" should be understood as a reference to any molecule, aggregate or complex of molecules, compound or other entity which, when an antigen is associated with it, facilitates the induction of an immune response, and in particular a cytotoxic T-lymphocyte response, to the antigen. The subject carrier is "organic" and, in this regard, "organic" should be understood as a compound of carbon whether naturally, recombinantly or synthetically obtained or derived. In a particularly preferred embodiment the organic carrier is an adjuvant. By "adjuvant" is meant any organic molecule, aggregate or complex of organic molecules, compound or other entity which functions to stimulate, enhance or otherwise up-regulate any one or more aspects of the immune response. For example, the adjuvant may induce inflammation thereby attracting immune response cells to the site of antigen localisation. Alternatively, the adjuvant may slowly release the antigen thereby providing on-going stimulation of the immune system.
Examples of adjuvants suitable for use in the present invention include, but are not limited to, saponin, saponin complexes, any one or more components of the immunostimulating complex of saponin, cholesterol and lipid known as ISCOMATRIXTM (for example the saponin component and/or the phospholipid component), liposomes or oil-in-water emulsions. [The composition and preparation of ISCOMATRIXTM is described in detail in International Patent Application Number WO 87/02250, Australian Patent Numbers 558258 and 632067 and European Patent Publication No. 0 180 564.

Further examples of adjuvants include, but are not limited to, those detailed in the publication of Cox and Coulter, 1992, 1997 and 1999. It should be understood that the subject organic carrier may be naturally occurring or it may be synthetically or recombinantly derived.

Accordingly, the present invention still more preferably provides an immunogenic complex comprising a negatively charged adjuvant and a positively charged protein which adjuvant and protein are electrostatically associated.

Preferably, said adjuvant comprises saponin or a saponin complex. More preferably, said saponin complex is ISCOMATRIXTM.

The organic carrier of the present invention may also be, in its initial or natural form, negatively charged, positively charged or neutral. Increasing the degree of negative charge (for example, where the organic carrier is only weakly negatively charged) or converting a neutral or positively charged organic carrier to a negatively charged organic carrier may also be achieved by any suitable method known to those skilled in the art. For example, where the organic carrier is an oil-in-water emulsion, incorporation of any anionic surfactant with a non-polar tail will impart an overall negative charge to the emulsion due to insertion of the tail of the surfactant into the oil droplet which thereby leaves the negatively charged head group exposed. The negative charge of a saponin complex adjuvant may be increased, for example, by the addition of negatively charged lipid during complex formation.

Examples of detergents which can increase the negative charge of a carrier include, but are not limited to cholic acid, deoxycholic acid, taurocholic acid and taurodeoxycholic acid.
Examples of lipids which can increase the negative charge of a carrier include, but are not limited to, phospholipids (preferably phosphatidyl inositol, phosphatidyl serine, phosphatidyl glycerol and phosphatidic acid and most preferably cardiolipin) and bacterial lipids (preferably monophosphoryl lipid A(MPL) and most preferably diphosphoryl lipid A
such as OM174 as described in International Patent Puhlicatinn Nn WO 95/14I16) WO 00/48630 PCT/AU00/00110_ Without limiting the present invention in any way, the inventors have determined that where the subject charged organic carrier and charged antigen are naturally negatively and positively charged, respectively, the object of the invention can be achieved.
However, a still more effective immunogenic complex is achieved if the subject naturally negatively charged organic carrier is rendered more negatively charged (preferably by addition of cardiolipin or diphosphory lipid A) and/or the subject naturally positively charged antigen is rendered more positively charged (preferably by addition of a polylysine tail).
Preferably, both the naturally negatively charged organic carrier is rendered more negatively charged and the naturally positively charged antigen is rendered more positively charged.

Accordingly, in one preferred embodiment there is provided an immunogenic complex comprising a negatively charged adjuvant and a positively charged protein, wherein said negatively charged adjuvant is a naturally negatively charged adjuvant which has been modified to increase the degree of its negative charge, which adjuvant and protein are electrostatically associated.

In another preferred embodiment there is provided an immunogenic complex comprising a negatively charged adjuvant and a positively charged protein, wherein said positively charged protein is a naturally positively charged protein which has been modified to increase the degree of its positive charge, which adjuvant and protein are electrostatically associated.

In a most preferred embodiment that is provided an immunogenic complex comprising a negatively charged adjuvant and a positively charged protein, wherein said negatively charged adjuvant is a naturally negatively charged adjuvant which has been modified to increase the degree of its negative charge and said positively charged protein is a naturally positively charged protein which has been modified to increase the degree of its positive charge, which adjuvant and protein are electrostatically associated.

WO 00/48630 PCT/AU00/00110_ Reference to an adjuvant or protein being "naturally" negatively or positively charged, respectively, should be understood as a reference to the charge which the molecule bears upon its creation - whether that be by natural, recombinant or synthetic means.
Modification to increase the degree of charge can be achieved by any suitable technique as hereinbefore discussed. Preferably, the subject protein is rendered more positively charged via the addition of a polylysine tail and the subject adjuvant is rendered more negative via the addition of cardiolipin or diphosphoryl lipid A.

Reference to "derivative and equivalents" should be understood as a reference to fragments, parts, portions, chemical equivalents, mutants, homologs and analogs from natural, synthetic or recombinant sources. Where the subject antigen or carrier is a protein, derivatives may be derived from insertion, deletion or substitution of amino acids.
Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids.
Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which one residue in the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins. "Equivalents" can act as a functional analog of the subject carrier or antigen. Chemical equivalents may not necessarily be derived from the subject carrier of antigen but may share certain conformational similarities.
Alternatively, chemical equivalents may be designed to mimic certain physiochemical properties of the subject carrier or antigen. Equivalents may be chemically synthesized or may be detected following, for example, natural product screening. Homologs contemplated herein include, but are not limited to, molecules derived from different species.

The present invention is predicated, in part, on the formation of immunogenic complexes via the electrostatic association, preferably, of a negatively charged organic carrier with a positively charged antigen. The administration of such a complex to a subject facilitates the induction of a significantly better immune response than would be achieved were the adjuvant and antigen administered simultaneously but in a non-associated form.
In particular, the administration of an antigen associated with an adjuvant, according to the present invention, facilitates the induction of a cytotoxic T-lymphocyte response to the antigen. However, humoral and other cellular responses can also be enhanced.

Without limiting the present invention to any one theory or mode of action, it is thought that the complexing of the adjuvant with the antigen facilitates co-delivery of the adjuvant and the antigen to the same antigen presenting cell thereby facilitating the induction of immune responses which either would not occur or would not occur as effectively were these molecules not co-delivered. For example, the induction of some CD8 +
cytotoxic T-lymphyocyte responses are thought to occur where the adjuvant induces endosomal escape of the antigen in the antigen presenting cell. This necessarily requires co-delivery of the antigen and the adjuvant to the antigen presenting cell.
A further aspect of the present invention therefore relates to the use of the invention to induce an immune response in a mammal including, but not limited to, a humoral and/or cell mediated immune response.

Accordingly, another aspect of the present invention relates to a vaccine composition comprising as the active component an immunogenic complex comprising a charged organic carrier and a charged antigen which organic carrier and antigen are electrostatically associated together with one or more pharmaceutically acceptable carriers and/or diluent.

Preferably, said organic carrier is an adjuvant, and even more preferably a saponin or a saponin complex. Preferably said saponin complex is ISCOMATRIXTM.

Still more preferably, said antigen is a protein.

Preferably said organic carrier is negatively charged and said antigen is positively charged.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The organic carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
Such compositions and preparations should contain at least 1 % by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.

The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 gg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

Without limiting the operation of the present invention in any way, the co-delivery of the immunogenic complex of the present invention is particularly useful for inducing an immune response and, in particular, a cytotoxic T-lymphocyte response to an antigen said immune response may be a specific (T cell and/or B cell) and/or non-specific immune response.

Accordingly, still another aspect of the present invention relates to a method of eliciting, inducing or otherwise facilitating, in a mammal, an immune response to an antigen said method comprising administering to said mammal an effective amount of an immunogenic complex or a vaccine composition as hereinbefore described.
Preferably said immune response is a cytotoxic T-lymphocyte response.

It should be understood that the subject cytotoxic lymphocyte response may occur either in isolation or together with a helper T cell response, a humoral response or other specific or non-specific immune response.

A further aspect of the present invention relates to the use of the immunogenic complex of the invention in relation to the therapeutic and/or prophylactic treatment of disease conditions. Examples of disease conditions which can be treated in accordance with the method of the present invention include, but are not limited to, any disease condition which results from a microbial infection or a cancer. Examples include HIV, Hepatitis B, Hepatitis C, melanoma, prostate cancer, breast cancer, tuberculosis and parasitic conditions.

Accordingly, yet another aspect of the present invention relates to a method of treating a disease condition in a mammal said method comprising administering to said mammal an effective amount of an immunogenic complex or a vaccine composition as hereinbefore described wherein administering said composition elicits, induces or otherwise facilitates an immune response which inhibits, halts, delays or prevents the onset or progression of the disease condition.

An "effective amount" means an amount necessary at least partly to attain the desired immune response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

The term "mammal" includes humans, primates, livestock animals (eg. horses, cattle, sheep, pigs, donkeys), laboratory test animals (eg. mice, rats, rabbits, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. kangaroos, deer, foxes).
Preferably, the mammal is a human or laboratory test animal. Even more preferably, the mammal is a human.

The mammal undergoing treatment may be human or an animal in need of therapeutic or prophylactic treatment of a disease condition or a potential disease condition.

In yet another aspect the present invention relates to the use an immunogenic complex or vaccine composition as hereinbefore defined in the manufacture of a medicament for inhibiting, halting, delaying or preventing the onset or progression of a disease condition.
Yet another aspect of the present invention relates to an agent for use in inhibiting, halting, delaying or preventing the onset or progression of a disease condition. Said agent comprising an immunogenic complex or vaccine composition as hereinbefore defined.
Further features of the present invention are more fully described in the following non-limiting Examples.

Reference to "ISCOPREPTM 703" should be understood as a reference to a saponin preparation comprising from 50-90% by weight of Fraction A of Quil A and 50%
to 10%
by weight of Fraction C of Quil A. Fractions A and C are prepared from the lipophilic fraction of Quil A. Fractions "A" and "C", their method of preparation and the method of preparing 703 are detailed in International Patent Publication No. W096/11711.

PREPARATION OF STANDARD AND MODIFIED ISCOMATRIXT'"
ISCOMATRIXTM (Immunostimulating complex without antigen) was prepared essentially by the method of Morein et al. (1989). Briefly, to 1.76 ml PBS pH 7.2 was added 0.16 ml of a solution containing 10 mg/ml tritiated (3H) cholesterol and 10 mg/ml lipid in 20%
MEGA-10 detergent (w/v) then 0.08 ml of a solution containing 100 mg/ml ISCOPREPTM
703 in PBS. The solution was held at 25 C for 1 hour with gentle mixing.
During subsequent dialysis against PBS/azide, ISCOMATRIXTM containing cholesterol, DPPC
and ISCOPREPTM was formed. All the ISCOMATRIXTM formulations were of typical appearance by electron microscopy.

Lipids:

Standard DPPC dipalmitoylphosphatidylcholine CDL modified cardiolipin DPL modified diphosphoryl lipid A

MPL modified monophosphoryl lipid A
DPA modified phosphatidic acid DPPG modified dipalmitoylphosphatidyl glycerol After formulation, preparations were purified on a sucrose gradient (10 to 50%
w/v) and fractions analysed for lipid and cholesterol. Cholesterol was detected by 3H
cpm of 100 l sample in lml scintillant and lipid was detected using diphenylhexatriene (DPH) which fluoresces when associated with lipid. Briefly, DPH was dissolved at lmg/ml in acetone then diluted 1 in 50 in PBS pH7.2, then 50 l mixed with 501tl of each fraction in a microtitre plate.
Following incubation for 150 mins at 20-25 C the plate was read in a fluorometer using excitation 355nm and emission 460nm. The DPH and 3H peaks coincided for all formulations and the gradient profiles of the modified formulations were similar to the standard formulation indicating incorporation of the lipid into the ISCOMATRIXTM (Figure 1).

PREPARATION OF ANTIGEN ASSOCIATED ISCOMATRIXTM
WITH A NATURALLY POSITIVELY CHARGED PROTEIN: H.pylori family E protein (HpE) The HpE protein has a pl of 9.24 making it a postively charged protein at pH8.
Solubility of the HpE was maintained using 0.5M Tris, 0.5M NaCl, 0.1 % 1,2-Diheptanoyl-sn-Glycero-3-phosphocholine (DHPC) pH8. The HpE associated ISCOMATRIXTM
formulations were prepared by mixing at a 1:5 ratio of protein to ISCOPREPTM
as ISCOMATRIXTM for 60 minutes at 20-25 C. The ISCOMATRIXTM formulations used were DPPC, CDL, DPL and DPPG.
After formulation, preparations were purified on a sucrose gradient (10 to 50%
sucrose w/v) and fractions analysed for HpE, association between HpE and ISCOMATRIXTM
and ISCOMATRIXTM (Figure 2). HpE was detected by adsorbing fractions diluted 1 in 10 in PBS to wells of an EIA plate then detecting with a Horse radish peroxidase (HRP) conjugated monoclonal antibody to HpE. Association was determined by EIA using a monoclonal antibody to HpE to capture and a HRP conjugated monoclonal antibody to ISCOPREPTM to detect. ISCOMATRIXTM was determined by detecting 3H cholesterol.
The HpE protein, when not mixed with ISCOMATRIXTM, was found in fractions 3-10 by EIA. (Figure 2E). When mixed with DPPC ISCOMATRIXTM the HpE was found predominantly in fractions 2-8 but some was found in fractions 12-20 coinciding with the ISCOMATRIXTM and association peaks which indicates that association occurred (Figure 2A). When mixed with CDL or DPL ISCOMATRIXTM the HpE was found predominantly in fractions 7-16 coinciding with the ISCOMATRIXTM and association peaks which indicates that almost complete association occurred (Figure 2B&C). There was very little, if any, free HpE found in fractions 2-8. When mixed with DPPG ISCOMATRIXTM the results were similar to the DPPC ISCOMATRIXTM (Figure 2D).

These results indicate that DPPG and standard DPPC ISCOMATRIXTM can associate weakly with antigens that are positively charged and the capacity to associate can be substantially increased by using CDL or DPL ISCOMATRIXTM.

PREPARATION OF ANTIGEN ASSOCIATED ISCOMATRIXTM
WITH A NATURALLY POSITIVELY CHARGED PROTEIN:
NY-ESO-1 (ESO) The ESO protein has a pI of 9.1 making it a positively charged protein at pH7.
Solubility of the ESO was maintained using 8M Urea, 50mM Tris, 50mM NaH2PO4.2H201 0. 15M
NaCl pH7. The ESO associated ISCOMATRIXTM formulations were prepared by mixing at a 1:5 ratio of protein to ISCOPREPTM as ISCOMATRIXTM for 60 minutes at 20-25 C.
The ISCOMATRIXTM formulations used were DPPC and DPL.

After formulation, preparations were purified on a sucrose gradient (10 to 50%
sucrose w/v) and fractions analysed for ESO, association between ESO and ISCOMATRIXTM
and ISCOMATRIXTM (Figure 3). ESO was detected by adsorbing fractions diluted 1 in 10 in PBS to wells of a EIA plate then detecting with a HRP conjugated monoclonal antibody to ESO. Association was determined by EIA using a monoclonal antibody to ESO to capture and a HRP conjugated monoclonal antibody to ISCOPREPTM to detect. ISCOMATRIXTM
was determined by detecting 3H cholesterol.

The ESO protein, when not mixed with ISCOMATRIXTM, was found in fractions 1-6 by EIA. (Figure 3C). When mixed with standard DPPC ISCOMATRIXTM ESO was found in fractions 1-6 and 12-16 (Figure 3A). The presence in fractions 12-16 coincided with the ISCOMATRIXTM and association peaks indicating there was association but a large proportion of the ESO was not associated as indicated by the presence in fractions 1-6.
When mixed with DPL ISCOMATRIXTM the ESO was found predominantly in fractions 12 to 16 coinciding with the ISCOMATRIXTM and association peaks which indicates that association occurred (Figure 3B).

These results show there was some association of a positively charged protein with standard DPPC ISCOMATRIXTM but the capacity to associate was substantially increased by use of DPL ISCOMATRIXTM

IMMUNISATION OF MICE WITH ESO ASSOCIATED STANDARD
ISCOMATRIXTM
Antibody Responses:

Ten BALB/c mice were immunised, on days 0 and 28, subcutaneously in the scruff of the neck with 0. Ind of ESO containing 5 g protein or ESO associated ISCOMATRIXTM
containing 5 g protein and 5 g ISCOPREPTM. The mice were bled on day 35 and the sera analysed for antibodies to ESO by indirect EIA. Briefly, ESO was adsorbed to a microtitre plate in PBS pH7.2 then the plate blocked with a 0.1 % casein solution and dried. Dilutions of sera were incubated for 1 hour at 20-25 C then the plates washed.
HRP conjugated goat anti mouse IgG, IgG, or IgG2a was added and plates incubated for 1 hour at 20-25 C then washed. TMB substrate was added and incubated for 10 mins at 20-25 C followed by addition of 0.5M H2SO4 to stop the reaction. Plates were read at OD450nm and end point titres calculated.

There was a greater than 20 fold increase in the IgG and IgG, responses to ESO
when associated with ISCOMATRIXTM and a thousand fold increase in IgG2a titre (Figure 4).
Cytotoxic T Lymphocyte (CTL) Responses:

Five HLA A2 transgenic HHD mice were immunised subcutaneously at the base of the tail with 0. lml of ESO containing 5 g protein or ESO associated ISCOMATRIXTM
containing 51tg protein and 51tg ISCOPREPTM. After 14 days splenocytes were harvested and cells restimulated in 24-well plates with EL4HHD cells sensitised with ESO
peptide (10 g/ml for 1 hour 37 C), irradiated and washed twice. Cells were cultured in RPMI
media supplemented with 10% foetal calf serum, 2mM glutamine, 5X 10-5 MR-mercaptoethanol, 1001tg/ml strepomycin and 1001U/ml pencillin and incubated at 37 C for 6 days in 5 %CO2. On day 4 lml of medium was added containing 5U/ml recombinant human IL-2. On day 6 the cultures were used as effectors in standard 6 hour 51Cr release assays against EL4HHD cells sensitised as for restimulation.

CTL were not detected in mice immunised with ESO alone but when associated with ISCOMATRIXTM, CTL was detected in all mice (Figure 5).

These results indicate that association is required for optimal induction of cellular immune responses.

PREPARATION OF ANTIGEN ASSOCIATED ISCOMATRIXTM WITH A
NATURALLY NEGATIVELY CHARGED PROTEIN: HPV E6E7 (E6E7) The E6E7 protein has a pl of 5.9 making it a negatively charged protein at pH6.9.
Solubility of the E6E7 was maintained using 8M Urea, 50mM Tris, 50mM
NaH2PO4.2H2O1 150mM NaCl pH6.9. The E6E7 associated ISCOMATRIXTM

formulations were prepared by mixing at a 1:5 ratio of protein to ISCOPREPTM
as ISCOMATRIXTM for 60 minutes at 20-25 C. The ISCOMATRIXTM formulations used were DPPC, CDL, DPL, MPL, DPA and DPPG.

After formulation, preparations were purified on a sucrose gradient (10 to 50%
sucrose w/v) and fractions analysed for E6E7, association between E6E7 and ISCOMATRIXTM
and ISCOMATRIXTM (Figure 8). E6E7 was detected by EIA using two non-competing monoclonal antibodies to E7. Association was determined by EIA using a monoclonal antibody to E7 to capture and a HRP conjugated monoclonal antibody to ISCOPREPTM
703 to detect. ISCOMATRIXTM was determined by detection of 3H cholesterol.

The E6E7 protein alone was found in fractions 10-22 by EIA (Figure 6G). When mixed with standard DPPC ISCOMATRIXTM most of the E6E7 found was in fractions 14-20 with little association detected (Figure 6A). When mixed with CDL, DPL and DPA
ISCOMATRIXTM the E6E7 was found predominantly in fractions which coincided with the association and the ISCOMATRIXTM peaks which indicated that almost complete association occurred (Figure 6B,C, E). When mixed with MPL and DPPG
ISCOMATRIXTM the protein was found in fractions 9-14 coinciding with the association and ISCOMATRIXTM peaks indicating association but a significant amount found not associated in fractions 17-22 (Figure 6D,F).

These results indicate that a negatively charged protein binds poorly to standard DPPC
ISCOMATRIXTM and the capacity to associate increases by using CDL, DPL, MPL, DPA
or DPPG to varying degrees.

MODIFIED ISCOMATRIXTM

Three C57BL/6 mice were immunised, on day 0 and day 21, subcutaneously with 0.
lml of E6E7 associated ISCOMATRIXTM containing 101tg protein and 6 g ISCOPREPTM.
After 7 days splenocytes were harvested and 20X 106 cells restimulated in 8mL in a T25 tissue culture flask with E7 transfected EL4 cells (C2) mytomycin-C treated and washed three times. Cells were cultured in RPMI media supplemented with 10% foetal calf serum, 2mM glutamine, 5.5X10-5 M(3-mercaptoethanol, 501tg/ml gentamicin and incubated at 37 C for 5 days in 5 %CO2. On day 6 the cultures were used as effectors in a standard 4 hour 51Cr release assays against C2 cells.

The E6E7 associated DPL ISCOMATRIXTM induced a CTL response in 2 out of 3 mice (Figure 7A). The E6E7 associated with standard DPPC ISCOMATRIXTM failed to induce a CTL response in any mice (Figure 7B). The negative mouse in the DPL

ISCOMATRIXTM group had insufficient cells for optimal readout and would not comply with criteria for a valid response. All other mice fulfilled criteria for valid responses.
These results show that the greater the association the better the CTL
response.

PREPARATION OF ANTIGEN ASSOCIATED ISCOMATRIXTM
WITH A NATURALLY NEGATIVELY CHARGED PROTEIN: H.pylori family C protein (HpC) The HpC protein has a pI of 5.05 making it negatively charged, at pH7.2. The protein was soluble in PBS pH7.2. The HpC associated ISCOMATRIXTM formulations were prepared by mixing at a 1:5 ratio of protein to ISCOPREPTM as ISCOMATRIXTM for 60 minutes at 20-25 C. The ISCOMATRIXTM formulations used were DPPC and DPL.

After formulation, preparations were purified on a sucrose gradient (10 to 50%
sucrose w/v) and fractions analysed for HpC, association between HpC and ISCOMATRIXTM
and ISCOMATRIXTM (Figure 8). HpC was detected by adsorbing fractions diluted 1 in 10 in PBS to wells of an EIA plate then detecting with a HRP conjugated monoclonal antibody to HpC. Association was determined by EIA using a monoclonal antibody to HpC
to capture and a HRP conjugated monoclonal antibody to ISCOPREPTM to detect.

ISCOMATRIXTM was determined by either detection of 3H cholesterol or DPH as described in example 1.

HpC alone was found in fractions 1-5 and when mixed with standard DPPC

ISCOMATRIXTM the HpC was found predominantly in fractions 1-5 and not associated.
When mixed with DPL ISCOMATRIXTM a significant proportion of the HpC was found in fractions 11-17 coinciding with the ISCOMATRIXTM and association peaks indicating association.

These results indicate that a negatively charged protein binds poorly to standard DPPC
ISCOMATRIXTM and the capacity to associate increases by using DPL ISCOMATRIXTM

PREPARATION OF ANTIGEN ASSOCIATED ISCOMATRIXTM WITH A
NATURALLY POSITIVELY CHARGED PROTEIN UTILISING pH TO GIVE A
POSITIVE CHARGE: E6E7.

The E6E7 protein has a pI of 5.9 making it a negatively charged protein at pH7.2. It contains a hexa histidine sequence at the N terminus which will be positively charged at pH6. Solubility of the E6E7 was maintained using 8M urea, 50mM Bis Tris, 0.
15M NaCl pH6. The E6E7 associated ISCOMATRIXTM formulation was prepared by mixing equal mass of E6E7 with ISCOPREPTM as ISCOMATRIXTM for 60 minutes at 20-25 C, dialysing against 50mM Bis Tris, 0. 15M NaCl pH6 to remove the urea then centrifugation at 10,000 g for 5 mins to remove any precipitate.

After formulation, preparations were purified on a sucrose gradient (50 to 10%
sucrose w/v) and fractions analysed for protein, association between E6E7 and ISCOMATRIXTM
and ISCOMATRIXTM (Figure 9). Protein was detected using a sandwich EIA for E7.
Association was determined by EIA using a monoclonal antibody to E7 to capture and a HRP conjugated monoclonal antibody to ISCOPREPTM to detect. ISCOMATRIXTM was determined by detection of 3H cholesterol or DPH as described in example 1.

E6E7 was found in fractions 10-22 when run alone (Figure 9C). When mixed with DPPC
ISCOMATRIXTM at pH7.2 the E6E7 was predominantly found in fractions 16-22 with little evidence of association (Figure 9B). When mixed with standard DPPC
ISCOMATRIXTM at pH6 the E6E7 was predominantly found in fractions 12-16 coinciding with the ISCOMATRIXTM and association peaks which indicates association(Figure 9A).
These results show that pH can be used to increase the capacity of standard DPPC
ISCOMATRIXTM to associate with naturally negatively charged proteins.

IMMUNISATION OF MICE WITH pH MODIFIED E6E7 ASSOCIATED DPPC
ISCOMATRIXTM
Six C57BL/6 mice were immunized, on days 0 and 21, subcutaneously in the scruff of the neck with 0.1 ml of E6E7 associated ISCOMATRIXTM containing 61tg ISCOPREPTM
and 6 g E6E7.

Antibody Responses:

Mice were bled on day 26 and sera analysed for antibodies to E7 by indirect EIA.
Purified GSTE7 was adsorbed to a microtitre plate in 0.1M Carbonate pH9.6 then the plate blocked with a 0.1 % casein solution and dried. Dilutions of sera were incubated for 1 hour at 20-25 C then the plates washed. HRP conjugated goat anti mouse IgG
was added and plates incubated for 1 hour at 20-25 C then washed. TMB substrate was added and incubated for 10 mins at 20-25 C followed by addition of 0.5M H2SO4 to stop the reaction. Plates were read at OD450nm and end point titres calculated.

The E6E7 associated ISCOMATRIXTM group had a GMT of 949. Typically E6E7 with Al(OH)3 gives GMT of approximately 100.

Cytokine Responses:

On day 27 splenocytes from each of 3 mice were harvested and pooled and 2.5X
106 cells restimulated in 48-well plates with GSTE7 at 1 and 5 g with ConA and RPMI as controls. Cells were cultured in RPMI media supplemented with 10% foetal calf serum, 2mM glutamine, 5X 10-5 M(3-mercaptoethanol, 100 g/ml streptomycin and 100IU/ml pencillin and incubated at 37 C for 2 days in 5%CO2. The supernatant was harvested and yIFN and IL5 detected by EIA using reagents from Endogen.

The E6E7 associated ISCOMATRIXTM induced up to 7.4 ng/ml yIFN and 140 pg/ml (Table 2). Typically E6E7 with Al(OH)3 induces no detectable yIFN (<30 pg/ml) or IL5 (< 4 pg/ml).

These results show that pH modified E6E7 associated ISCOMATRIXTM were immunogenic in mice and induced a Thl type response.

PREPARATION OF CHELATING (CHL) ISCOMATRIXTM

CHL ISCOMATRIXTM was prepared by the method of Macfarlan and Malliaros, (1998) International Patent Publication No. WO 98/36772). Briefly, to 1.6 ml 50mM
Tris, 150mM
NaCl, 0.6mM CuC12 pH 7.2 (Buffer A) was added 0.2 ml of a solution containing 10 mg/ml cholesterol, 9 mg/ml DPPC, 1.074 mg/ml dipalmitoyl-rac-glycerol-3(8-(3,6-dioxy) octyl-l-amino-N,N-diacetic acid) (DPIDA) in 20% MEGA-10 detergent (w/v) then 0.2 ml of a solution containing 50 mg/ml ISCOPREPTM 703 in Buffer A. The solution was held at 25 C
for 90 mins with gentle mixing. Dialysis was then performed firstly against Buffer A
overnight with 2 changes of buffer then against 50mMTris, 50mM NaH2PO4.2H2O, 150mM
NaCl pH6.9 for 2 days with two changes of buffer. During dialysis CHL
ISCOMATRIXTM
containing cholesterol, DPPC, DPIDA and ISCOPREPTM was formed. The CHL
ISCOMATRIXTM formulation was of typical appearance by electron microscopy.

GENERATION, EXPRESSION AND PURIFICATION OF HEXAHISTIDINE (6H) HEXALYSINE (6K) HpC.

The HpC protein has a pI of 5.05 making it negatively charged at pH7.2.
Addition of 6K
would change the pI to 7.68 and give a positively charged tail. Two clones were constructed to give HpC plus 6H, for purification, and with and without 6K.

DNA (HpC13 with a C-terminal 6H in the vector pGexStop as described in Edwards et al.

1998) was used as the template for PCR amplification to generate a C-terminal 6K. The PCR product was cloned into the EcoRI-BglII sites of the expression vector pGexStoplV, creating tandem C-terminal 6K followed by 6H tags. This was generated in the E. coli strain ER1793 and designated CSL 1424.

One litre cultures were induced at A6,=2 with 0.5mM IPTG and harvested 5 hours post induction. Soluble recombinant protein was purified utilising the C-terminal 614 tag for metal (nickel) affinity chromatography. Eluted protein was dialysed against PBS.

PREPARATION OF ANTIGEN ASSOCIATED ISCOMATRIXT"' WITH 6H AND 6K TAGS : HpC.

The HpC protein with 6H has a pI of 5.85 making it negatively charged at pH7.2.
Addition of a 6K to this protein gives a pI of 7.68 making it positively charged at pH7.2.
Both forms of the protein were soluble in PBS pH7.2. The HpC associated ISCOMATRIXTM formulations were prepared by mixing at a 1:5 ratio of protein to ISCOPREPTM as ISCOMATRIXTM for 60 minutes at 20-25 C. The ISCOMATRIXTM
formulations used were DPPC and CHL. CHL ISCOMATRIXTM technology was used as a standard method for associating 6H proteins with ISCOMATRIXTM

After formulation, preparations were purified on a sucrose gradient (10 to 50%
sucrose w/v) and fractions analysed for HpC, association between HpC and ISCOMATRIXTM
and ISCOMATRIXTM (Figure 10). HpC was detected by adsorbing fractions diluted 1 in 10 in PBS to wells of an EIA plate then detecting with a HRP conjugated monoclonal antibody to HpC. Association was determined by EIA using a monoclonal antibody to HpC
to capture and a HRP conjugated monoclonal antibody to ISCOPREPTM to detect.
ISCOMATRIXTM was determined by detection of DPH as described in example 1.

When mixed with standard DPPC ISCOMATRIXTM the 6H-HpC was found in fractions 1-6 and with little evidence of association (Figure 10C). When 6K6H-HpC was mixed with DPPC ISCOMATRIXTM a significant amount of HpC was in fractions 7-11 coinciding with the association and ISCOMATRIXTM peaks indicating association (Figure 10A).
When 6H-HpC was mixed with CHL ISCOMATRIXTM most of the HpC was in fractions 7-14 coinciding with the ISCOMATRIXTM and association peaks indicating association (Figure 10B).

These results show that addition of a 6K to a negatively charged protein increased its capacity to associate with standard DPPC ISCOMATRIXTM and the association achieved was comparable to that using 6H with CHL ISCOMATRIXTM.

PREPARATION OF SYNTHETIC POLYTOPE ISCOMTM AND ASSOCIATED
ISCOMATRIXT'I WITH PALMITIC ACID (PAL), 6H, 6K AND NO
FORMULATION TAGS.

The polytopes were synthesised and purifed by Chiron Technologies on Multipin (TM) crowns, as described by Valerio et al., using the Fmoc alpha-amino protection scheme for the amino acids. After sidechain deprotection and cleavage in a trifluoracetic acid/scavenger solution, peptides were precipitated with ether and dried. The redissolved peptide was purified by preparative reverse phase HPLC using elution with a gradient of acetonitrile. Fractions containing material of the correct molecular mass, as determined by ion spray mass spectrometry, were pooled and dried.

The polytope was as follows:
Tag-YPHFMPTNLRPQASGVYMTYQRTRALVSYIPSAEKI-OH (< 400 > 3) containing four known BALB/c restricted epitopes, YPHFMPTNL (< 400 > 4), RPQASGVYM
(< 400 > 5), TYQRTRALV (< 400 > 6) and SYIPSAEKI (< 400 > 7). The tags used were PAL, 6H, 6K or H (No tag).

For the PAL polytope association was achieved by incorporation into ISCOMTM
(Immunostimulating complex) according to the method of Morein et al. (1989).
Briefly, to 4 mg of polytope solubilised in 1.76 ml 10% MEGA-10 detergent (w/v), 50%
Acetonitrile in PBS was added 0.16 ml of a solution containing 10 mg/mi cholesterol and 10 mg/ml DPPC in 20% MEGA-10 detergent (w/v) then 0.08 ml of a solution containing 100 mg/ml ISCOPREPTM 703 in PBS. The solution was held at 25 C for 1 hour with gentle mixing. During subsequent dialysis against PBS/azide ISCOMSTM
containing palmityfied polytope, cholesterol, DPPC and ISCOPREPTM were formed. These ISCOMSTM were of typical appearance by electron microscopy.

The 6H polytope was solubilised in 8M urea then mixed with CHL ISCOMATRIXTM
and the 6K and no tag polytopes were solubilised in PBS then mixed with standard DPPC
ISCOMATRIXTM. All formulations were prepared at a ratio of 1:8 protein to ISCOPREPTM as ISCOMATRIXTM and incubated for 60 mins at 20-25 C.

The preparations were purified on a sucrose gradient (10 to 50% sucrose w/v) and fractions analysed for protein and ISCOMATRIXTM. Protein was detected using CBQCA
(< 400 > 8) from Molecular Probes according to the manufacturers instructions or by Coomassie according to the method of Bradford (1976). Briefly 1001tl of each fraction was added to a microplate followed by addition of 1001il Coomassie reagent then the plate read at 595nm. ISCOMATRIXTM was detected by DPH as described in example 1.

The polytope alone was found in fractions 1-5 (Figure 11E). The protein, as detected by CBQCA (< 400 > 8), in the PAL polytope ISCOMTM was found predominantly in fractions 11-13 coinciding with the ISCOMATRIXTM peak indicating incorporation (Figure 11A).
The protein, as detected by Coomassie, in the 6K polytope associated ISCOMATRIXTM

was found predominantly in fractions 1-5 and was probably not associated with ISCOMATRIXTM (Figure 11C). A significant proportion of polytope was found in fractions 12-14 coinciding with the ISCOMATRIXTM peak indicating association.
The protein, as detected by Coomassie, in the 6H polytope associated CHL
ISCOMATRIXTM
was found predominantly in fractions 4-10 coinciding with the ISCOMATRIXTM
peak indicating association (Figure 11B). There was a significant proportion of 6H
polytope found in fractions 1-3 which was probably not associated. The protein, as detected by Coomassie, in the no tag associated ISCOMATRIXTM was almost all found in fractions 1-5 and probably not associated with ISCOMATRIXTM.

These results show that a tag was required for association of the polytope tested with ISCOMATRIXTM and that 6Kpolytope association with standard DPPC ISCOMATRIXTM
was comparable to incorporation of hydrophobic PAL polytope into ISCOMsTM but not as good as 6H polytope association with CHL ISCOMATRIXTM

IMMUNISATION OF MICE WITH SYNTHETIC POLYTOPE ISCOMTM AND
ASSOCIATED ISCOMATRIXTM FORMULATIONS

Three BALB/c mice were immunized subcutaneously at the base of the tail with 0.1 ml of polytope ISCOMTM or associated ISCOMATRIXTM containing 6 g ISCOPREPTM and between 3.5 g and 5 g protien.

CTL assays were performed according to the method of Elliott et al. (1999).
Briefly, splenocytes from each spleen were removed on day 14 and cultured in 1 ml medium at 5X106 cell/ml, in a 24 well plate, together with 1 g/ml of the individual peptides (4 peptides/spleen) in a humidifed incubator at 37 C. On day 3, 1 ml of fresh media was added and then further in vitro restimulation performed on day 7 by adding irradiated (800 rad) peptide sensitised (10 g/m1, 1 hr 37 C, 2 washes) P815 cells at a responder to stimulator ratio of 20:1 to 2X 106 effectors/well. The procedure was repeated twice more at 7 day intervals and the bulk cultures were used as effectors 6 days later in a standard 6 hr chromium release assay. Medium contained RPMI 1640 supplemented with 10%
FCS
(QIMR), 5X10"5 M 2-mercaptoethanol, 2mM glutamine and pen/strep antibiotics.
Target cells were "Cr labelled peptide sensitised and unsensitised (control) P815 cells. The ratio of effector:target was 50, 10 and 2 to 1. The assays were performed in 96 well round bottom plates in duplicate.

The PAL polytope ISCOMTM induced CTL responses against all 4 epitopes with 3/3 mice for TYQ, 1/3 for SYI, 2/3 for YPH and 2/3 for RPQ (Figure 12A). The 6H
polytope associated CHL ISCOMATRIXTM induced CTL responses against 3/3 mice for all 4 epitopes (Figure 12B). The 6K polytope associated DPPC ISCOMATRIXTM induced CTL
responses against all 4 epitopes with 3/3 for TYQ, YPH and RPQ and 2/3 for SYI
(Figure 12C). The no tag polytope associated DPPC ISCOMATRIXTM induced a weak CTL
response in 2/3 mice for RPQ but there was no CTL response detected to any of the other epitopes (Figure 12D). The SYI sequence is known to be a weak epitope and this was the case for all formulations.

These results show that association of polytope with the ISCOMTM or ISCOMATRIXTM
was required for optimal CTL induction and that association using 6K was as effective as 6H
with CHL ISCOMATRIXTM or classical incorporation of hydrophobic proteins (PAL
polytope ISCOMTM ) GENERATION, EXPRESSION AND PURIFICATION OF RECOMBINANT (r) 6H
6K POLYTOPE.

Pstmpdv DNA(supplied by QIMR) was used as the template for PCR amplification of the murine polytope, YPHFMPTNLTSSGPSNTPPEIFAPGNYPALSYIPSAEKIEEGAIVGEI
RPQASGVYM (<400>9), to enable generation with and without a C-terminal 6K (CSL
1430 and 1426 respectively). PCR products were cloned into the BamHI-Xhol sites of the expression vector pET24b (Novagen) generating an N-terminal T7-tag (for identification) and tandem C-terminal 6K followed by 6H (for purification).

Clones were generated in the E. coli strain ER1793 and subsequently transformed into the expression strain BL21(DE3). One litre cultures were induced at A600=2 with 0.5mM
IPTG and harvested 4 hours post induction. Soluble recombinant protein was purified utilising the C-terminal 6H tag for metal (nickel) affinity chromatography.
Eluted protein was dialysed against PBS.

PREPARATION OF rPOLYTOPE ASSOCIATED ISCOMATRIXTM 6H AND 6K.
The murine polytope with 6H has a pI of 5.85 making it negatively charged at pH7.2.
Addition of a 6K to this gives a p1 of 7.68 making it positively charged at pH7.2. Both forms of the protein were soluble in PBS pH7.2. The polytope associated ISCOMATRIXTM formulations were prepared by mixing at a 1:5 ratio of protein to ISCOPREPTM as ISCOMATRIXTM for 60 minutes at 20-25 C. The ISCOMATRIXTM
formulations used were DPPC, CDL, DPL and CHL. Formulation at pH4.3 to be below the p1 of glutamic acid (E) was investigated as there were a number of E's in the sequence which could potentially interfere with association.

After formulation, preparations were purified on a sucrose gradient (10 to 50%
sucrose w/v) and fractions analysed for protein and ISCOMATRIXTM (Figure 13). Protein was detected by adsorbing fractions diluted 1 in 10 in PBS to wells of an EIA
plate then detecting with a HRP conjugated monoclonal antibody to 6H. ISCOMATRIXTM was determined by of detection 3H cholesterol or DPH as described in example 1.

The protein in the r6H6K polytope alone was found in fractions 1-6 (Figure 13K). The protein in the r6K6H polytope mixed with standard DPPC ISCOMATRIXTM at pH7 was found predominantly in fractions 1-9 with little evidence of association (Figure 13 A). The protein in the r6K6H polytope mixed with CDL and DPL ISCOMATRIXTM at pH 7 was found predominantly in fractions coinciding with ISCOMATRIXTM indicating association (Figure 13 C, E). There was a significant proportion of protein found in fractions 1-3 and 1-6, for CDL and DPL respectively, and probably not associated. The protein in the r6K6H polytope mixed with standard DPPC ISCOMATRIXTM at pH4.3 was found predominantly in fractions 1-9 with some evidence of association in the coinciding ISCOMATRIXTM peak in fractions 10-12 (Figure 13 B). The protein in the r6K6H
polytope mixed with CDL and DPL ISCOMATRIXTM at pH4.3 was almost all found in fractions coinciding with ISCOMATRIXTM indicating almost complete association (Figure 13 D, F). The r6H polytope mixed with DPPC ISCOMATRIXTM was found predominanlty in fractions 1-7 with little evidence of association. The r6H polytope mixed with CHL
ISCOMATRIXTM showed similar patterns of association for standard DPPC and CDL

ISCOMATRIXTM at both pH7 and pH4.3. The protein was found in about equal amounts in fractions 1-4 non-associated and 5-10 coniciding with the ISCOMATRIXTM peak indicating association (Figure 13 H, I, J).

These results show that the rpolytope used here would not associate with standard ISCOMATRIXTM even with the addition of 6K. Association could be achieved using modified ISCOMATRIXTM and the capacity to associate with these formulations was increased by utilising low pH. The combination of modified ISCOMATRIXTM and low pH resulted in as good as, or better, association than with 6H CHL
ISCOMATRIXTM
which was not increased by use of modified ISCOMATRIXTM or low pH.

WO 00/48630 PCT/AU00/00110_ IMMUNISATION OF MICE WITH rPOLYTOPE ASSOCIATED ISCOMATRIXTM
FORMULATIONS
Three BALB/c mice were immunized subcutaneously at the base of the tail with 0.1 ml of associated ISCOMATRIXTM containing 6 g ISCOPREPTM 703 and between 3.51tg and 5 g protein.

CTL assays were performed according to the method of Elliott et al. (1999).
Briefly, splenocytes from each spleen were removed on day 14 and cultured in 1 ml medium at 5X106 cell/ml, in a24 well plate, together with 1 g/ml of the individual peptides (4 peptides/spleen) in a humidifed incubator at 37 C. On day 3, 1 ml of fresh media was added and then further in vitro restimulation performed on day 7 by adding irradiated (800 rad) peptide sensitised (101tg/ml, 1 hr 37 C, 2 washes) P815 cells at a responder to stimulator ratio of 20:1 to 2X106 effectors/well. The procedure was repeated twice more at 7 day intervals and the bulk cultures were used as effectors 6 days later in a standard 6 hr chromium release assay. Medium contained RPMI 1640 supplemented with 10%
FCS
(QIMR), 5X10"5 M 2-mercaptoethanol, 2mM glutamine and pen/strep antibiotics.
Target cells were 51Cr labelled peptide sensitised and unsensitised (control) P815 cells. The ratio of effector:target was 50, 10 and 2 to 1. The assays were performed in 96 well round bottom plates in duplicate.

The r6K6H polytope associated CDL ISCOMATRIXTM pH4.3 induced CTL responses in mice for the SYI, YPH and RPQ epitopes and in 1/3 for the TYQ epitope (Figure 14A). The r6H associated CHL ISCOMATRIXTM pH7 induced CTL responses in 3/3 mice for the SYI, YPH and RPQ epitopes and in 2/3 for the TYQ epitope (Figure 14B). Both formulations induced very low responses to the TYQ epitope.

These results show that CTL responses can be induced using r6K6H polytope associated CDL
ISCOMATRIXTM pH4.3 and are comparable to responses with r6H associated CHL
ISCOMATRIXTM pH7.

PREPARATION OF DPPC AND DPL LIPOSOMES WITH A NATURALLY
NEGATIVELY CHARGED PROTEIN: E6E7 Liposomes were prepared according to the method of Talsma and Crommelin (1992).
Briefly, 3H cholesterol was dissolved in methanol, chloroform then lipid added and liposomes allowed to form by solvent evaporation in a rotaflask with gentle swirling. The lipids used were the standard DPPC and the negatively charged DPL. E6E7 was then added to the liposomes and the mixture sonicated then extruded through a 26G
needle. The liposomes were of typical appearance by electron microscopy.

After formulation, preparations were purified on a sucrose gradient (10 to 50%
sucrose w/v) and fractions analysed for protein and ISCOMATRIXTM (Figure 15). Protein was detected by sandwich EIA for E7 using monoclonal antibodies. ISCOMATRIXTM was determined by detection 3H cholesterol.

The E6E7 in the DPPC liposomes was found predominanlty in fractions 1-3 but very little was present on the gradient which indicated the protein had precipitated (Figure 15A). The protein that was present was probably not associated with the liposome which was found in fractions 2-4. The E6E7 in the DPL Liposome was found throughout the gradient coinciding with the liposome which was also found throughtout the gradient (Figure 15B).
The spread of the formulation throughout the gradient was probably indicative of a range of sizes of liposomes but almost all of the protein seems to be associated with the liposomes.

These results show that negatively charged lipids can be used in liposomes to allow association with a negatively charged protein which would not associate with standard liposomes.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Table 2 Cytokine Analysis of E6E7 Associated ISCOMATRIXTI

Stimulated with Concentration ,ug Cytokine pg/ml aIFN IL5 ConA 0.4 2130 74 RPMI - <30 4 REFERENCES

Bradford, M.M. (1976) Anal. Biochem. 36:207-212.

Cox, J.C. and Coulter, A.R. Advances in Adjuvant Technology and Application in Animal Parasite Control Utilising Biotechnology. Chapter 4. Editor Yong, W.K. CRC
Press, 1992.
Cox, J.C. and Coulter, A.R. (1997) Vaccine 15(3):248-256.

Cox, J.C. and Coulter, A.R. (1999) BioDrugs 12(6):439-453.

Edwards, S.J., Margetts, M.B., Hocking, D.M., Moloney, M.B.H., Rothel, L.J.
and Webb, E.A. (1998). Design of a candidate recombinant therapeutic vaccine or cervical cancer. In:
Recent Research Developmens in Biotechnology & Bioengineering. Editor S.G.
Pandalai, Research Signpost, India, 343-356.

Elliot, S.L., Pye, S., Le, T., Mateo, L., Cox, J., Macdonald, L., Scalzo, A.A., Forbes, C.A. and Suhrbier, A. (1999) Vaccine 17:2009-2019.

Morein, B., Lovgren, K. and Hoglund, S., (1989), Immunostimulating complex (ISCOM).
In "Vaccines: Recent Trends and Progress." G. Gregoriadis, A.C. Allison and G.
Poster (Eds), Plenium Press, New York, p153.

Talsma, H. and Crommelin, D.J.A. (1992) BioPharm, October, 36-47.

Valerio, R.M., Bray, A.M. and Maeji, N.J., Int. J. Pept. Prof. Res. 44:158-165 (1994).

<110> CSL LIMITED

<120> Immunogenic Complexes and Methods Relatng Thereto <130> 2257451/TDO

<140> International <141> 2000-02-17 <150> PP8735/99 <151> 1999-02-17 <150> PQ1861/99 <151> 1999-02-17 <160> 9 <170> Patentln Ver. 2.0 <210> 1 <211> 11 -<212> PRT
<213> mammalian <400> 1 Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu <210> 2 <211> 9 <212> PRT
<213> mammalian <400> 2 Ser Leu Leu Met Trp Ile Thr Gln Cys WO 00/48630 PCT/AU00/00110_ <210> 3 <211> 9 <212> PRT
<213> synthetic construct <400> 3 Tyr Pro His Phe Met Pro Thr Asn Leu <210> 4 <211> 36 <212> PRT
<213> synthetic construct <400> 4 Tyr Pro His Phe Met Pro Thr Asn Leu Arg Pro Gln Ala Ser Gly Val Tyr Met Thr Tyr Gln Arg Thr Arg Ala Leu Val Ser Tyr Ile Pro Ser Ala Glu Irys Ile <210> 5 <211> 9 <212> PRT
<213> synthetic construct <400> 5 Arg Pro Gln Ala Ser Gly Val Tyr Met <210> 6 <211> 9 <212> PRT
<213> synthetic construct <400> 6 Thr Tyr Gln Arg Thr Arg Ala Leu Val <210> 7 <211> 9 <212> PRT
<213> synthetic construct <400> 7 Ser Tyr Ile Pro Ser Ala Glu Lys Ile <210> 8 <211> 5 <212> PRT
<213> synthetic construct <400> 8 Cys Asx Gln Cys Ala <210> 9 <211> 57 <212> PRT
<213> synthetic construct <400> 9 Tyr Pro His Phe Met Pro Thr Asn Leu Thr Ser Ser Gly Pro Ser Asn Thr Pro Pro Glu Ile Phe Ala Pro Gly Asn Tyr Pro Ala Leu Ser Tyr Ile Pro Ser Ala Glu Lys Ile Glu Glu Gly Ala Ile Val Gly Glu Ile Arg Pro Gln Ala Ser Gly Val Tyr Met

Claims (19)

CLAIMS:

1. An electrostatically-associated immunogenic complex, comprising;
(A) a negatively-charged organic complex that comprises a saponin and a sterol, and (B) a positively-charged antigen, where said organic complex and antigen are associated only by electrostatic interaction.

2. The immunogenic complex according to claim 1 wherein said antigen is a protein or comprises a peptide region.

3. The immunogenic complex according to claim 1 wherein the organic complex further comprises a phospholipid.

4. The immunogenic complex according to claim 3 wherein said phospholipid is a phosphoglyceride.

5. The immunogenic complex according to claim 4 wherein the phosphoglyceride is selected from the group consisting of phosphatidyl inositol, phosphatidyl glycerol, phosphatidic acid and cardiolipin.

6. The immunogenic complex according to claim 3 wherein said phospholipid is lipid A.

7. The immunogenic complex according to claim 6 wherein the lipid A is selected from the group consisting of diphosphoryl lipid A and monophosphoryl lipid A.

8. The immunogenic complex according to claim 1, wherein said immunogenic complex induces a cytotoxic T-lymphocyte response when administered to a mammal.

9. The immunogenic complex according to claim 1, wherein the antigen has been modified to increase the degree of its positive charge.

10. The immunogenic complex according to claim 1, wherein said organic complex has been modified to increase the degree of its negative charge.

11. The immunogenic complex according to claim 1, wherein said antigen is a naturally positively charged antigen which has been modified to increase the degree of its positive charge.

12. The immunogenic complex according to claim 9, wherein said antigen has been modified by the addition of polylysine and/or arginine.

13. The immunogenic complex according to claim 10, wherein said organic complex is a naturally negatively charged complex which has been modified to increase the degree of its negative charge.

14. The immunogenic complex according to claim 10, wherein said organic complex has been modified with an anionic surfactant and/or negatively charged lipid.

15. The immunogenic complex according to claim 1, wherein said antigen is a non-amphipathic antigen.

16. The immunogenic complex according to claim 1, wherein said antigen does not comprise a hydrophobic region.

17. The immunogenic complex according to claim 1, wherein said complex does not comprise a liposome.

18. The use of the immunogenic complex of any one of claims 1 to 17, to elicit or facilitate an immune response in a mammal.

19. The use of the immunogenic complex of any one of claims 1 to 17 in a vaccine.