WO2009118523A1 - Adjuvant - Google Patents

Abstract

An adjuvant composition comprising a polyacrylic acid containing polymer. In particular, the adjuvant composition enhances a Th1 biased immune response.

Description

ADJUVANT

The invention relates to an adjuvant composition and to uses of said composition in enhancing a specific immune response, in particular, but not exclusively, enhancing a specific Thl-biased immune response.

Developing vaccines which require a predominant induction of a cellular response is a major challenge. T cells are the main effector cells of the cellular immune response. These cells recognise antigens that are synthesized in pathogen-infected cells, therefore, successful vaccination requires the synthesis of immunogenic antigens in cells of the subject being vaccinated. One approach is the use of live- attenuated vaccines, however this presents significant limitations. For example, there is a risk of infection, either when the subject being vaccinated is immunosuppressed, or when the pathogen itself can induce immunosuppression (e.g. Human Immunodeficiency Virus, HIV) . Furthermore, some pathogens are difficult or impossible to grow in cell culture (e.g. Hepatitis C Virus, HCV) . Other existing vaccines such as inactivated whole-cell vaccines or alum-adjuvanted, recombinant protein subuηit vaccines are notably poor inducers of cellular immune responses. The induction of cellular immune responses is associated with a Thl-bias in the immune response. Conversely, responses predominantly associated with antibodies are known as Th2-biased responses. Currently-licensed adjuvants, typified by alum, induce a Th2-type biased response, and are weak inducers of cellular immunity.

There is therefore a great need for an effective adjuvant/vaccine formulation, in particular, one which enhances a specific immune response of ThI cytokines. An additional ability of this adjuvant to induce an antibody response would also be desirable. According to a first aspect of the invention, there is provided an adjuvant composition comprising a polyacrylic acid containing polymer.

According to a further aspect, there is provided the use of a polyacrylic acid containing polymer as an adjuvant.

The adjuvant composition may be for use with, or in, an immunogenic composition, such as a vaccine, for eliciting an immune response to an antigen in the immunogenic composition.

In use the adjuvant and antigen may be administered simultaneously, sequentially or separately.

According to another aspect, the invention provides an immunogenic composition, such as a vaccine composition, comprising: an antigen which elicits an immune response against a human pathogen; and an adjuvant comprising a polyacrylic acid containing polymer. Preferably the antigen elicits an immune response against a human pathogen and the immunogenic composition is therefore intended for use with a human subject.

The presence of a polyacrylic acid containing polymer in an immunogenic composition of the present invention, or the use of a polyacrylic acid containing polymer in conjucntion with an immunogenic composition, surprisingly results in enhancing a specific ThI biased immune response, in particular of ThI cytokines.

Preferably, an adjuvant comprising a polyacrylic acid containing polymer stimulates at ThI response and may be referred to as a ThI adjuvant. References to "ThI adjuvant" include references to an adjuvant which specifically enhances the immune response of cytokines specific to ThI cells. Such cytokines include IL-2, GM-CSF, IFN-γ, IL-12 (p70) and TNF-α. An increase in IgG2a may also be an indication of a ThI response.

The adjuvant composition may, in use, stimulate a Th₁ (type 1 - a cytotoxic T cell response) immune response. Alternatively, the adjuvant composition may, in use, stimulate a Th₁ and a Th₂ (a B cell antibody response) immune response.

References to "a poly acrylic acid containing polymer" refer to a polymer having the structure shown in formula (I) :

(D

Preferably the polyacrylic acid containing polymer has a molecular weight in the range of from about 1x10⁵ daltons to about 1x10¹⁰ daltons, more preferably from about 7x10⁵ daltons to 4x10⁹ daltons.

The polyacrylic acid containing polymer may comprise polymers of acrylic acid cross-linked with polyalkenyl ethers or divinyl glycol. Preferably the polymer is a carbomer. The skilled man will understand that the Carbopol® polymers are examples of carbomers.

Preferably the polymers of acrylic acid are cross linked using one or more of allyl ether of sucrose, allyl ether of penta erythritol and allyl ether of propylene. The polymers of arylic acid may have a viscosity in the range of between about 3000 and about 40000 cP (0.5wt% at ρH7.5), more preferably from between about 4000 and about 11000 cP or from between about 25000 and about 4000OcP

In one embodiment, the poly acrylic acid containing polymer is a Carbopol® polymer.

References to "Carbopol® polymer" include references to polymers of acrylic acid cross-linked with polyalkenyl ethers or divinyl glycol.

Carbopol® polymers are manufactured by a cross-linking process.

Depending upon the degree of cross-linking and manufacturing conditions, various grades of Carbopol® are commercially available. The readily water-swellable Carbopol® polymers are used as excipients in a diverse range of pharmaceutical applications, such as: controlled release tablets; bioadhesion in buccal, ophthalmic, intestinal, nasal, vaginal and rectal applications; thickening at very low concentrations to produce a wide range of viscosities and flow properties in topical lotions, creams and gels, oral suspensions and transdermal gel reservoirs; suspensions of insoluble ingredients in oral suspensions and topical formulations; and emulsifying topical oil-in-water systems without the need for irritating surfactants .

However, there has been no disclosure of polymers of acrylic acid cross linked with polyalkenyl ethers or divinyl glycol, such as Carbopol® polymers, being used as an adjuvant in human vaccine compositions.

In one embodiment, the Carbopol®polymer is Carbopol® 934P, Carbopol® 71G, Carbopol® 971P, Carbopol® 974P or Polycarbophil. In a further embodiment, the Carbopol® polymer is Carbopol® 974P. Carbopol® 934 P may be prepared by cross-linking with allyl sucrose and is polymerized in benzene as a solvent. Carbopol® 71G, 971P and 974P may be prepared by cross-linking with allyl penta erythritol followed by polymerization in ethyl acetate. Polycarbophil may be prepared by cross- linking the polymer in divinyl glycol and benzene as a solvent. All of the polymers are typically fabricated in ethyl acetate and neutralized by 1-3% potassium hydroxide.

The antigen may be a nucleic acid, a protein, a peptide, a glycoprotein, a polysaccharide or other carbohydrate, a fusion protein, a lipid, a glycolipid, a peptide mimic of a polysaccharide, a cell or a cell extract, a dead or attenuated cell or extract thereof, a tumour cell or an extract thereof, or a viral particle or an extract thereof, or any combination thereof.

The antigen may be derived from a human or non-human animal, a bacterium, a virus, a fungus, a protozoan or a prion.

In one embodiment, the antigen which elicits an immune response against a human pathogen is derived from HIV-I (such as gag or fragments thereof, such as p24, tat, nef, envelope glycoproteins such as gpl20, gpl40 or gplόO, or any fragments thereof), human herpes viruses (such as gD or derivatives thereof or Immediate Early protein such as ICP27 from ¹ HSVl or HSV2) , cytomegalovirus (esp Human, such as gB or derivatives thereof) , Rotaviral antigen, Epstein Barr virus (such as gp350 or derivatives thereof) , Varicella Zoster Virus (such as gpl, Il and IE63) , or from a hepatitis virus such as hepatitis B virus (for example Hepatitis B Surface antigen or a derivative thereof) , or antigens from hepatitis A virus; hepatitis C virus and hepatitis E virus, or from other viral pathogens, such as paramyxoviruses: Respiratory Syncytial virus (such as F G and N proteins or derivatives thereof), parainfluenza, measles virus, mumps virus, human papilloma viruses (for example HPV 6, 11 , 16, 18, ) flaviviruses (for example, Yellow Fever Virus, Dengue Virus, Tick- borne encephalitis virus, Japanese Encephalitis Virus) or orthomyxoviruses including Influenza virus purified or recombinant proteins thereof such as HA, NP, NA, or M proteins, or combinations thereof) , or derived from bacterial pathogens such as Neisseria spp, including N. gonorrhea and N. meningitidis (for example, transferrin- binding proteins, lactoferrin binding proteins, PiIC, adhesins) ; S. pyogenes (for example M proteins or fragments thereof, C5A protease,), S. agalactiae, S. mutans; H. ducreyi; Moraxella spp, including M catarrhalis, also known as Branhamella catarrhalis (for example high and low molecular weight adhesins and invasinsj ; Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium spp. , including M. tuberculosis (for example ESAT6, Antigen 85A, -B or -C) , M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L pneumophila; Escherichia spp, including enterotoxic E. coli (for example colonization factors, heat-labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof) , enterohemorragic E. coli, enteropathogenic E. coli Vibrio spp, including V. cholera (for example cholera toxin or derivatives thereof); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y. enterocolitica (for example a Yop protein) , V. pestis, Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (for example toxins, adhesins and invasins) and C. coli; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp. , including L monocytogenes; Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin) ; Pseudomonas spp, including P. aeruginosa; Staphylococcus spp. , including S. aureus, S. epidermidis; Enterococcus spp. , including E. faecalis, E. faecium; Clostridium spp. , including C. tetani (for example tetanus toxin and derivative thereof) , C. botulinum (for example botulinum toxin and derivative thereof) , C. difficile (for example Clostridium toxins A or B and derivatives thereof); Bacillus spp. , including B. anthracis (for example botulinum toxin and derivatives thereof); Corynebacterium spp. , including C. diphtheriae (for example diphtheria toxin and derivatives thereof) ; Borrelia spp. , including B. burgdorferi (for example OspA, OspC, DbpA, DbpB) , B. garinii (for example OspA, OspC, DbpA, DbpB) , B. afzelii (for example OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC, DbpA, DbpB) , B. hermsii; Ehrlichia spp. , including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp. , including C. trachomatis (for example MOMP, heparin- binding proteins), C. pneumoniae (for example MOMP, heparin-binding proteins,) , C. psittaci; Leptospira spp. , including L interrogans; Treponema spp., including T. pallidum (for example the rare outer membrane proteins,) , T. denticola, T. hyodysenteriae; or derived from parasites such as Plasmodium spp. , including P. falciparum; Toxoplasma spp. , , in eluding T. gondii (for example SAG2, SAG3, Tg34) ; Entamoeba spp. , including E. histolytica; Babesia spp. , including B. microti; Trypanosoma spp. , including T. cruzi; Giardia spp., including G. lamblia; Leshmania spp., including L. major; Pneumocystis spp. , including P. carinii; Trichomonas spp. , including T. vaginalis; Schispstoma spp. , including S. mansoni, or derived from yeast such as Candida spp. , including C. albicans; Cryptococcus spp. , including C. neof ormans .

In one embodiment, the antigen which elicits an immune response against a human pathogen is derived from HIV-I (such as gag or fragments thereof, such as p24, tat, nef, envelope glycoproteins such as gpl20, gpl40 or gplόO, or any fragments thereof) . In a further embodiment, the antigen which elicits an immune response against a human pathogen is derived from HIV-I (e.g. envelope glycoproteins such as gpl20, gpl40 or gpl60, or any fragments thereof) .

In an alternative embodiment, the antigen which elicits an immune response against a human pathogen is derived from Influenza virus purified or recombinant proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof.

In an alternative embodiment, the antigen which elicits an immune response against a human pathogen is derived from N. meningitidis (for example, capsular polysaccharides, transferrin-binding proteins, lactoferrin binding proteins, PiIC, adhesins) .

The antigen may be naturally produced (e.g. purified from the pathogen) , recombinantly produced (e.g. from a genetically-engineered expression system) or a synthetic product. The antigen may be a modified form of a natural product, for example the antigen may include modifications such as deletions, insertions, additions and substitutions, so long as the antigen elicits an immunological response that would recognise both the modified and the natural product.

The composition may comprise more than one antigen derived from the same or different pathogens .

The amount of antigen in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical subjects being vaccinated. Such amount will vary depending upon which specific immunogen/antigen is employed and how it is presented. Generally, it is expected that each human dose will comprise 0.1-1000μg of antigen, preferably 0.1-500μg, more preferably 0.1-lOOμg, most preferably 0.1 to 50μg. An optimal amount of an antigen for a particular vaccine may be ascertained by standard studies involving observation of appropriate immune responses in vaccinated subjects. Following an initial vaccination, subjects may receive one or several booster immunisations adequately spaced. Such a vaccine formulation may be applied to a subject in either a priming or boosting vaccination regime; or alternatively be administered systemically, for example via the transdermal, subcutaneous or intramuscular routes. In one embodiment, the formulation is applied via the subcutaneous or intramuscular routes. In a further embodiment, the formulation is applied via the intramuscular route'.

Generally, the polyacrylic acid containing polymer, such as a Carbopol® polymer, will be present within the adjuvant or immunogenic composition in an amount of 0.1-5% (w/w) . In one embodiment, the Carbopol® polymer is present within the composition in an amount of 0.2-1% (w/w).

It will be appreciated that adjuvant and immunogenic compositions, such as vaccine compositions, of the present invention may be used for both prophylactic and therapeutic purposes.

According to a further aspect of the invention, there is provided an immunogenic composition, such as a vaccine composition, as described herein for use in therapy. In a further embodiment there is provided a method of treatment of an individual susceptible to or suffering from a disease by the administration of a composition as described herein. Preferably the adjuvant composition, or immunogenic composition, is for use in immune activation and/or modulation. The adjuvant may be used in a composition to stimulate an immune response, for example, in a vaccine composition or in an antiviral or anticancer composition or drug. The adjuvant may also be used in a composition to modulate or control an immune response, for example, in a composition to control an allergic reaction. The adjuvant composition may be used alone, without a specific antigen, to control an immune response. The antigen may be an environmental antigen, such as pollen, nuts or other allergens. To control an allergic reaction the adjuvant composition may stimulate a Th₁ response. Alternatively, or in addition, the composition may stimulate a Th₂ response.

According to a further aspect of the invention, there is provided a method to prevent an individual from contracting a disease selected from the group comprising infectious bacterial and viral diseases; parasitic diseases, particularly intracellular pathogenic diseases; proliferative diseases such as prostate, breast, colorectal, lung, pancreatic, renal, ovarian or melanoma cancers; non-cancer chronic disorders, such as allergy, asthma, or other hypersensitivity-related immune disorders; comprising the administration of a composition as substantially described herein to said individual. In one embodiment, the disease is viral (e.g. HIV or influenza) or bacterial (e.g. meningitis) .

According to a further aspect of the invention, there is provided a use of a composition as defined herein in the manufacture of a medicament for the treatment of one or more of the above disorders.

According to a further aspect of the invention, there is provided a pharmaceutical composition as defined herein for use in the treatment of the above disorders. According to a further aspect of the invention there is provided a method of inducing a ThI antigen specific or biased immune response in a mammal, such as a human, comprising administering to said mammal a composition of the invention.

According to a further aspect of the invention there is provided a process for preparing a vaccine as described herein comprising admixing an antigen which elicits an immune response against a human pathogen with an adjuvant comprising a polyacrylic acid polymer.

In one embodiment, the admixing of the antigen with an adjuvant may be performed at low pH (eg. pH 2, 3, 4, 5 or 6) to reduce viscosity for administration (e.g. injection) purposes. Upon administration, the polyacrylic acid polymer (e.g. a Carbopol® polymer) is believed to re- equilibrate to neutral pH and recover viscosity, a feature that may be helpful for antigen retention and therefore adjuvant activity.

In one embodiment, the adjuvant, immunogenic or vaccine composition of the invention may additionally comprise one or more pharmaceutically acceptable excipients. In a further embodiment, the pharmaceutically acceptable excipients include carriers, diluents, binders, lubricants, preservatives, stabilizers, dyes, antioxidants, suspending agents, coating agents, solubilising agents and flavouring agents.

Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.

Examples of suitable diluents include ethanol, glycerol, water and the like. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.

Examples of suitable preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid and the like.

The adjuvant, immunogenic or vaccine composition may also comprise one or more additional adjuvants in addition to a polyacrylic acid containing polymer. In one embodiment, the one or more additional adjuvants may be selected from the group consisting of metal salts, oil in water emulsions, Toll like receptor ligands (in particular Toll like receptor 2 ligand, Toll like receptor 3 Hg and, Toll like receptor 4 ligand (such as monophosphoryl lipid A, an alkyl glucosaminide phosphate or 3 Deacylated monophoshoryl lipid A (3 D - MPL)), Toll like receptor 7 ligand, Toll like receptor 8 ligand and Toll like receptor 9 ligand) , saponins (e.g. Qs21) , polyethyleneimine (PEI) or combinations thereof.

According to another aspect, the invention provides the use of an adjuvant composition according to the invention in the preparation of a composition for eliciting an immune response. The composition for eliciting an immune response may be a vaccine.

According to a yet further aspect, the invention provides the use of a polyacrylic acid containing polymer in the preparation of a composition for eliciting an immune response. Preferably the composition is a vaccine, Preferably the composition also comprises one or more antigens.

According to a yet further aspect, the invention provides a vaccine composition comprising an adjuvant according to the invention and one or more antigens.

According to a yet further aspect, the invention provides an antiviral and/or an anti-cancer and/or an immuno-modulating composition comprising an adjuvant according to the invention and one or more antigens .

A vaccine composition, or a composition for eliciting an immune response, according to the invention may be for oral, systemic, parenteral, topical, mucosal, intramuscular, intradermal, subcutaneous, intranasal, intravaginal, sublingual, or inhalation administration.

Preferably, a vaccine composition, or a composition for eliciting an immune response, according to the invention is intended for administration to a human.

A composition according to the invention may be administered to a subject in the form of a pharmaceutical composition. A pharmaceutical composition preferably comprises one or more physiologically effective carriers, diluents, excipients or auxiliaries which facilitate processing and/or delivery of the antigen and/or adjuvant.

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

According to another aspect, the invention provides an immunogenic composition capable of eliciting an immune response when administered to a human or non-human animal comprising an adjuvant according to the invention. Preferably the immunogenic composition also comprises one or more antigens.

The non-human animal may be a mammal, bird or fish.

According to yet another aspect, the invention provides a method for inducing or enhancing immunogenicity of an antigen in a human or non- human animal to be treated comprising administering to said subject one or more antigens and an adjuvant composition according to the invention in an amount effective to induce or enhance the immunogenicity of the antigen in the subject. The adjuvant and antigen may be administered simultaneously, sequentially or separately.

Preferably the method produces an immune reaction sufficient to vaccinate a subject against a pathogen from which the antigen is derived.

The subject may be a human or non-human animal, including mammals, birds and fish.

The skilled man will appreciate that any of the preferable/optional features discussed above can be applied to any of the aspects of the invention.

The terms adjuvant and adjuvant composition are intended to have the same meaning and are used interchangeably. Similarly, the terms vaccine and vaccine composition are intended to have the same meaning and are used interchangeably.

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

Figure 1: (A) Serum-gpl40-specific total murine IgG response during the time course of the experiment described in Example 1. Each individual point represents the median endpoint titre for each individual mouse group at each time point. Error bars represent the range. Serum gpl40-specific total IgG response 3 weeks after the prime (B), 2 weeks after the boost (C) and 9 and a half weeks after the boost (D). Where a statistical significant difference is detected the p value is shown. The dotted line represents the detection limit, that is the lowest serum dilution tested for gpl40-specific total IgG

, response. Vaccine formulations are shown on the x-axis of each graph. Statistical analysis was done with non-parametric tests. The

Kruskall-Wallis test with Dunn's multiple comparison test was used for inter-group analysis comparing the group that received gpl40 alone to the group that received Carbopol® + gpl40 or , Alum + gpl40.

Figure 2: Serum-gpl40-specific murine IgGl response 3 weeks after the prime in the experiment described in Example 1 (A) , 2 weeks after the boost (B) and 9 and a half weeks after the boost

(C) . Serum gpl40-sρecific IgG2a response 3 weeks after the prime

(D), 2 weeks after the boost (E) and 9 and a half weeks after the boost (F) also shown. Where a statistical significant difference is detected the p value is shown. The dotted line represents the detection limit, that is the lowest serum dilution tested for a gpl40- specific total IgG, IgGl, and IgG2a response. Vaccine formulations are shown on the x-axis of each graph. The Kruskall- Wallis test with Dunn's multiple comparison test was used for inter-group analysis comparing the group that received gpl40 alone to the group that received Carbopol® +gpl40 or Alum + gpl40.

Figure 3: Ratio of murine serum IgG2a/IgGl in each individual mouse group 3 weeks after the prime in the experiment described

, in Example 1 (A), 2 weeks after the boost (B) and 9 and a half weeks after the boost (C). The IgG2a/IgGl was calculated for each individual mouse by dividing the endpoint IgG2a titre by the endpoint IgGl titre. The Mann-Whitney test was used for inter- group analysis comparing the IgG2a/igGl ratio in the group that received Carbopol® + gpl40 to the IgG2a/IgGl ratio in the group that received Alum + gρl40. Vaccine formulations are shown on the x-axis.

Figure 4: Cytokine stimulation index of immune murine splenocytes in the experiment described in Example 1 for day 1 (A) and day 5 (B). The stimulation index was calculated based on the following formula. Stimulation index = (cytokine produced in pg/mL in presence of gpl40)/ (cytokine production in pg/mL in the absence of gpl40) . The Kruskall- Wallis test with Dunn's multiple comparison test was used for inter-group analysis comparing the group that received gpl40 alone to the group that received

Carbopol® + gpl40 or Alum + gpl40 as vaccine formulations. Cytokines tested are shown on the x-axis of each graph. SPLN refers to splenocytes.

Figure 5: Serum gpl40-specific total murine serum IgG response 3 weeks after the prime in the experiment described in Example 2 (A) , 2 weeks after the boost (B) and 11 weeks after the boost (C) . Where a statistical significant difference is detected the p value is shown. The dotted line represents the detection limit, that is the lowest serum dilution tested for gpl40-specific total IgG response for each individual time point. The Kruskall-Wallis test with

Dunn's multiple comparison test was used for inter-group analysis comparing the group that received gpl40 alone to the group that received 1% Carbopol® + gpl40 or 0.2% Carbopol® + gpl40 or FCA + gρl40. "C" indicates Carbopol® 974P; FCA indicates Freund's Complete Adjuvant. Vaccine formulation is shown on the x-axis.

Figure 6: Serum-gpl40-specific murine serum IgGl response for the experiment described in Example 2 (A) and IgG2 response (B) 3 weeks after the prime. Where a statistically significant difference is detected the p value is shown. The dotted line represents the detection limit, that is the lowest serum dilution tested for a gpl40- specific IgGl and IgG2a response. The Kruskall-Wallis test with Dunn's multiple comparison test was used for inter-group analysis comparing the group that received gρl40 alone to the group that received 1% Carbopol® + gp 140 or 0.2% Carbopol® + gpl40 or FCA + gρl40. "C" indicates Carbopol® 974P; FCA indicates Freund's Complete Adjuvant. Vaccine formulation is shown on the x-axis of each graph.

Figure 7: Ratio of murine serum IgG2a/IgGl in each individual group 3 weeks after the prime for the experiment described in Example 2. The IgG2a/IgGl was calculated for each individual mouse by dividing the endpoint IgG2a titre over the endpoint IgGl titre. The Kruskall-Wallis test with Dunn's multiple comparison test was used for inter-group analysis comparing the group that received that received 1% Carbopol® + gpl40 or 0.2% Carbopol® + gpl40 to the group that received FCA + gpl40.

Figure 8: Rabbit serum gpl40-specific total IgG response 4 weeks after the I^s' immunisation in the experiment described in Example 3

(A) and 2 weeks after the 2^nd immunisation (B). Vaccine formulation and route of immunisation is shown along the x-axis.

Figure 9: Ability of each individual rabbit serum sample to neutralise MW965.23 pseudoviruses in the experiment described in

Example 3. Results expressed as the reciprocal serum ID₅₀ titre, i.e. the reciprocal serum dilution that was effective for 50% virus neutralisation. SC: subcutaneous; IM: intramuscular, C: Carbopol® 974P; P: PEL

Figure 10: Rabbit serum gpl40-specific total IgG response for the experiment described in Example 4 (A), IgGl response (B), and IgG2a response (C) 3 weeks after the 1^st immunisation. Where a statistical significant difference was observed the p value is shown. Results analysed by a non-parametric Mann- Whitney test. Blue colour: WT 129/SvEv; red colour: Myd88-/- 129/SvEv. On the x- axis for each graph the vaccine formulations are shown. Detection limit is also shown.

Figure 11; Demonstrates that Carbopol® 974P as an adjuvant results in a 10 fold dose reduction for the antigen gpl40 in mice. Groups of four mice were subcutaneously immunized on weeks 0 and 4 with 10, 1 or 0.1 μg of gpl40 either alone or with Carbopol® 975P adjuvant or Imject Alumm following the manufacturers instructions. The mean reciprocal endpoint IgG, IgGl and IgG2a titres 2 weeks after the boost are respectively shown in (A) , (B) and (C) . Error bars show SD. Antigen doses are indicated on the X-axes. Any serum antibody reciprocal endpoint titres of < 1 in 50 were considered negative and were assigned a value of 1. Evaluation of statistical differences between data obtained from the Carbopol® 974P adjuvanted group and the Imject Alum adjuvanted group was carried out using the Mann- Whitney test. * /? < 0.05.

Figure 12: Compares Carbopol® 974P, Imject Alum, Alhydrogel and FCA a s adjuvants in mice. (A) Weight (g) of Balb/c mice subcutaneously injected twice at 3 week interval (immunizations indicated by arrows) with gpl40, 1% Carbopol® 974P/gpl40, lmg Imject Alum/gpl40, lmg Alhydrogel/gpl40, or FCA (prime)-FIA (boost)/gpl40 over the course of the experiment. (B) Photos of the site of injections from a representative mouse from each individual group. White circles indicate the site of injection. Treatment is shown under each individual photo.

Figure 13: Shows the antibody responses in mice subcutaneously vaccinated with gpl40 in different adjuvants. (A) Induction of serum anti-gpl40 IgG 3 weeks after immunization with gpl40 in either 1% Carbopol® 974P, lmg Imject Alum, lmg Alhydrogel or FCA. (B) Serum gpl40-specific IgG 2 weeks post boost. Anti- gpl40 antibody levels are presented as box and whiskers plots of the value of the reciprocal endpoint IgG titre. Treatments are indicated on the X-axes. Any serum antibody reciprocal endpoint titres of < 1 in 50 were considered negative and were assigned a value of 1. A nonparametric 1-way analysis of variance (Kruskal- Wallis test) followed by a Dunn's multiple comparison post test for inter-group analysis were used for statistics. * /> < 0.05, **/? < 0.01 ***p < 0.001.

Figure 14: S hows reciprocal IgGl and IgG2a titres after immunization with gpl40 admixed with 1% Carbopol® 974P, lmg

Imject Alum, lmg Alhydrogel or FCA. The sera from week 3 post prime (A and B) and week 2 post boost (C and D) were titrated by an ELISA for IgGl (A and C) or IgG2a (B and D) . Treatments are indicated on the X-axes. Any serum antibody reciprocal endpoint titres of < 1 in 50 were considered negative and were assigned a value of 1. A nonparametric 1-way analysis of variance (Kruskal- Wallis tesfollowed by a Dunn's multiple comparison post test for inter-group analysis were used for statistics. */> < 0.05, ** /> < 0.01 *** p < 0.001, n.d: not determined.

Figure 15: Shows the serum anti-HA specific reciprocal endpoint IgG (A), IgGl (B) and IgG2c (C) titre 1 day prior to challenge with a lethal dose of PR8 virus. C57B1/6 mice were immunized by the subcutaneous route with 1 μg BHA with 1% Carbopol® 974P or Imject Alum as described in Materials and Methods. Serum antibody titres of five mice from each group were measured by ELISA using HA as the detecting antigen. Treatments are indicated on the X-axes. Any serum antibody reciprocal endpoint titres of < 1 in 50 were considered negative and were assigned a value of 1. A nonparametric 1-way analysis of variance (Kruskal-Wallice test) followed by a Dunn's multiple comparison post test for comparison of antibody responses between the two adjuvanted groups and the antigen only immunized group were used for statistics.

Figure 16: Illustrates the weight loss (A) and survival rate (B) of mice immunized with different regimes following challenge with a lethal dose of fully virulent PR8. (A) Each symbol represents the weight loss for each group as a percentage of the median weight before challenge versus after challenge. Survival and weight loss was observed for 12 days after challenge. The cross indicates the time point were all mice in that group succumbed to disease

Figure 17: Demonstrates the correlation of survival or weight loss with the serum HA-specific IgGl and IgG2c titre. (A and B) The Log₁₀ of the median reciprocal endpoint IgGl and IgG2c titre for each group 1 day before challenge is plotted against the survival rate (%) . (C and D) The Log™ of the reciprocal endpoint IgGl and IgG2c titre for each mouse 1 day before challenge is plotted against the weight loss on day 6 post challenge. The weight loss is expressed as a percentage of the median original body weight. The correlation coefficients (r) and P values are from Spearman rank analysis and the line is from linear regression analysis.

Figure 18: Demonstrates reduced tumor growth after vaccination with B16F10-Carbopol® 974P mixtures. C57B1/6 mice were immunized via the subcutaneous route with 1 x 10⁷ irradiated

^' B16F10 cells in PBS or admixed to 0.5% Carbopol® 974. 4 weeks later all mice were subcutaneously challenged with 2 x 10⁵ live tumor cells. Differences in tumor growth of B16F10/Carbopol®

974p- vaccinated animals and that of conrol mice (Bl 6F10 only) at different time points is assessed by a non-parametric Mann-

Whitney test. Lines represent the mean *: p < 0.05, **: p < 0.01.

Figure 19: Illustrates the cytokine (A) and chemokine (B) concentration (pg/mL) in Carbopol® 974P-stimulated BMDCs. BMDCs were incubated with PBS or Carbopol® 974P (0.001%) or LPS (1 μg/rnL) for 15 hours. BMDC culture supernatants were analysed for cytokine/chemokine production in a Luminex-based assay. The data are presented as mean plus SD. Pooled data from two independent experiments are shown. Detection of each individual cytokine/chemokine is shown along the x-axis.

Figure 20: Shows neutrophil infiltration following i.p treatment with 0.1% Carbopol® 974P. Neutrophil infiltration was determined by FACS analysis. Cells were stained with Ly-6G-PE and F4/80-APC as described in Methods. Expression of Ly-6G and

F4/80 on gated cells in PBS-, Carbopol® 974P-, and Zymosan- injected mice is shown. A gate (R2) was constructed around the

. neutrophil population (Ly-6G + ve, F4/80 -ve). Representative

FACS plots for each treatment group at 1 hour, 4 hour and 24 hours after i.p injection are shown (A-I) . The number in FACS plots shows the percentage of Ly-6G + ve-F4/80 -ve cells among all cells. Pooled data are shown in H as bar chart.

Figure 21: Illustrates Il-lα (A) , IL-I β (B) , IL-6 (C), IL-12 (p40) (D) , KC (E) , RANTES (F) and TNF-α (G) concentration (pg/ml) in i.p lavage at 1 hour, 4 hours and 24 hours following treatment with either 0.01% Carbopol® 974P, zymosan (10 μg/mouse) or . vehicle control (PBS) . Each point shown the mean cytokine/chemokine production in each group at each individual time point and the error bars show the SD .

Example 1

In vivo immunogenicity of Carbopol® 974P-gpl40 mixtures

The aim of this experiment was to investigate the effect of subcutaneous priming with gpl40 in the presence of Carbopol® 974P and to evaluate the cytokine profile of splenocytes stimulated in vitro with gpl40. Carbopol® 974P is commercially available and was obtained from Lubrizol.

On day 0, 3 groups of female Balb/c mice (5 mice/group) were subcutaneously (sc) immunised as follows:

Group A received 0.7% Carbopol® 974P + gpl40; Group B received gpl40 alone; Group C received Alum + gpl40.

On day 21, group A and B were sc boosted with the same formulation used in priming and Group C was sc boosted with gpl40 alone.

Blood samples were collected at different time points, i.e. 3 weeks after the prime 2 weeks after the boost and 9 and a half weeks after the boost, for analysis of serum gpl40-specific total IgG, IgGl, and IgG2a responses.

At the end of the experiment, mice were sacrificed, spleens were excised, and splenocytes from each individual mouse were stimulated in vitro for 5 days with either gpl40, medium alone (-ve control) and PMA/Ionomycin ( + ve control). On day 1 and 5, supernatants were collected for analysis of cytokine production.

(a) Results (serum gpl40-specific total-IgG responses):

A robust serum gpl40-specific total IgG response was detected in group A that received 0.7% Carbopol® 974p + gpl40 as a vaccine formulation (Fig IA) . Although weaker, gpl40-specific total IgG responses are detected in groups B and C that received gρl40 and Alum + gρl40 as a vaccine formulation respectively. Assessment of the responses at each individual time point showed that priming and boosting with 0.7% Carbopol® 974P + gpl40 formulation induced a significantly higher response as compared to the one induced by gpl40 alone (Fig IB, C, D) .

(b) Results (serum gpl40-specific IgGl and IgG2a responses):

At 3 weeks after the prime (Fig 2 A, 2D) , 2 weeks after the boost (Fig 2B, 2E) and 9.5 after the boost (Fig 2C, 2F) both the serum gρl40-specific IgGl and IgG2a responses were significantly higher in the 0.7% Carbopol® 974P + gpl40 immunised group as compared to the gpl40 alone immunised group.

(c) Results (IgG2a/IgGl):

At all time points (3 weeks after the prime/Fig 3 A, 2 weeks after the boost/Fig 3B, 9.5 weeks after the boost/Fig 3C) the IgG2a/IgGl ratio in group A (0.7% Carbopol® 974P + gpl40) was significantly higher than the IgG2a/IgGl ratio in group C (Alum + gpl40).

(d) Results (Cytokine profile of splenocytes stimulated in vitro with gpl40):

As shown in Fig 4, on day 1 and day 5 the median stimulation index of gpl40-induced IL-2, 4, 5, 10, 12 (p70), GM-CSF, IFN-γ and TNF-α production was significantly higher in the splenocyte cultures from the Carbopol® 974P + gpl40 immunised group as compared to the corresponding median stimulation index of gpl40-induced cytokine production in the splenocyte cultures from the gpl40 alone immunised group. Of importance is the significantly higher production of ThI cytokines such as IL-2, GM-CSF, IFN-γ and TNF-α.

Conclusions: Carbopol® was able to increase significantly the responses (total IgG, IgGl and IgG2a) towards gpl40 as compared to the responses induced upon of administration of gpl40 alone.

As an adjuvant, Carbopol® appears to be much stronger than Alum with a marked significant increase in the induction of total IgG, IgGl and especially IgG2a responses.

The significant difference of the IgG2a/IgGl ratio between Carbopol® + gpl40 and Alum + gpl40 immunised groups gives the first clear evidence for the Thl-biasing effect of Carbopol®.

In vitro gpl40-stimulation of splenocytes from the Carbopol® + gpl40 immunised group resulted in a significant increase of all cytokines tested, especially ThI cytokines such as IFN-γ, TNF-α, IL-2, IL-12 (p70) , as compared to the cytokines produced from the splenocytes from the gpl40 immunised group in response to gpl40 stimulation.

In summary, the data presented in Example 1 demonstrate that Carbopol® 974P'is a strong ThI adjuvant.

Example 2

In vivo immunogenicity of different concentrations of Carbopol®

974P-gpl40 mixtures The aim of this experiment was to investigate the effect of subcutaneous (sc) priming with gpl40 in the presence of different concentrations of Carbopol® 974P and to compare the adjuvanticity of different concentrations of Carbopol® 974P-gpl40 mixtures with Freund's Complete Adjuvant (FCA)-gpl40 mixtures. On day O, 4 groups of female Balb/c mice (5 mice/group) were sc immunised as follows:

Group A received 1% Carbopol® 974P ("C") + gρl40; Group B received 0.2% Carbopol® 974P ("C") + gpl40; Group C received FCA + gρl40 followed by FIA + gρl40;

Group D received gpl40 alone.

On day 21, groups A, B, and D were sc boosted with the same formulation used in priming, whereas group C received a sc boost with Freund's Incomplete Adjuvant (FIA) + gpl40.

Blood samples were collected at different time points, i.e. 3 weeks after the prime, 2 weeks after the boost and 11 weeks after the boost, for analysis of serum gpl40-specific total IgG, IgGl , and IgG2a responses.

(a) Results (serum gpl40-specific total-IgG responses):

At all time points tested (Fig 5 A, B, C), the total IgG response was higher in group A (1% "C" + gρl40) and C (FCA + gpl40) as compared to the response in group D (gpl40 alone) . These differences reached statistical significance.

Direct comparison of the response in the FCA + gρl40 immunised group with either of the two l%/0.2%"C" + gpl40 immunised group revealed no statistically significant difference (Mann- Whitney test, p > 0.05) .

(b) Results (serum gpl40-speeific IgGl and IgG2a responses 3 weeks after prime):

The serum gρl40-specific IgGl response (Fig 6A) was significantly higher in the FCA + gpl40 immunised group as compared to the gpl40 alone immunised group. Serum gpl40-specific IgG2a responses were significantly higher in the two Carbopol® (1% and 0.2%) + gpl40 immunised groups as compared to the group that was immunised with gpl40 alone.

(c) Results (IgG2a/IgGl):

The IgG2a/IgGl ratio in group A (1% "C" + gpl40) was significantly higher than the IgG2a/IgGl ratio in the FCA + gpl40 group (Fig 7) .

Direct comparison of the IgG2a/IgGl ratio in the 0.2% "C" + gpl40 group with the IgG2a/IgGl ratio in the FCA + gpl40 group showed no significant difference.

Conclusions:

The two different Carbopol® concentrations tested induced significantly higher total IgG, IgGl and IgG2a responses as compared to the corresponding responses induced following gpl40 immunisation.

The total-IgG and IgGl responses were statistically indistinguishable between the 1% Carbopol® + gpl40, 0.2% Carbopol® + gpl40 and FCA + gpl40 immunised groups.

Although not statistically significant, the gpl40-specific IgG2a response in the two Carbopol® + gpl40 immunised groups was higher than that in the FCA + gpl40 vaccinated group.

The significant difference in the IgG2a/IgGl ratio between the 1% Carbopol® + gpl40 group and the FCA + gpl40 group, together with the strong IgG2a-induced response following immunisation with Carbopol® 974P shows the strong ThI adjuvant effect of Carbopol® 974P. Example 3

Evaluation of the in vivo immunogenicity of Carbopol® 974P-gpl40 mixtures in a different animal model

The aim of this experiment was to evaluate the immunogenicity of Carbopol® 974P + gpl40 mixtures in rabbits and evaluate the immunogenicity of Carbopol® 974P + gpl40 mixtures by different immunisation routes (subcutaneous-sc and intramuscular-im) .

8 New-Zealand White rabbits were assigned in 4 groups and treated as follows:

Group (A) (n = 2) SC injection with 1% Carbopol® 974P + gρl40 at weeks 0 and 4;

Group (B) (n = 2) IM injection with 1% Carbopol® 974P + gpl40 at weeks 0 and 4;

Group (C) (n = 2) SC injection with 0.5mg PEI + gpl40 at weeks 0 and 4; and

Group (D) (n = 2) IM injection with 0.5mg PEI + gpl40 at weeks 0 and 4

At weeks 0, 4 and 6, blood samples were collected for analysis of gpl40- specific serum total IgG responses.

(a) Results (serum gpl40-specifϊc total-IgG responses): 4 weeks after the first immunisation (Fig 8A) and 2 weeks after the second immunisation (Fig 8B), the mean serum gpl40-specific total IgG response was comparable in all groups, irrespective of the adjuvant used in the vaccine formulation and the route of immunisation.

(b) Results (TZM.bl neutralisation assay for MW965.23): High titre neutralising antibody responses were detected in all rabbit groups irrespective of the route of immunisation and the adjuvant used (Fig 9) . The mean ID₅₀ titre in the 2 groups that either received sc 1% Carbopol® + gpl40 or im 1% Carbopol® + gpl40 was higher than the mean ID₅₀ titre in the corresponding two groups that received PEI as an adjuvant.

Conclusions:

The total-IgG responses induced following immunisation with Carbopol® + gp 140 shows that Carbopol® 974P is able to exert its adjuvanticity in a different animal model such as rabbits.

Subcutaneous and intramuscular administration of Carbopol® + gpl40 mixtures induced comparable total-IgG responses. Since human vaccines are preferably administrated by the im route, these results make Carbopol® 974P a more attractive adjuvant for human application.

All vaccine formulations and routes of administration used induced high titres of neutralising antibodies .

Example 4

Role of Myd88 in the adaptive immune response elicited by

Carbopol® 974P + gpl40 immunisation

Myeloid differentiation primary response gene 88 (Myd88) is a universal adapter protein used by all TLRs (except TLR3 and an alternative pathway used by TLR4), IL-IR, IFN-γR and IL-18R. As an adapter protein Myd88 is crucial in the signalling cascade initiated upon TLR engagement leading to activation of transcription factor NfKB. The use of

Myd88 knock-out mice (Myd88-/-) provided an opportunity to dissect and further identify any molecules/pathways that are involved in the initiation of an immune response by Carbopol® 974P + gpl40 immunisation.

WT (129/SvEv) and Myd88-/- (129/SvEV) received an initial immunisation on day 0 as follows:

Group A (WT129/SvEv-5 mice) : 0.2% Carbopol® 974P ' ("C") + gpl40;

Group B (Myd88-/-/129/SvEv-5 mice) : 0.2% Carbopol® 974P ("C") + gpl40; Group C (WT129/SvEv-4 mice) : CpG/PEI + gpl40;

Group D (Myd88-/-/129/SvEv-4 mice) : CpG-PEI + gp 140.

(a) Results (serum gpl40-specific total IgG, IgGl, IgG2a response):

The total IgG response in the two Myd88-/- groups were significantly reduced as compared to the response in the corresponding WT groups (A vs B and C vs D) (Fig 10A) .

The IgGl response was comparable in all groups (Fig 10B) .

Similar to the total IgG response, the serum gpl40-specific IgG2a response was significantly reduced in both the Myd88-/- groups as compared to the WT groups (Fig 10C) .

Conclusions: The strong gpl40-specific total-IgG, IgGl , and IgG2a responses detected in the WT group immunised with 0.2% Carbopol® 974P + gpl40 show that Carbopol® can act as an adjuvant in a different, more Thl/Th2 balanced mouse strain such as 129. The total IgG, IgGl , and IgG2a response were comparable between the two WT groups that received either 0.2% Carbopol® +gpl40 or CpG/PEI + gpl40.

So far, the significant reduction in the total-IgG and IgG2a responses in both of the Myd88-/- groups as compared to their WT counterparts implies that Myd88-/- is implicated in the induction of the total-IgG and IgG2a responses upon vaccination with either 0.2% Carbopol® + gpl40 or CpG/PEI + gpl40. However, since Myd88-/- is involved in signalling through other receptors other than TLRs (ie IL-IR, IL-18R and IFN-γR) we cannot yet define which specific signalling pathway is used by Carbopol® to initiate an immune response.

Example 5 The utility of Carbopol® 974P for dose reduction of antigen for a gpl40-containing vaccine

The aim of this experiment was to demonstrate that by using Carbopol® 974P as an adjuvant lower doses of antigen could be used.

Balb/c mice were immunized twice on weeks 0 and 4 with 10, 1 or 0.1 μg gpl40 either alone or in combination with 0.5% Carbopol® 974P or Imject Alum.

9 groups of 6-9 weeks old mice female Balb/c mice (4 mice/group) were subcutaneously immunized twice in a 4 week interval as follows: 3 groups received as a prime and a boost gpl40 (10 μg/mouse) either alone or admixed to 0.5% Carbopol® 974P or Imject Alum according to the manufacturers instructions. The second set of 3 groups received gpl40 (1 μg/mouse) either alone or in combination with 0.5% Carbopol® 974P or Imject Alum whereas the remaining 3 groups received gpl40 alone (0.1 μg/mouse) in the presence or absence of 0.5% Carbopol® 974P or Imject Alum. At different time points blood was collected for analysis of gpl40-specific IgG, IgGl an IgG2a responses.

Results

The combination of Carbopol® 974P or Imject Alum adjuvants with gpl40 (10 μg and 1 μg) resulted in comparable IgG responses as measured by an ELISA (Figure 11 A) .

The ELISA used to detect gpl40-specific antibody response followed the following protocol. Ninety-six well, flat-bottomed plates were coated directly with gpl40 (0.5 μg/ml) in PBS overnight at 4 °C. Coated plates were washed thrice (300 μl/well) with wash buffer (WB, PBS supplemented with 0.05% Tween 20) followed by blocking with either 2% skimmed milk supplemented with 0.05% Tween 20/PBS or 1% BSA supplemented with 0.05% Tween 20/PBS for 2 hours at room temperature (RT) . Following washing as before serum samples were serially diluted in sample buffer (SB, 1% BSA in PBS supplemented with 0.05% Tween 20) in 96-well v-bottomed plates and transferred (50 μl/well) to the already blocked gpl40-coated plates for 2 hours at RT. Bound antibodies were detected using one of the following HRP-conjugated secondary antibodies (50 μl/well) following washing as before: anti-mouse IgG (used 1:10000 in SB for 1 hour at RT), anti-mouse IgGl (used 1 : 1000 in SB for 1 hour at RT) , anti-mouse IgG2a (used 1:1000 in SB for 1 hour at RT) . After washing, gpl40-specific antibodies were detected using TMB substrate (50 μl/well) . Before reading the optical density (OD) at 450 nm the reaction was stopped with 0.5M H₂SO4 (50 μl/well) . Serum hemagglutinin (HA) -specific antibody responses were detected following the protocol described above with only minor modifications. In particular, ninety-six well, flat-bottomed plates were coated directly with HA (0.5 μg/ml) in 0.1 M NaHCO₃ pH 8.5 buffer overnight at 4 ⁰C. Washed plates were blocked with 1% BSA supplemented with 0.05% Tween 20/PBS for 2 hours at RT.

Importantly, immunization with a 10-fold lower doses of gpl40 using Carbόpol® 974P adjuvant resulted in significantly increased IgG titres as compared to those achieved by the same antigen dose in Imject Alum adjuvant (Figure 11 A). Likewise, the IgGl response induced with 10 or lμg of gpl40 with Carbopol® 974P were similar in magnitude to those achieved with 10 or 1 μg of gpl40 in Imject Alum, whereas a significant difference between the two groups became apparent with the lowest gpl40 dose (0.1 μg) (Figure HB) . Analysis for IgG2a responses revealed that the Carbopol® 974P adjuvanted vaccines induced antibody responses at all doses of gpl40 investigated, but most importantly these responses were consistently higher than those achieved by the Imject Alum adjuvanted vaccines (Figure HC) .

Example 6

Comparison of Carbopol® 974P vs Imject Alum, Alhydrogel and FCA as adjuvants in mice

Having shown that Carbopol® 974P exhibits strong adjuvant activity in vivo leading to enhanced gpl40-specific IgG, IgGl and IgG2a responses an experiment was designed to compare its immunostimulatory capabilities with those of known adjuvants that are routinely used in human vaccines and in day to day animal experiments. The adjuvants chosen for comparison were 1% Carbopol® 974P, lmg Imject Alum, a mixture of aluminium hydroxide and magnesium hydroxide, 1 mg Alhydrogel, and FCA, one of the most common adjuvants used in animal experiments .

6-8 weeks old female Balb/c mice (5 groups; 7 mice/group) were immunized subcutaneously with gpl40 (2 μg/mouse) either alone or formulated in either of the following adjuvants: 1% Carbopol® 974P, lmg Imject Alum, lmg Alhydrogel, or FCA. A subcutaneous boost was administrated 3 weeks later using the same antigen/ adjuvant formulations as in prime apart from the FCA/gpl40-primed group. The latter was boosted with gpl40 (2 μg/mouse) in FIA. Two weeks following the boost spleens were collected for assessing of gpl40-specific T-cell responses. The mice weight as well as the site of injection for any local adverse effects were monitored during the entire course of the experiment. At the end of the experiment, prior to collection of spleens, a picture of the site of injection was taken.

Results

As shown in Figure 12A no weight loss was observed in either the control group (gpl40 only) or the adjuvant/gpl40 treated groups. As expected no signs . of adverse effects at the site of injection were observed in the group that received only the protein antigen (Figure 12B) . On the other hand administration of gpl40 plus Imject Alum or Alhydrogel resulted in the formation of subcutaneous nodules at the site of injections, an adverse effect that has been reported with aluminium-containing adjuvants.

Following immunization with gpl40 in the potent mineral oil adjuvants

FCA of FIA, the injection sites at the end of the experiment were characterized by abscess formation, a clear indication that these adjuvants are not easily absorbed. In case of Carbopol® 974P/gpl40 administration macroscopic examination of the injection sites did not show any obvious adverse effects including swelling, granuloma and abscess formation. Blood was collected at week 3 post prime, week 2 post boost and assessed for IgG, IgGl and IgG2a responses. All mice immunized with either the 1% Carbopol® 974P/gpl40 or the FCA/gpl40 preparation were seropositive after only a single immunization, with the responses being comparable between the two groups (Figure 13A) . In contrast, only a fraction of mice immunized with gpl40 or Imject Alum/gpl40 or Alhydrogel/gpl40 had a detectable IgG response. Most importantly the gpl40-specific IgG response detected in both, the Imject Alum/gpl40- immunized group and then Alhydrogel/gpl40-immunized group, was significantly lower than the corresponding response in the 1% Carbopol® 974p/gpl40 immunized group. After the booster immunization the IgG response increased in all groups irrespective of the administrated formulation (Figure 13B) . Similarly as seen after the prime the median anti-gpl40 antibody titre in the Carbopol® 974P/gpl40 group was consistently higher that in groups receiving gpl40 in either Imject Alum or Alhydrogel respectively. However at this time point only the Carbopol® 974P/gpl40 and the Alhydrogel/gpl40 groups differed significantly in terms of their gpl40-specifc IgG response. Using a multiple comparison test for intergroup analysis showed no significant difference between the Carbopol® 974/gpl40 and the Imject Alum/gpl40 and FCA/gpl40 groups. Nevertheless, when a Mann-Whitney test was used , for comparison the Carbopol® 974P/gpl40 group differed significantly in terms of IgG from both groups (Carbopol® 974P/gpl40 vs Imject Alum/gpl40: P < 0.01; Carbopol® 974P/gpl40 vs FCA/gpl40: P < 0.05) .

As seen in previous experiments the subclass analysis of the anti-gpl40 antibodies 3 weeks post prime revealed a strong and rapid induction of IgGl and IgG2a in response to Carbopol® 974P/gpl40 immunization. For both, IgGl (Figure 14A) and IgG2a (Figure 14B) , a significant difference was detected between the Carbopol® 974 and the Imject Alum and Alhydrogel groups (for IgGl : Carbopol® 974P/gpl40 vs Imject Alum/gρl40: P < 0.05; Carbopol® 974P/gρl40 vs Alhydrogel/gρl40: P < 0.05, for IgG2a: Carbopol® 974P/gpl40 vs Imject Alum/gpl40: P < 0.01; Carbopol® 974P/gpl40 vs Alhydrogel/gpl40: P < 0.001) . Lack of IgG2a production by Imject Alum and Alhydrogel was not unexpected since both of then are known to induce a Th2 type of an immune response that is characterized by a dominated IgGl response. The unexpected finding was to see the superiority of Carbopol® 974 at inducing IgG2a in comparison to a strong Thl/Th2 adjuvant such as FCA (Figure 14B) . Although the IgG2a response between these two groups was strikingly different a multiple comparison test showed no significant difference. Nevertheless, direct comparison by a Mann- Whitney test showed the significantly higher Carbopol® 974P-induced IgG2a response (Carbopol® 974P/gpl40 vs FCA/gpl40: P < 0.01) . After the booster immunization the difference in the IgG2a titre between the Carbopol® 974 and the Imject Alum and Alhydrogel groups was still significant (Figure 14D) , whereas the IgGl response was significantly different only between the Carbopol® 974P, and the Alhydrogel group (Figure 14C) . As seen after prime, the IgG2a response in the Carbopol® 974P/gpl40 primed and boosted group was significantly higher than that in the FCA/gpl40 group (P < 0.05, Mann Whitney test) (Figure 14D) .

Example 7

Subcutaneous administration of Carbopol® 974P-combined inflenza virus BHA vaccine protects mice from a lethal PR8 virus infection To compare the adjuvant effects of Carbopol® 974P and Imject Alum with BHA, the IgG, IgGl and IgG2c responses to BHA protein were assesed in C57B1/6 mice.

7 weeks old female C57B1/6 mice (4 groups; 5 mice/group) were systemically primed on week 0 and boosted on week 4 by the subcutaneous route with one of the following formulations: BHA (1 μg/mbuse) , 1% Carbopol® 974P/BHA (1 μg/mouse) , Imject Alum/BHA (1 μg/mouse) . One group was left unimmunized during the entire course of the experiment. Six weeks later all of the previous immunized groups were boosted with BHA alone (1 μg/mouse) . Three weeks later all mice, under ether anaesthetization, were intranasally challenged with a lethal dose ^' (0.1 units/mouse) of fully virulent A/PR8/34 (HlNl) virus. Following challenge mice were monitored for mortality and weight loss until the termination of the experiment.

Results

Among the adjuvants used, Carbopol® 974P induced the highest serum IgG (Figure 15A) , and IgG2c (Figure 15C) antibody titres to HA. In terms of IgG and IgG2c, the groups differed significantly (P < 0.05 Kruskal-Wallice test), however, a Dunn's multiple comparison post test for intergroup analysis showed no significant increase of IgG and IgG2c responses between the two adjuvanted groups and the BHA alone vaccinated group, despite the fact that the median IgG and IgG2c responses in the Carbopol® 974P/BHA-immunized group were respectively ~ 16 fold and ~ 30 fold higher than the corresponding responses in the group recieving no adjuvant. The serum HA-specific IgGl response was comparable and statistically indistinguishable between the groups (Figure 15B). Example 8

Protective efficacy of different vaccine formulations to influenza challenge

To test the efficacy of the different vaccine formulations, mice were challenged with a lethal dose of A/PR/8/34 influenza virus (HlNl) and monitored daily for weight loss and mortality. Weight loss for each animal is expressed as a percentage of the original body weight 1 day before challenge versus days after challenge.

As shown in Figure 16A unimmunized mice showed a drastic loss of body weight, with all the mice losing more than 20% of their original body weight by day 6 after challenge. Similarly, by day 6 after challenge all of the BHA-immunized mice (100%) and 4 out of 5 (80%) of the Imject Alurn/BHA-immunized mice did drop to less than 80% of their original weight. In contrast, the Carbopol® 974P/BHA-immunized mice lost the least weight, with none of the immunized mice losing more than 10% of their original body weight at any point of the experiment (Figure 16A) . Among the different vaccine formulations, only the Carbopol® 974P- containing formulation provided complete protection against viral infection (Figure 16B). In contrast, 60% and 40% of the Imject Alum/BHA and the BHA-immunized mice died following challenge with the lethal PR8 virus respectively. As expected none of the unimmunized mice survived the challenge (Figure 16B) .

Example 9

Correlation between HA-specific antibody production and protection from weight loss or death

Induction of cell-mediated immune responses, which have been shown to be important in conferring protection against influenza viral infection, were not evaluated in this experiment. Nevertheless, analysis of IgG subtypes is indicative of the T cell responses induced. Therefore the titre of HA-specific antibodies (IgGl or IgG2c) was documented in correlation with the survival rate (Figure 17 A and B) or the percentage of the original weight after challenge (Figure 17C and D) .

As shown in Figure 17B a good positive correlation was observed between the induction of antibodies of the IgG2c subtype and protection against PR8 challenge, however, due to the small number of data points this correlation was not significant (correlation coefficient = 1.000, P > 0.05) . In contrast, a negative correlation was observed between the survival rate and the induction of an IgGl response (correlation coefficient = -0.5000, P > 0.05) (Figure 17A) . Plotting the percentage of initial body weight on day 6 after challenge against the IgG2c response allowed for more rigorous analysis, highlighting the importance of IgG2c induction following immunization. As shown in Figure 17D the IgG2c titre was significantly correlated with reduced weight loss (correlation coefficient = 0.7383, P = 0.0017), whereas unlike IgG2c, the level of IgGl did not correlated in any way with the percentage of original body weight (Figure 17C) .

Example 10

Suppression of tumor growth by immunization with B16F10 formulated in Carbopol® 974P adjuvant

As a general purpose adjuvant Carbopol® should be readily applicable and effective in different settings. Therefore, a prophylactic study was conducted to examine the efficacy of B16F10/ Carbopol® 974P mixtures against subsequent B17F10 challenge. Mice were immunized and subsequently challenged with live B16F10 tumor cells. More specifically, 6-9 weeks old C57B1/6 mice were immunized subcutaneously as follows: Group A (n = 9) received 1 x 10⁷ of irradiated B16F10 cells in PBS and group B (n = 12) received 1 x 10⁷ of irradiated B16F10 cells in presence of 0.5% Carbopol® 974P. 4 weeks later all mice were challenged by the subcutaneous route with 2 x 10⁵ live B16F10 tumor cells. Tumor growth was evaluated on day 5, 12, 18 and 21 and the tumor size was calculated as follows: a 2mmx 2mm tumor is plotted as 2 mm x 2 mm x 2 mm = 8mm³-

Results

Interestingly, tumors that developed in mice immunized with Bl 6F10/ Carbopol® 974P mixtures were significantly smaller than those of control mice (day 5 post challenge: p < 0.01, day 12 post challenge: p < 0.05) (Figure 18) .

Example 11

Maturation of dendritic cells as assessed by upregulation of activation markers and secretion of cytokines/chemokines

To test whether Carbopol® 974P had any direct effect on dendritic cell function, bone marrow derived dendritic cells (BMDC) were incubated with Carbopol® 974P for 15 hours. The maturation status of BMDCs was determined by the expression of cell surface activation markers, such as MHC class II, CD40, CD80 and CD86 on CDllc-positive cells. BMDCs incubated with Carbopol® 974P showed no increased expression of either of these surface activation markers (data not shown) . As an additional measure of BMDC activation secretion of pro-inflammatory cytokines and chemokines in response to Carbopol® 974P stimulation was evaluated in a Luminex-based assay. In the Luminex-based assay cytokine standards were reconstituted in 500 μl of the appropriate matrix (RPMI 1640 with L-glutamine supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin), left at 4 -C for 30 min, and then serially diluted in the appropriate matrix. 50 μl of 0.5x anti-cytokine labeled beads (prepared in bio-plex assay buffer) were added in each well and washed twice with bio-plex wash buffer (100 μl/well) using a vacuum manifold pump. Samples of medium from test ceslls (50 μl/well) and standards (50 μl/well) were then added in the plate. Two control wells where anti- cytokine beads were incubated in matrix alone were also included to obtain a background signal for the assay. The assay plate was sealed with film and covered with aluminum foil before shaking at 1100 rpm for 30 seconds and 300 rpm for 30 minutes at room temperature. The wells were then washed three times in bio-plex wash buffer as before and 25 μl of 0.5x of detection antibody (prepared using antibody diluent) was added to each well. The plate was then sealed and incubated as before. Following three washes as previously 50 μl of 0.5x streptavidin-PE antibody (prepared in bio-plex assay buffer) were added in each well and the plate was sealed and incubated for 10 minutes at room temperature as before. The plate was then washed three times as before followed by resuspension of the beads in 125 μl of bio-plex assay buffer. Prior to loading into the Luminex machine the plate was shaken at 1100 rpm for 30 seconds.

Bone marrow-derived dendritic cells were generated as follows. Briefly, female Balb/c mice were sacrificed using rising CO₂ concentration and femurs and tibias were removed and purified from the surrounding muscle tissue. Following cutting of both ends of the bones with scissors the bone marrow was flushed with handling medium (HM; RPMI 1640 medium with L-glutamine supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin) using a 5 ml syringe with a 26-gauge needle. Bone marrow cells were centrifuged (1250 rpm for 4 minutes at 4°C) , counted using trypan blue, resuspendend in culture medium (RPMI medium with L-glutamine supplemented with 10% heat- inactivated fetal bovine serum, 2 mM L-glutamine, 1% penicillin/streptomycin, 0.00035% 2-mercaptomethanol and 20 ng/ml recombinant mouse granulocyte macrophage colony-stimulating factor) at a final concentration of 1 x 10⁶ cells/ml and placed into a 24-well flat-bottom plate (1 ml/well) . Cells were .cultured in a standard incubator (5% CO2 at 37 °C) for a total of 7 days. Culture medium was replaced by fresh culture medium (1 ml/well) on day 3. On day 7 immature bone marrow-derived dendritic cells were incubated with LPS (1 μg/ml) , or Carbopol® 974P (Oi 001% corresponding to 10 μg/ml) overnight. PBS treated dendritic cells were also - incubated as a negative control. After overnight incubation supernatant was collected and stored at -80 °C until analysis for cytokine/chemokine production. Dendritic cells were harvested using HM, pelleted (1250 rpm for 4 minutes at 4°C) and counted using trypan blue. Cells (2 x 10⁵) were then transferred into a 96-well v-bottom plate and washed once with ice cold FACS wash buffer (FWB; 1% BSA in PBS) by centrifugation (1250 rpm for 4 minutes at 4°C) . Following incubation with Fc-γ block antibody for 15 minutes on ice to block binding to Fc receptors cells were labeled with the following monoclonal antibodies for 30 minutes on ice: PE-conjugated anti-CDllc, FITC- conjugate anti-MHC class II, FITC-conjugated anti-CD80, PE-conjugated anti-CD40 antibodies, PE-conjugated anti-CD86 and the corresponding isotype control. Cells were then washed twice with FWB and once with PBS as before. Following fixing with 1% formaldehyde/PBS (100 μl/well) cells were washed twice with PBS as before, resuspended in FWB and subjected to fluorescence-activated cell sorting (FACS) analysis. As shown in Figure 19 A, incubation of BMDCs with Carbopol® 974P greatly increased the production of a variety of cytokines including IL- lα, IL-lβ, IL-4, IL-12 (p40) and IL18. Similarly chemokine production was also upregulated in response to Carbopol® 974P stimulation, with the level of all chemokines tested being higher than the baseline level in the PBS-stimulated BMDC cultures (Figure 19B) .

Example 12

Carbopol® 974P mechanism of action using a murine peritonitis model

Although Carbopol® 974P is capable of inducing a strong ThI response in vivo, direct stimulation of antigen presenting cells such as dendritic cells fails to induce IL-12 production, a key cytokine known to be crucial for ThI polarisation in vivo . Nevertheless, Carbopol® 974P stimulation of BMDCs lead to an increase of proinflammatory cytokines and chemokines that are known to induce recruitment of various cell types in vivo. It is therefore possible that in vivo production of these cytokine/chemokines following Carbopol® 974P administration could induce infiltration of various cell types that could in turn either directly or indirectly shape the type of the induced immune response. The effects of Carbopol® 974P administration on cell infiltration and intraperitoneal (i.p) lavage cytokine/chemokine levels in a murine model of peritonitis was therefore examined.

To investigate the imniunostimulatory properties of Carbopol® 974P in vivo, a time-course experiment was performed extending over 24 hours, in which 6-9 weeks old female Balb/c mice were administrated 500 μl of 0.1% Carbopol® 974P or zymosan (10 μg/mouse) or vehicle control (PBS) intraperitoneally. After 1, 4 and 24 hours mice were sacrificed and peritoneal exudates were collected by peritoneal lavage with 4 ml of sterile RPMI 1640 medium with L-glutamine supplemented with 2 mM EDTA. Total cell counts were determined, and the cellular composition (neutrophils) of peritoneal lavage fluid was obtained by FACS analysis following the same protocol as described above using PE-conjugated anti- mouse Ly-6G (neutrophil marker) and APC -conjugated anti-mouse F4/80 (monocyte/macrophage marker) antibodies.

It was interesting to see that administration of Carbopol® 974P i.p produced a gradual exravasation of neutrophils into the peritoneal cavity (Figure 20 B, E, H and J). Unlike Carbopol® 974P, zymosan injection brought the peak neutrophilia at 4 hours (Figure 20 C, F, I and J) .

The cytokine/chemokine profile of i.p lavage samples from each individual mouse was examined. Analysis revealed that Carbopol® i.p injection lead to the production of a number of pro-inflammatory mediators such as Il-lα, IL-lβ, IL-6, IL-12 (p40) , KC, RANTES and TNF-α (Figure 21A-G) . Production of most of these mediators peaked at 4 hours post injection and by 24 hours their level was reduced back to baseline. Only IL-12 (p40) and RANTES production appeared to increase with time. These data are in agreement with the strong neutrophil extravasation following Carbopol® 974P i.p treatment since IL-lβ and KC are known recruiters of neutrophils .

Claims

1. An adjuvant composition comprising a polyacrylic acid containing polymer.

2. Use of a polyacrylic acid containing polymer as an adjuvant.

3. An immunogenic composition comprising: an antigen which elicits an immune response; and an adjuvant comprising a polyacrylic acid containing polymer.

4. A composition according to claim 3 for use as a vaccine.

5. The composition of claim 1, 3 or 4, or the use of claim 2, wherein the adjuvant enhances a ThI biased immune response.

6. The composition or use of claim 5 wherein the ThI biased response enhances the level of one or more of the cytokines selected from the group comprising IL-2, GM-CSF, IFN-γ, IL-12 (p70) and TNF-α.

7. . The composition or use of any preceding claim wherein the polyacrylic acid containing polymer comprises polymers of acrylic acid cross linked with polyalkenyl ethers or divinyl glycol.

8. The composition of claim 7 wherein the cross linking agent is selected from the group comprising allyl ether of sucrose, allyl ether of penta erythritol and allyl ether of propylene.

9. The composition or use of any preceding claim wherein the polyacrylic acid containing polymer is a carbomer.

10. The immunogenic composition of claim 3 or any claim dependent thereon wherein the antigen is derived from a human pathogen.

11. The immunogenic composition of claim 10 wherein the antigen is derived from HIV-I (such as gag or fragments thereof, such as p24, tat, nef, envelope glycoproteins such as gpl20, gpl40 or gplόO, or any fragments thereof) , Influenza virus purified or recombinant proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof) or N. meningitidis (for example, capsular polysaccharides, transferrin- binding proteins, lactoferrin binding proteins, PiIC, adhesins) .

12. The immunogenic composition of claim 3, or any claim dependent therepn, wherein the composition contains 500μg, more preferably 0.1- lOOμg, most preferably 0.1 to 5O_jUg of antigen per dose.

13. The composition of any preceding claim wherein the adjuvant is present in an amount of 0.1-5% (w/w) .

14. The composition of any preceding claim for use in therapeutic or prophylactic treatments or both.

15. Use of an adjuvant according to claim 1 , or any claim dependent thereon, in the preparation of a composition for eliciting an immune response.

16. The use of claim 15 wherein the composition is a vaccine.

17. The use of claim 15 or 16 wherein the composition also comprises one or more antigens.

18. An anti- viral and/or an anti-cancer and/or an immuno-modulating composition comprising an adjuvant according claim 1, or any claim dependent thereon.

19. A pharmaceutical composition comprising an adjuvant according to claim 1 or any claim dependent thereon, or a composition according to claim 3 or any claim dependent thereon, and one or more physiologically effective carriers, diluents, excipients or auxiliaries.

20. ■ A method for inducing or enhancing immunogenicity of an antigen in a human or non-human animal subject to be treated, comprising administering to said subject one or more antigens and an adjuvant composition according to claim 1, or any claim dependent thereon, in an amount effective to induce or enhance the immunogenicity of the antigen in the subject.

21. The method of claim 20 wherein the adjuvant and antigen are administered simultaneously, sequentially or separately.

22. , The method of claim 20 or claim 21 wherein the immune reaction produced is sufficient to vaccinate a subject against a pathogen from which the antigen is derived.

23. A method of treatment or prophylaxis of an individual suffering from a disease by the administration of a composition as defined in any of claims 1, 3 to 14, 18 or 19.

24. The method of treatment as defined in claim 23 wherein said disease is selected from the group comprising infectious bacterial and viral diseases; parasitic diseases, particularly intracellular pathogenic disease; proliferative diseases such as prostate, breast, colorectal, lung, pancreatic, renal, ovarian or melanoma cancers; non-cancer chronic disorders including allergies, asthma and other hypersensitivity-related disorders.

25. The method of treatment as defined in claim 24 wherein said disease is a viral disease such as HIV, meningitis or influenza.

26. Use of a composition as defined in any of claims 1 , 3 to 14, 18 or 19 in the manufacture of a medicament for the treatment of a viral disorder such as HIV or influenza or a bacterial disorder such as meningitis .

27. A method of inducing a ThI specific immune response in a mammal, comprising administering to said mammal a composition as defined in any of claims 1, 3 to 14, 18 or 19.

28. A process for preparing a composition as defined in claim 3 or any claim dependent thereon which comprises admixing an antigen which elicits an immune response with an adjuvant comprising a polyacrylic acid containing polymer.

29. A process as defined in claim 28 wherein said admixing step is performed at a low pH, such as pH 2, 3, 4, 5 or 6.