An adjuvant complex composed of bacterial outer membrane protein proteosomes complexed to bacterial liposaccharide is prepared to contain the component parts under a variety of conditions. The complex can be formulated with antigenic material to form immunogenic compositions, vaccines and immunotherapeutics. An induced immune response includes protective antibodies and/or type 1 cytokines is shown for a variety of protocols.

Description of the Invention

FIELD OF INVENTION

This invention relates to adjuvants for enhancing the immunogenicity and improvement of the immune response of antigens and to methods and compositions for preparing and using them.

BACKGROUND OF THE INVENTION

The ability of antigens to induce protective immune responses in a host can be enhanced by combining the antigen with immunostimulants or adjuvants. Alum-based adjuvants are almost exclusively used for licensed injectable human vaccines, however, while alum enhances certain types of serum antibody responses (Type 2), it is poor at enhancing other types of antibody responses (Type 1) and is a poor activator of cellular immune responses that are important for protection against intracellular pathogens and for therapeutic vaccines for cancer and allergy. Furthermore, alum enhances allergic reactions due to production of IgE. Although numerous substances have been tested and shown to be potent adjuvants for antibody and cellular (Type 1) immune responses in animal models, very few have proved to be suitable for use in humans due to unacceptable levels of reactogenicity and/or disappointing immuno-enhancing abilities. Furthermore, there are currently no licensed adjuvants capable of enhancing immune responses at mucosal surfaces where the majority of infectious agents enter the host. Indeed, development of the most promising nasally delivered mucosal adjuvants, the bacterial enterotoxins (e.g. mutated cholera and heat-labile toxins), have been halted in North America due to their ability to be transported to, and cause inflammation in the olfactory bulb region of the CNS of rodents. There is a need for potent adjuvants that are safe in humans and capable of inducing protective systemic and mucosal humoral and cellular immune responses.

Lipopolysaccharides (LPS) from gram negative bacteria are potent adjuvants. LPS activates the innate immune system causing production of inflammatory cytokines such as IL-1, TNF-.quadrature., IL-10 and IL-12 from macrophages and dendritic cells; IL-1, IL-6 and IL-8 from endothelial cells and IL-8 from epithelial cells. In addition, LPS is a B cell activator in mice and, to a certain extent in humans, as evidenced by B cell mitogenicity and stimulation of polyclonal antibody secretion. LPS mediates it's effects by binding to CD14 molecules and activation of toll like receptors (TLR) on the surface of antigen presenting cells leading to the initiation of a transcriptional cascade, gene expression and secretion of pro-inflammatory molecules.

Despite the adjuvant potential of LPS, its use in humans has been restricted due to the associated endotoxicity mediated by the lipid A portion of the molecule. Chemical modification of the lipid A region of LPS was shown to substantially detoxify lipid A (e.g. monophosphoryl lipid A or MPL-A or e.g. alkali-detoxification to remove certain fatty acids) while maintaining certain adjuvant properties (see Qureshi et. al. J. Biol Chem 1982; 257:11808-15). While MPL-A exhibited potent adjuvant activity in animals, the experience in humans has been inconsistent, showing poor adjuvant activity with some antigens and unacceptable reactogenicity overall in many situations.

Non-covalent proteosome-LPS complexes, containing proteosomes from Neisseria meningitidis and purified LPS from Shigella flexneri or Plesiomonas shigelloides, have been administered to humans intranasally and orally in phase 1 and phase 2 clinical trials in the context of stand-alone vaccines. These vaccines induce protective immune responses against Shigella flexneri or S. sonnei infection, respectively, in animals (Mallet et. al. Infect and Immun 1995; 63:2382-86) and humans (Fries et. al. Infect Immun. 2001; 69:4545-53) when given via the intranasal route. Further, these complexes were well-tolerated via the nasal or oral routes in humans at very high doses (up to 1.5 mg of proteosomes along with comparable amounts of LPS given intranasally and up to 2 mg of each of the proteosome and LPS components given orally) (Fries et. al. 2000) and showed no olfactory bulb or other CNS associated toxicity in small animal toxicity studies. Proteosomes consist predominantly of porin proteins and other outer membrane proteins. Evidence suggests that proteosome porins may also induce IL-12 from dendritic cells and induction of CD8+ T cells (Jeannin et. al. Nature Immunology 2000; 1:502-509) and activation of Hela cells to produce IL-8 (Pridmore et. al. J. Infect Dis 2000; 10:183). Proteosome porins also upregulate B7.2 (CD28) co-stimulatory molecules on antigen presenting cells via the activation of the toll-like receptor 2 (Massari et. al. J. Immunol. 2002, 168:1533-1537).

Dalseg et. al. (in Vaccines 96 pp. 177-182 (Cold Spring Harbor laboratory Press, 1996)) report the use of meningococcal outer membrane vesicles (OMV's) as a mucosal adjuvant for inactivated whole influenza virus. Dalseg and his associates and collaborators have reported that the OMV's they prepare employ a process that retains 6% to 9% of endogenous lipooligosaccharide (LOS) remaining compared to the amount of total OMV protein by weight. These OMV preparations have also been reported to specifically retain 16% of detergent (deoxycholate) in their OMV's, an amount that may be unhealthy or toxic in toxicity studies or in humans.

BRIEF DESCRIPTION OF INVENTION

The instant invention (IVX-908) describes compositions of and processes for production of novel formulations that are adjuvants for antigens and result in adjuvanted vaccines or immunotherapeutics when the invention and antigen(s) are combined by simple mixing and the adjuvanted vaccines or immunotherapeutics are delivered by a parenteral or mucosal route. The adjuvant consists of two major components. The first component is an outer membrane protein preparation of proteosomes prepared from gram-negative bacteria including, but not limited to Neisseria meningitidis. The second component is a preparation of liposaccharide. Liposaccharide includes native or modified lipopolysaccharide (LPS) and lipooligosaccharide derived from S. flexneri or Plesiomonas shigelloides or other gram-negative bacteria including, but not limited to, Shigella, Plesiomonas, Escherichia or Salmonella species. The two components may be formulated at specific initial ratios by processes described, so as to optimize interaction between the components resulting in stable non-covalent complexes of the components to each other. The processes generally involve the mixing of the components in a selected detergent solution (e.g. Empigen BB, Triton X-100, and/or Mega-10) and then effecting complexing of the components while removing detergent by dialysis or, preferably, by diafiltration/ultrafiltration methodologies. Mixing, co-precipitation and/or lyophilization of the two components may also be used to effect adequate complexing or association.

The end result of the process is the production of an adjuvant that when administered together with antigens forms an adjuvanted vaccine or immunotherapeutic that can be delivered by a mucosal route (such as nasal, oral, oropharyngeal, ocular, geniturinary mucosal including vaginal, sublingual, intrapulmonary, intratracheal or rectal) or a parenteral route (such as intramuscular, subcutaneous, intravenous, intraperitoneal, submucosal, intradermal) or a transdermal, topical or transmucosal route to induce enhanced levels of serum and/or mucosal antibodies and/or type 1 cellular immune responses against the antigen compared with the antigen alone given by the same routes. In the following examples, mixtures containing proteosome-LPS (using LPS from either Shigella or Plesiomonas or Escherichia or Salmonella) and a mono or multivalent split or purified recombinant influenza antigen and delivered by liquid or spray or by injection as an adjuvanted influenza vaccine induced specific anti-influenza immune responses including, for example one or more of the following: a) serum IgG antibodies or serum antibodies measured in functional assays including, but not limited to, hemagglutination inhibition (HAI) antibodies; it is noted that HAI responses are significant since their induction is known to correlate with protection against influenza in humans; b) mucosal antibodies including IgA in mucosal secretions collected from the respiratory, gastrointestinal or genitourinary tracts including, but not limited to the nasopharynx, lungs and vagina and c) correlates of cell-mediated immunity (CMI) including the switch or decrease from higher or predominant type 2 responses to result in mixed, balanced, increased or predominant type 1 responses, for example, as measured by the induction of cytokines such as IFN-.gamma. without comparable increases in induction of certain type 2 cytokines such as IL-5 whose levels may, for example, be maintained, decreased, or absent. Such Type 1 responses are predictive of the induction of other CMI associated responses such as development of cytotoxic T cells (CTLs) indicative of Th1 immunity.

The ability of the adjuvant given nasally or intramuscularly to elicit these three types of responses against the antigen indicate that the vaccine can provide immunity against infectious diseases since functional serum antibodies (including HAI antibodies) and virus specific lung antibodies are generated. Also, the induction of vaginal IgA for mucosally administered adjuvanted vaccines using the adjuvant of the instant invention supports utilization against mucosal infections or allergies distal from the site of immunization such as at the gastrointestinal or genitourinary tracts. Furthermore, the induction of type 1 of responses assists the elimination of residual or intracellular virus, parasite or certain bacterial pathogens. In addition the ability of the adjuvant to produce type 1 immune responses against the antigen will be beneficial for producing effective therapeutic vaccines for example against cancer, autoimmune diseases and allergy where CTL and Th1 cytokine responses are important.

For example, allergic rhinitis can often be effectively controlled by immunotherapy --a series of injections with increasing doses of the substance against which the individual is allergic. Allergic rhinitis can be cured in approximately 50% of individuals who undergo classic immunotherapy. Successful immunotherapy is associated with one or more of the following: a switch from T cell responses that result in the production of type 2 cytokines (e.g. IL-5 and IL-4) to those that produce type 1 cytokines (e.g. IFN-.gamma.) and/or an increase in IgG and/or reduction in IgE specific for the allergen. However, in order to achieve these results, up to three years of repeated immunizations are required. The use of allergens together with adjuvants that promote type 1 immune responses may enhance the effectiveness of such immunotherapy and reduce the number of immunizations required.

In the following example we show the results of studies in mice immunized intranasally with IVX-908 together with rBet v 1a as a recombinant protein representing the major allergen of Birch tree pollen or Birch tree pollen extract. The results for both the recombinant protein and allergen extract demonstrate that IVX-908 converts T cell cytokine production against Bet v 1a from a type 2 to a predominately type 1 phenotype. Furthermore, the type 1 response is associated with the increased production of allergen-specific serum IgG compared with the allergen alone, and a reduction in Bet v 1a-specfic serum IgE compared with allergen administered with aluminum phosphate, a depot and Type 2 adjuvant known to sensitize mice for allergic responses against an allergen. Importantly, the increase in the type 1 cytokine, IFN .gamma. was also observed following the immunization of allergic mice with the same allergen given with IVX-908. The pre-allergic state of the mice mimics the situation in allergic humans, suggesting that IVX-908/allergen formulations may be candidates for therapeutic allergy vaccines.

It is noted that the instant invention can readily adjuvant vaccines containing single, monovalent or multi-component antigens such as peptides, proteins, toxoids, glycoproteins, glycolipids, carbohydrates and/or polysaccharides, isolated from biologic organisms of the animal or plant kingdom that may be infectious organisms, such as parasites, viruses and bacteria, or may be extracts or purified or chemically modified extracts of allergens derived from unicellular or multicellular organisms or may be chemical material. It is also envisioned that whole or disrupted microorganisms including viruses, bacteria or parasites, attenuated or inactivated could be used as antigen. These materials may also be produced by synthetic or recombinant molecular procedures to induce immunity to and protect against several strains of a particular organism or multiple organisms or disease-causing agents or against allergies, cancer or auto-immune diseases. The utility in human and veterinary fields is proposed. Furthermore, the invention can be used to enhance immunity when given together with the antigen as an adjuvanted vaccine or immunotherapeutic as priming or boosting immunizations prior to or subsequent to administering the antigen (by mucosal or parenteral routes) without the instant invention.

For parenteral, nasal, oral or suppository use, the adjuvant may be given together with amounts of a variety of excipients or other adjuvants including oils, emulsions, nano-emulsions, fats, waxes, buffers, or sugars, as diluents or vehicles customary in the art to provide stable delivery of the product in the desired delivery format.

Of particular note, it is emphasised that using the instant invention as an adjuvant is particularly novel since it may, in a preferred embodiment, combine the adjuvant effect of proteosomes together with the immunostimulatory potential of LPS. This complex would not have been predicted to be effective from prior art since it contains full-length LPS that is normally toxic when given alone. As a stable proteosome complex LPS is non-toxic by the nasal and parenteral routes in the given examples as verified by both pre-clinical safety, immunogenicity and toxicity as well as in clinical studies in FDA-approved phase I and phase II clinical trials.

The instant invention may be composed of purified or recombinant bacterial outer-membrane proteins from gram-negative bacteria species including but not limited to Neisseria meningitidis strains. The LPS can be derived from gram negative bacteria such as, but not limited to Shigella or Plesiomonas or Escherichia or a salmonella species and can be from the same or different species of the bacteria used to provide the outer membrane protein proteosomes. In the preferred embodiment the final liposaccharide or LPS content by weight as a percentage of the total proteosome protein can be between about 13% and 300% and, depending on the specificity of the application and route of administration may be effective and practical for use at liposaccharide or LPS percentages of 20% to 200%, or may be further distinguished in a particular application at a liposaccharide percentage of between 30% to 150%. The instant invention together with antigen is designed to deliver adjuvanted vaccines by mucosal (nasal, sub-lingual, oral or rectal) or parenteral (intramuscular, subcutaneous, intradermal or transdermal) routes for use in the prevention or treatment of cancer, autoimmune, viral or microbial diseases or against certain toxins or biologic threat agents or allergies whether acquired by mucosal routes such as and specially by inhalation, or by ingestion or sexual transmission, or by parenteral routes such as transdermal, intradermal or subcutaneous or intramuscular.

An embodiment of the instant invention is a process for preparing proteosomes with endogenous lipooligosaccharide (LOS) content of between 0.5% up to about 5% of total protein. Another embodiment of the instant invention specifies a process for preparing proteosomes with endogenous liposaccharide of between about 12% to about 25%, and in a preferred embodiment, between 15% and 20% of total protein.

The instant invention specifies a composition containing liposaccharide derived from any gram negative bacterial species which may preferably be naturally or recombinantly different from or the same as the gram negative bacterial species which is the source of the proteins in the invention. The composition of the present invention may be optimised, specifically specified by the formulators and varied at will to contain amounts of proteosomes and liposaccharide such that the resultant composition of the instant invention contains liposaccharide to an amount that is at least about 13% by weight of the weight of total proteosome protein and in a preferred embodiment, may be from 15% to 300% and may be further preferred, depending on the application, to be between 20% to 200% of the total protein on a weight:weight basis or even between 30% and 150% of the total protein.

A most preferred embodiment of the instant invention is the adjuvant composition wherein the proteosomes are prepared from Neisseria meningitides and the liposaccharide is prepared from Shigella flexneri or Plesiomonas shigelloides and the final liposaccharide content is between 50% to 150% of the total proteosome protein by weight.

DETAILED DESCRIPTION OF THE INVENTION

Results show the following activities of IVX-908 adjuvant when mixed with recombinant and split antigens from influenza virus:

A. By the Injectable Route:

1. Induces up to eight-fold increases in serum HAI and IgG compared with injectable split flu influenza vaccine alone 2. Shifts elicited immune responses to Type 1 (CMI) responses compared to split flu influenza vaccine alone B. By the Nasal Route: 1. Induces>100-fold increases in serum HAI and IgG responses, compared with split flu influenza antigen alone given by the nasal route 2. Induces up to 10-fold higher specific serum HAI and IgG compared with split flu given by injection 3. Induces>100-1000 fold higher specific IgA in lung and/or nose compared with split flu influenza antigen alone given nasally or by injection 4. Induces up to 160-fold higher specific IgA in genital tract compared with split flu influenza antigen alone given nasally or by injection 5. Shift to Type 1 (CMI) responses compared to split flu alone 6. Amounts of IVX-908 as little as 0.3 ug to lug are sufficient to achieve optimal enhancement of serum IgG responses against split-flu HA 7. Recombinant influenza HA co-administered with IVX-908, induces responses which are protective against mortality and morbidity, and superior to those induced by injection or i.n. administration of the antigen alone 8. IVX-908 prepared at protein:LPS ratios of 3:1 to 1:3 using LPS from Shigella, Escherichia and Salmonella species were effective.

The results show that respiratory or parenteral immunization with the instant invention and influenza split flu antigen induces enhanced specific anti-influenza HA antibody formation in each of the serum and mucosal bio-samples compared to immunizing with the influenza split product without adjuvant.

Results show the following activities of IVX-908 adjuvant when mixed with rBet v 1a, the major allergen from Birch pollen as either recombinant allergen or Birch pollen allergen extract and administered via the nasal route. 1. The nasal IVX-908 and rBet v 1a mixture enhanced induction of the type 1 cytokine, IFN-.gamma. by 50- and 74-fold compared with Bet v 1a alone and Bet v 1 a formulated in aluminium phosphate respectively. The nasal IVX-908 and Birch pollen extract (BPEx) mixture enhanced induction of the type 1 cytokine, IFN-.gamma., by >44- and 3-fold compared with Bet v 1a alone and Bet v 1a formulated in aluminium phosphate respectively. 2. The increases in IFN-.quadrature. production by the IVX-908/Bet v 1a and IVX-908/BPEx mixtures were not associated with an increase in IL-5 secretion, indicating that IVX-908 directed the immune response against Bet v 1a towards a type 1-biased T cell response. 3. Serum IgE induced by the IVX-908 Bet v 1a and IVX-908/BPEx mixtures were approximately 37- and 44-fold lower than that induced by the allergens given with aluminium phosphate respectively. 4. Allergen-specific serum IgG was increased by >400-fold and 22-fold for mice immunized with the IVX-908/Bet v 1a and IVX-908/BPEx mixtures compared with Bet v 1a and BPEx alone, respectively. 5. In mice sensitized with Bet v 1a plus alum, the production of the type 1 cytokine, IFN-.gamma.was increased by 4.7- and 33-fold following immunization with IVX-908/rBet v 1a and IVX-908/BPEx respectively compared with the corresponding allergens alone. In these mice, the levels of the type 2 cytokine, IL-5 were reduced compared to the corresponding allergens alone. 6. In mice immunized nasally with IVX-908/allergen mixtures and subsequently given a sensitizing injection with Bet v 1a plus alum the type 1 cytokine, IFN-.gamma. increased by 10-fold compared with birch pollen extract alone. In these mice, the levels of the type 2 cytokine, IL-5, were not similarly elevated and indeed were somewhat reduced compared to birch pollen extract alone.

The results demonstrate that IVX-908/allergen formulations induce strong type 1 cytokine responses in allergen naive and sensitized mice, suggesting that these formulations prepared with purified or recombinant proteins or extracts of allergens may be used as vaccines or therapeutics for specific immunotherapy for allergic diseases. Results show the following activities of IVX-908 adjuvant when mixed with ovalbumin (OVA), a known poor immunogen and given by the nasal or injectable route. 1. Enhances OVA-specific serum IgG titers by greater than 60- and 75-fold via the nasal and injectable routes respectively compared with antigen alone, 2. Enhances the secretion of OVA-specific IFN-.gamma. and IL-5 from re-stimulated splenocytes compared with antigen alone resulting in a balanced type of immune response.
 

Claim 1 of X Claims

1. An immunogenic composition for inducing an immunological response to an antigen, said composition comprising the antigen and an effective amount of proteosome-lipopolysaccharide adjuvant, wherein the proteosome-lipopolysaccharide adjuvant enhances the immunological response to the antigen, wherein the antigen and the proteosome-lipopolysaccharide adjuvant are separate chemical entities, and wherein the proteosome-lipopolysaccharide adjuvant is formed from an outer membrane proteosome complexed with a lipopolysaccharide preparation, wherein both the proteosome and the lipopolysaccharide preparation are from gram-negative bacteria, which proteosome-lipopolysaccharide adjuvant has a final lipopolysaccharide content by weight as a percentage of the total proteosome protein of at least 13%.

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