Saponin double mutant thermally labile toxin (dmLT) adjuvants and related uses

Abstract

A composition for a vaccine, preferably an immunologic adjuvant system, contains a saponin component and a dmLT component. The combination of the saponin component and the dmLT component shows a synergistic effect in the treatment of a variety of conditions, diseases, conditions. Methods of use for prophylactic use or therapeutic treatment are disclosed. Exemplary adjuvant compositions include a double mutant thermally labile toxin adjuvant derived from Escherichia coli enterotoxins and a saponin, optionally with additional vaccine components (e.g., antigens), particularly when used in vaccines.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 415,823, filed on October 13, 2022, the disclosure of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to an immunoadjuvant system for vaccines, which contains a saponin component and a double mutant heat-labile toxin (dmLT) component, and methods of use thereof for prophylactic use or therapeutic treatment. Exemplary adjuvant compositions include double mutant heat-labile toxin adjuvants and saponins derived from Escherichia coli enterotoxin, optionally having additional vaccine components (such as antigens), particularly when used in vaccines. Background Art

[0004] The adjuvant dmLT, or more precisely LT(R192G / L211A), is an 84 kDa multimeric protein with an AB5 structure, consisting of a pentameric B subunit (composed of five 11.5 kDa monomers) non-covalently bound to an enzymatically active A subunit (28 kDa). dmLT differs from its parental molecule, heat-labile enterotoxin (LT), by the substitution of two residues in the A subunit, namely arginine with glycine at amino acid 192 (R192G) and leucine with alanine at amino acid 211 (L211A). The ribbon diagram of dmLT can be inferred from the crystal structure of partially cleaved LT toxin (References 1, 2), although there may be unresolved variations in the three-dimensional (3D) structure due to the amino acid substitutions in the A subunit. dmLT is an adjuvant that enhances vaccine-specific systemic and mucosal immune responses after mucosal or parenteral delivery. Studies have shown that dmLT has four main characteristics compared to other adjuvant systems.

[0005] (1) dmLT promotes immunity against antigens that are co-delivered after simply mixing dmLT and the antigen in an aqueous buffer. Thus, unlike many depot-type adjuvants (such as aluminum hydroxide), formulating antigen / adjuvant vaccines does not require pre-preparation or absorption. dmLT can be formulated with antigens at the point of manufacture or delivery.

[0006] (2) Through the combined action of the immunostimulatory properties of dmLT and its general cell binding, the uptake of co-delivered antigens is enhanced, promoting mucosal immunity. This enables immunological preparations (most notably subunit vaccines) to be delivered at sites that were previously inaccessible, such as oral (p.o.), sublingual (s.l.), transcutaneous (t.c.i.), etc. delivery. Many of these methods are needle-free, potentially improving the convenience and compliance of administration and reducing the risk of disease outbreaks caused by unsafe injections (3 - 6).

[0007] (3) Different from other adjuvants, such as aluminum hydroxide or many Toll-like receptor (TLR)-based adjuvants (e.g., monophosphoryl lipid A [MPL] and CpG), dmLT induces strong interleukin-17 (IL-17) recall cytokine secretion and antigen-specific Th17 responses after parenteral or mucosal immunization (7-15). This is a new branch of the adaptive immune response and is crucial in protecting against pathogens, especially in preventing mucosal tissue infections and controlling bacterial infections (16). In addition, the secretion of IL-17 increases the availability of mucosal antibodies by upregulating the level of polymeric Ig receptor in epithelial cells, increasing the transport of secretory IgA (sIgA) into the lumen of mucosal tissues, and promoting the differentiation of T-independent B cells into IgA-secreting cells (17-20).

[0008] (4) Finally, dmLT promotes the development of mucosal immune responses after parenteral immunization.

[0009] The adjuvant effect of saponins was first reported in 1925, when studies showed that the addition of breadcrumbs, cassava flour, saponins, and "starch oil" to antigen preparations greatly enhanced the antibody response to diphtheria or tetanus. In 1951, Espinet used a commercially available crude saponin preparation to enhance the potency of foot-and-mouth disease vaccine. In addition, in 1974, Dalsgaard successfully isolated the saponin Quil A from the bark of the South American tree Quillaja saponaria Molina and found that Quil A stimulated both humoral and cellular immunity and induced different antibody isotypes. Since then, Quil has been commercialized and widely used in veterinary vaccines and preclinical studies. Other studies have shown that when formulated with aluminum salts, liposomes, and water-in-oil emulsions, as well as with amphiphilic proteins and lipids, it forms detergent / lipid / saponin complexes called immunostimulating complexes (ISCOMs).

[0010] Quil A is a heterogeneous product consisting of up to 23 different saponin peaks detectable by HPLC, and its toxicity upon parenteral injection has precluded its use in human vaccines. Since Quil A is a mixture, Kensil et al. conducted further studies in 1991, in which 10 RP-HPLC fractions of ultrafiltered aqueous extracts of Quillaja saponaria bark were tested and found that fractions QS-7, QS-17, QS-18, and QS-21 were particularly effective. However, their toxicity varied greatly upon parenteral (injection) administration. QS-18, the major component of Quillaja saponaria, was found to be highly toxic to mice, while QS-7 and QS-21 exhibited much lower toxicity.

[0011] Saponin fractions, such as QS-21, are currently used in GSK Herpes zoster vaccine and malaria vaccine, saponin components A and C are currently used in the Novavax COVID-19 vaccine for humans as adjuvants. Saponin-based adjuvants are also being studied in many other human indications (i.e., tuberculosis, respiratory syncytial virus, cancer, etc.).

[0012] Purified saponins have been shown to be safe and effective when injected and have significantly improved the efficacy of some oral vaccines in clinical studies. The toxicity of food-grade saponins (FGS) extracted from Quillaja saponaria limits its use as a parenteral adjuvant; however, this toxicity is attenuated when taken orally.

[0013] FGS extracts are commonly used in the global food and beverage industry and are recognized as generally regarded as safe (GRAS) by the U.S. Food and Drug Administration (FDA) and the Food Additives and Nutrient Sources Group (ANS) of the European Food Safety Authority (EFSA). At the current levels of intake of 1.5 mg / kg / day for adults and infants or approximately 100 mg of pure saponin for adults and approximately 12 mg of pure saponin for infants, FGS is not toxic to humans. From this perspective, calculating the amount of QS-21 that is safely ingested as FGS in 100 mg of pure saponin is equivalent to approximately 2.8% or 2.8 mg of QS-21, which is approximately 60 times (50 mcg) the amount of QS-21 found in the vaccine and is 120 times (25 mcg) the amount of QS-21 found in the vaccine.

[0014] Food-grade saponin (FGS) extracts are defined as type 1 and type 2 extracts and have significant potential advantages for use with vaccines and provide a broad adjuvant effect due to multiple saponin components.

[0015] Despite the long history of saponins as adjuvants in the prior literature and the knowledge that several saponin components have been and are being used in commercial vaccines for humans, there is still a need for the vaccine adjuvant system described in the present disclosure, which provides a vaccine adjuvant system for vaccines derived from Quillaja saponaria and dmLT. Of particular note, all currently available vaccines using saponins are injectable vaccines, and there are currently no mucosal saponin adjuvants. In the present invention, the use of mucosal saponin adjuvants and dmLT adjuvants shows an unexpected synergistic effect that has not been described in the prior art. Summary of the Invention

[0016] The present disclosure provides compositions for treating a disorder or disease or condition and methods of using the compositions. As described herein, one or more compositions (e.g., a "system" as used herein) can include one or more or all saponins, and a heat-labile toxin (including, e.g., dmLT), and their uses and methods in prophylactic use or therapeutic treatment. Exemplary adjuvant compositions include a double mutant heat-labile toxin adjuvant derived from an Escherichia coli enterotoxin and a saponin, optionally having a vaccine or vaccine component (e.g., an antigen). In one or more embodiments, the composition can include (i) a multimeric protein containing at least a portion of dmLT, and (ii) a saponin compound derived from Quillaja saponaria. The phrase "at least a portion" means that the multimeric protein is a detoxified enterotoxin derived from Escherichia coli and has at least 80% identity to the amino acid sequence of dmLT, wherein the detoxified enterotoxin is an adjuvant that retains the immunological activity of dmLT.

[0017] In one embodiment, the present invention provides a vaccine adjuvant system comprising (i) a composition containing dmLT, (ii) a composition containing a saponin, and optionally (iii) a vaccine component. In other embodiments, the vaccine adjuvant system can include the above compositions, which include an effective dose of a saponin compound derived from Quillaja saponaria and an effective dose of at least a portion of dmLT.

[0018] In another embodiment, the present invention provides a vaccine containing the above-described synergistic vaccine adjuvant system, wherein the system contains an effective dose of a Quillaja saponaria composition and an effective dose of dmLT. Alternative embodiments of the vaccine will further include the above-described vaccine adjuvant system, which contains or encodes at least one antigen. The vaccine can also contain polypeptides, nucleic acids, polysaccharides, polysaccharide-polypeptide conjugates, attenuated bacteria or inactivated bacteria, toxoids, attenuated or inactivated viruses, virus-like particles, viral vectors, and / or combinations thereof. Some embodiments of the vaccine can also contain or encode bacterial, viral, or fungal antigens.

[0019] In another embodiment, a kit can be provided that contains any of the above vaccine adjuvant systems or vaccines.

[0020] In another embodiment, a method of generating an immune response in a subject is disclosed, the method comprising administering to the subject the above-described vaccine adjuvant system or vaccine. In at least one embodiment, the administering step is by an oral route or a sublingual route. In at least one embodiment of the method, the immune response is a B cell response. In one or more other embodiments, the immune response is the generation of CD4+ T cells. In one or more other embodiments, the immune response is the generation of CD8+ T cells.

[0021] In another embodiment, the present disclosure provides a method of treating a disease, disorder, or condition, the method comprising administering a vaccine comprising a vaccine adjuvant system, the vaccine adjuvant system comprising: an effective dose of a saponin compound derived from Quillaja saponaria; an effective dose of at least a portion of dmLT; and optionally, an effective dose of one or more vaccine components related to the disease, disorder, or condition.

[0022] In a further embodiment, the present invention provides the use of a composition in a vaccine. In other embodiments, the present invention provides the use of a vaccine adjuvant system in a vaccine. In other embodiments, the present invention provides the use of a vaccine for treating a disease, disorder, or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawing(s) Figure 1 shows that oral immunization with a combination of dmLT and saponin adjuvant can enhance immunity to tetanus toxoid (TT) antigen. Anti-TT IgG was analyzed by ELISA, with the raw optical density values shown in the left panel and the compilation in the right panel. Significance was determined by Tukey's post hoc analysis of variance (ANOVA), and the P values are represented as * < 0.05, ** < 0.001. DETAILED DESCRIPTION

[0024] The present disclosure provides a composition, such as a synergistic vaccine adjuvant system, comprising a saponin compound derived from Quillaja saponaria and dmLT, for use in a vaccine and the like.

[0025] In one embodiment of the present disclosure, a synergistic vaccine adjuvant system for a vaccine is described, wherein the vaccine adjuvant system comprises an effective dose of a Quillaja saponaria compound and an effective dose of dmLT, and is used as an immunoadjuvant system for a vaccine. In one or more other embodiments of the present disclosure, the use of a vaccine adjuvant system is described, wherein the Quillaja saponaria compound and dmLT within the adjuvant system are used as an immunoadjuvant system for a vaccine. In one or more other embodiments of the present disclosure, a therapeutic treatment method is described, wherein the method comprises administering a vaccine comprising an effective dose of a Quillaja saponaria compound, an effective dose of dmLT, and an active component or vaccine component, such as a sterol, phospholipid, and / or antigen, for preventing or treating a disease, disorder, or condition. The vaccine adjuvant system described herein can be administered in any of a variety of ways, including but not limited to the following routes: oral, sublingual, subcutaneous, parenteral, rectal, otic, nasal, dermal, transdermal, or a combination thereof.

[0026] Saponin compounds are raw materials that can be obtained from the bark and, in some cases, from the whole biomass of the Quillaja saponaria tree. The term "saponin" as used herein includes glycosidic triterpenoid compounds (also known as triterpenoid glycoside compounds) that produce foam in aqueous solutions, are hemolytically active in most cases, and have immunoadjuvant activity. The term "saponin" also includes bioactive fragments of the above compounds. It should be understood that the term "QS" refers to Quillajasaponin.

[0027] Quillajasaponin is structurally different from saponins of other plant species. Two structural features that distinguish Quillajasaponin from saponins of other plant species are the fatty acid domain and the triterpene aldehyde at carbon 4 of the triterpene.

[0028] In the present disclosure, the Quillajasaponin compounds can be selected from, but not necessarily limited to: crude saponin extract, type 1 extract, type 2 extract, QS-7, QS-8, QS-17, QS-18, QS-21, QS-21 component A, QS-21 component C, or a combination thereof. The four most prominent Quillajasaponins that have been identified and purified are QS-7, QS-17, QS-18, and QS-21. As described above, these adjuvant saponins have been identified and purified from the aqueous extract of the bark of the South American tree Quillaja saponaria Molina, typically purified by HPLC and low-pressure silica gel chromatography, and have been found to have adjuvant activity, although there are differences in bioactivities such as hemolysis and toxicity. Recently, obtaining these saponins from the whole biomass of Quillaja saponaria Molina has significantly increased the available raw materials, improved production capacity, reduced costs, and made the product sustainable, as extracting the biomass does not cause the death of the tree or stress the native forests of Chile.

[0029] In addition to the saponin compounds of the present invention, another component of the adjuvant composition provided herein is a detoxified enterotoxin adjuvant derived from Escherichia coli, known in the literature as dmLT. The detoxified enterotoxin adjuvant exemplified herein was initially described in U.S. Patent No. 6,033,673. Referred to as "LT(R192G / L211A)" in that patent and also as "dmLT" herein, the detoxified enterotoxin adjuvant is a genetically distinct mutant of the Escherichia coli heat-labile enterotoxin (LT) that has lost the trypsin-sensitive site that links the Al and A2 subunits by modifying arginine at position 192 to glycine and leucine at position 211 to arginine, rendering the molecule non-toxic but still able to act as an immunoadjuvant.

[0030] dmLT can be produced by methods standard in the art. For example, plasmid pED403 described in Example 6.1 of U.S. Patent No. 603673 can be used to produce substantially pure LT(R192G / L211A) (dmLT) in E. coli. dmLT can be isolated from bacteria expressing the dmLT-encoding plasmid by agarose affinity chromatography. Alternative methods of purification standard in the art can be used to purify dmLT.

[0031] One of ordinary skill in the art can prepare other detoxified enterotoxins with equivalent immunological activity and use them in the compositions and methods disclosed herein. For example, detoxified enterotoxins having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the dmLT adjuvant are contemplated, which retain the immunological activity of dmLT.

[0032] The compositions provided herein, preferably adjuvant compositions, comprise a detoxified bacterial endotoxin adjuvant and a saponin compound adjuvant. The adjuvant composition can comprise a dmLT adjuvant and / or a Quillaja saponin compound, and Quillaja saponin compounds include, but are not limited to, crude saponin extract, type 1 extract, type 2 extract, QS-7, QS-8, QS-17, QS-18, QS-21, QS-21 fraction A, QS-21 fraction C, or combinations thereof.

[0033] The adjuvant composition, including the adjuvant system described herein, can be administered as a separate composition with or without a vaccine composition. In other embodiments, the adjuvant composition can be administered in the same / a single composition, or alternatively, separately from an optional one or more vaccine components.

[0034] The adjuvants provided herein are present in the composition in an amount in the range of 0.1 - 1000 μg / dose for each individual adjuvant (dmLT and saponin). The dose can be adjusted according to the weight, body surface area, weight, blood volume, or route of delivery of the subject. It will be apparent to one of ordinary skill in the art that the quantity and frequency of administration will depend on the response of the host. As described herein, the appropriate dose may also depend on the condition of the subject, i.e., the stage of the disease, general health, as well as age, gender, and weight, and other factors familiar to those skilled in the medical arts. Thus, the term "effective dose" refers to a dose that is considered suitable and sufficient by a person of ordinary skill in the art familiar with the individual subject or host to have a positive effect on treating the condition, disease, or symptom of the individual.

[0035] An adjuvant composition can be in any form that permits the composition to be administered to a subject. For example, the adjuvant composition can be in the form of a solid, liquid, or gas (aerosol). The pharmaceutical composition can be administered by any route. Typical routes of administration include, but are not limited to, oral, sublingual, buccal, topical, parenteral (including intradermal, subcutaneous, transdermal, intravenous, intramuscular, intrasternal, intracisternal, intra-aural, intratumoral, intracranial, intraspinal, or intraurethral injection or infusion), rectal, vaginal, intranasal (e.g., spray), and intrapulmonary administration. As used herein, the term "parenteral" includes, but is not limited to, iontophoresis, sonophoresis, thermotherapy, passive transdermal therapy, and microneedle administration, as well as intradermal / subcutaneous injection, intravenous injection, intramuscular injection, intrasternal injection, intracisternal injection, intracapsular injection, intranodal injection, intra-aural injection, intraurethral injection, intratumoral injection or infusion techniques. The adjuvant compositions provided herein can be administered intradermally by a technique selected from iontophoresis, microcavitation therapy, acoustophoresis, or microneedles.

[0036] The adjuvant composition can further comprise at least one physiologically (or pharmaceutically) acceptable or suitable excipient. Any physiologically or pharmaceutically suitable excipient or carrier known to those of ordinary skill in the art (i.e., non-toxic materials that do not interfere with the activity of the active components) can be used in the compositions provided herein. Exemplary excipients include diluents and carriers that maintain protein stability and integrity. Excipients for therapeutic use are well known.

[0037] "Pharmaceutically acceptable carriers" are also well known in the pharmaceutical art. For example, phosphate buffered saline at physiological pH and sterile saline can be used. Preservatives, stabilizers, dyes, and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid, and parabens can be added as preservatives. In addition, antioxidants and suspending agents can be used.

[0038] Similarly, "pharmaceutically acceptable salts" refer to salts of compounds derived from combinations of these compounds with organic or inorganic acids (acid addition salts) or organic or inorganic bases (base addition salts). The adjuvant compositions provided herein can be used in free base or salt form.

[0039] The adjuvant composition is formulated such that the active components contained therein are bioavailable upon administration of the composition to a patient. The composition to be administered to the patient is in the form of one or more dosage units, where, for example, a tablet can be a single dosage unit, and a container of one or more adjuvants in aerosol form can contain multiple dosage units.

[0040] The present disclosure can provide liquid adjuvant compositions, whether in solution, suspension, or other similar forms, and can include one or more of the following carriers or excipients: sterile diluents such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as squalene, squalane, mineral oil, mannitol monooleate, cholesterol, and / or synthetic monoglyceride or diglyceride of glycol such as polyethylene glycol, glycerol, propylene glycol, or other solvents that can be used as solvents or suspension media; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate, and agents for adjusting tonicity such as sodium chloride or glucose. Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0041] The adjuvant compositions provided herein can comprise a stable aqueous suspension of less than 0.2 μM and further comprise at least one component selected from phospholipids, fatty acids, surfactants, detergents, saponins, fluorinated lipids, and the like. Such stable aqueous formulations can be micellar formulations.

[0042] The adjuvant compositions provided herein can be formulated in an aerosolizable manner and can be powder or liquid formulations.

[0043] It may also be desirable to include other components in the adjuvant compositions, such as, including but not limited to, water-in-oil emulsions, biodegradable oil carriers, oil-in-water emulsions, liposomes, micellar components, microparticles, biodegradable microcapsules, and liposomes.

[0044] The adjuvant compositions provided herein can comprise stable oil-in-water emulsions and metabolizable oils. The meaning of the term "metabolizable oil" is well known in the art. Metabolizable can be defined as "able to be transformed by metabolism". The oil can be any vegetable oil, rapeseed oil, fish oil, animal oil, or synthetic oil that is non-toxic to the recipient and capable of being metabolically transformed. Nuts (such as peanut oil), seeds, and grains are common sources of rapeseed oil. Synthetic oils can also be used.

[0045] Other immunostimulatory substances may be included in the adjuvant compositions provided herein and may include N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), glucan, IL-12, GM-CSF, interferon-γ, and IL-12.

[0046] While any suitable carrier known to those of ordinary skill in the art can be used in the adjuvant compositions provided herein, the type of carrier will vary depending on the mode of administration and whether sustained release is desired. Biodegradable microspheres (such as poly(lactide-co-glycolide)) can also be used as carriers for the adjuvant compositions provided herein. Suitable biodegradable microspheres are known in the art. In this regard, the microspheres are preferably greater than about 25 microns.

[0047] The adjuvant compositions provided herein may also contain diluents such as buffers, antioxidants such as ascorbic acid, carbohydrates (including glucose, sucrose or dextrin), chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are exemplary suitable diluents. The adjuvant can be formulated as a lyophilizate using a suitable excipient solution (such as sucrose) as a diluent.

[0048] In one or more embodiments of the present invention, the adjuvant composition may also be included in a vaccine. In the case of providing a vaccine, the vaccine may also contain or encode an antigen.

[0049] The adjuvant compositions and methods provided herein are intended for use in subjects, including humans and other animals. Vaccines contemplated for use with the adjuvant compositions may contain or encode an antigen. Vaccines containing the adjuvant composition may also contain or encode, for example, bacterial, viral or fungal antigens.

[0050] By way of example and not limitation, antigens contemplated by the present disclosure include antigens from pathogenic strains: bacteria (including but not limited to Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Staphylococcus aureus, Bordetella pertussis, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Salmonella typhi, Treponema pallidum, Treponema pertenue, Treponema carateum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohaemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma, Rickettsia prowazekii, Rickettsia tsutsugamushi, Chlamydia, Helicobacter pylori); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entamoeba histolytica, Trichomonas tenax, Trichomonas hominis, Trichomonas vaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, malaria); or helminths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium and hookworms), presented to the immune system in the form of the whole organism or a portion isolated from a culture of the organism grown in a medium well known in the art for culturing said organism, or a protective antigen of said organism obtained by genetic engineering techniques or chemical synthesis.

[0051] Other antigens considered include, for example, antigens from pathogenic viruses (e.g., Poxviridae, Herpesviridae, Herpes simplex virus type 1, Herpes simplex virus type 2, Adenoviridae, Papillomaviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, Togaviridae, Rhabdoviridae, Arenaviridae, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, non-A / non-B hepatitis virus, Rhinoviridae, Coronaviridae, Rotaviridae, and human immunodeficiency virus) (e.g., rabies virus, herpes viruses such as Herpes simplex virus (HSV) type 2, Herpes simplex virus (HSV) type 1, human cytomegalovirus, Epstein - Barr virus, and varicella - zoster virus (VZV), human papillomavirus (HPV), human T - cell lymphotropic virus type 1, rotavirus, norovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, poliovirus, Japanese encephalitis virus, measles virus, mumps virus, rubella virus, yellow fever virus, varicella virus, dengue virus, rotavirus, paniovirus, human immunodeficiency virus - 1, Ebola viruses such as Ebola Sudan virus, Marburg virus, hantavirus, norovirus, Zika virus, West Nile virus, hantavirus, Lassa virus, lymphocytic choriomeningitis virus, Nipah virus, Rift Valley fever virus, Middle East respiratory syndrome coronavirus, SARS coronavirus, SARS virus 2, Crimean - Congo hemorrhagic fever virus, enterovirus, and norovirus), antigens isolated in whole or in part from media well - known in the art for culturing viruses, or obtained by genetic engineering techniques or chemical synthesis, are presented to the immune system.

[0052] Examples of vaccines contemplated include, but are not limited to, influenza vaccine, pertussis vaccine, combination of diphtheria and tetanus toxoids and pertussis vaccine, hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, hepatitis E vaccine, Japanese encephalitis vaccine, herpes vaccine, measles vaccine, rubella vaccine, mumps vaccine, combined measles and mumps and rubella vaccine, papillomavirus vaccine, parvovirus vaccine, respiratory syncytial virus vaccine, Lyme disease vaccine, polio vaccine, varicella vaccine, gonorrhea vaccine, schistosomiasis vaccine, rotavirus vaccine, mycoplasma vaccine, pneumococcal vaccine, meningococcal vaccine, campylobacter vaccine, helicobacter vaccine, cholera vaccine, enterotoxigenic E. coli vaccine, enterohemorrhagic E. coli vaccine, shigella vaccine, salmonella vaccine, etc. These are produced by known conventional processes. Generally, such vaccines contain whole organisms or viruses grown and isolated by techniques well known to those skilled in the art, or contain related antigens of these organisms or viruses produced by genetic engineering techniques or chemical synthesis.

[0053] It should be understood that other compositions provided herein may also be in the kit. In the kit, the components of the adjuvant composition may already be mixed together for administration, or these components may be separated in the kit and administered separately as indicated.

[0054] Finally, LT(R192G / L211A) or dmLT is the product of more than 35 years of research using bacterial ADP-ribosyl enterotoxins as adjuvants. dmLT has recently been successful in Phase 1 and 2 clinical trials for ETEC and poliovirus, although success with oral administration seems to depend on the immunogenicity of the antigen. These two adjuvants have been used separately for various vaccines and delivery routes, but have not been used together in any case. In preliminary studies, it has been observed that when dmLT and saponin are co-administered via oral and sublingual vaccines, unexpected synergistic adjuvant activity occurs. One of the projects under evaluation is to assess the combination of saponin and dmLT to provide a saponin-dmLT adjuvant (SDA) for oral or sublingual administration. Testing is currently underway to optimize SDA in relevant vaccines globally, including the adjuvanted subunit ETEC and campylobacter vaccine (ASEC) against bacterial intestinal infections and inactivated polio vaccine. The project is also exploring formulations for gastrointestinal delivery and conducting IND-enabling studies such as GLP and cGMP manufacturing, toxicology, and stability testing. It is believed that the present invention will contribute to the determination of an SDA adjuvant platform and formulation that provides potent systemic immunity and / or sustained mucosal immunity.

[0055] The following examples are exemplary in nature and the present invention is not limited thereto. Instead, as described above, the present invention relates to compositions for treating a disorder or condition, or disease or illness, and methods of using such compositions. One or more compositions (e.g., the "system" used herein) can include one or more quillajasaponins and heat-labile toxins, including, for example, dmLT, and their uses and methods in prophylactic use or therapeutic treatment.

[0056] Example 1

[0057] A combination of saponin and dmLT with tetanus toxoid was prepared for oral administration, wherein balb / c mice were immunized with the following groups on days 0, 7, and 14:

[0058] 1) 100 ug TT only

[0059] 2) 100 ug TT with 25 ug dmLT

[0060] 3) 100 ug TT with 500 ug food-grade type 2 saponin extract; and

[0061] 4) A combination of 100 ug TT with 25 ug dmLT and 500 ug food-grade saponin type 2 extract. Serum was collected on day 21 and anti-TT IgG was analyzed by ELISA.

[0062] As Figure 1 The results shown indicate that a synergistic and unexpected effect is produced when the two adjuvants are used in combination compared to when no adjuvant is used and when compared to the individual adjuvants.

Claims

1. A composition comprising (i) a multimeric protein containing at least a portion of dmLT, and (ii) a saponin compound derived from Quillaja saponaria.

2. The composition according to claim 1, wherein, the multimeric protein is a detoxified enterotoxin derived from Escherichia coli and has at least 80% identity with the amino acid sequence of dmLT, and the detoxified enterotoxin is an adjuvant that retains the immunological activity of dmLT.

3. The composition according to claim 1, wherein, the saponin compound is selected from the group consisting of a crude saponin extract, type 1 extract, type 2 extract, QS-7, QS-8, QS-17, QS-18, QS-21, QS-21 fraction A, QS-21 fraction C, and combinations thereof.

4. A vaccine adjuvant system comprising the composition according to claim 1, further comprising an effective dose of the saponin compound derived from Quillaja saponaria and an effective dose of at least a portion of dmLT.

5. A vaccine comprising the vaccine adjuvant system according to claim 4, the vaccine comprising or encoding at least one antigen.

6. The vaccine according to claim 5, further comprising a polypeptide, nucleic acid, polysaccharide, polysaccharide - polypeptide conjugate, attenuated bacterium or inactivated bacterium, toxoid, attenuated virus or inactivated virus, virus - like particle or viral vector.

7. The vaccine according to any one of claims 4 to 6, wherein, the vaccine comprises or encodes a bacterial, viral or fungal antigen.

8. The vaccine according to claim 7, wherein, The antigen is selected from the group consisting of the following antigens: (1) pathogenic bacterial strains, wherein the bacterial strains are selected from Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Staphylococcus aureus, Bordetella pertussis, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Salmonella typhi, Treponema pallidum, Treponema pertenue, Treponema carateum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohaemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma, Rickettsia prowazekii, Rickettsia tsutsugamushi, Chlamydia, and Helicobacter pylori; (2) pathogenic fungi, wherein the fungi are selected from the group consisting of Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum; (3) protozoa, which are selected from the group consisting of Entamoeba histolytica, Trichomonas tenax, Trichomonas hominis, Trichomonas vaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, malaria; (4) helminths, which are selected from the group consisting of Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and (5) hookworms.

9. The vaccine according to claim 7, wherein, The antigen is selected from antigens of pathogenic viruses, where the viruses include any one of the following viruses: Poxviridae, Herpesviridae, Herpes simplex virus type 1, Herpes simplex virus type 2, Adenoviridae, Papillomaviridae, Picornaviridae, Enterovirus, Parvoviridae, Reoviridae, Retroviridae, Influenza virus, Parainfluenza virus, Mumps, Measles, Respiratory syncytial virus, Rubella, Togaviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non-A / Non-B hepatitis virus, Rhinoviridae, Coronaviridae, Rotaviridae, and Human immunodeficiency virus, selected from Rabies virus, Herpes viruses such as Herpes simplex virus (HSV) type 2, HSV type 1, Human cytomegalovirus, Epstein-Barr virus, and Varicella-zoster virus (VZV), Human papillomavirus (HPV), Human T-cell lymphotropic virus type 1, Rotavirus, Norovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Influenza virus, Poliovirus, Japanese encephalitis virus, Measles virus, Mumps virus, Rubella virus, Yellow fever virus, Varicella virus, Dengue virus, Rotavirus, Paniovirus, Human immunodeficiency virus-1, Ebola viruses such as Ebola Sudan virus, Marburg virus, Hantavirus, Norovirus, Zika virus, West Nile virus, Hantavirus, Lassa virus, Lymphocytic choriomeningitis virus, Nipah virus, Rift Valley fever virus, Middle East respiratory syndrome coronavirus, SARS coronavirus, SARS virus 2, Crimean-Congo hemorrhagic fever virus, Enterovirus, and Norovirus.

10. A kit, which further comprises the vaccine according to any one of claims 5 to 9.

11. A method of generating an immune response in a subject, the method comprising administering to the subject the vaccine adjuvant system according to claim 4 or the vaccine according to any one of claims 5 to 9.

12. The method according to claim 11, wherein, the step of administration is by the oral route or the sublingual route.

13. The method according to claim 11 or 12, wherein, the immune response is a B cell response.

14. The method according to any one of claims 11 to 13, wherein, the immune response is the generation of CD4+ T cells.

15. The method according to any one of claims 11 to 13, wherein, the immune response is the generation of CD8+ T cells.

16. A method of treating a disease, disorder or condition, the method comprising administering a vaccine containing a vaccine adjuvant system, the vaccine adjuvant system comprising an effective dose of a saponin compound derived from Quillaja saponaria; an effective dose of at least a portion of dmLT; and, optionally, an effective dose of one or more vaccine components related to the disease, disorder or condition.

17. Use of the composition according to any one of claims 1 to 3 in a vaccine.

18. Use of the vaccine adjuvant system according to claim 4 in a vaccine.

19. Use of the vaccine according to any one of claims 5 to 9 in a vaccine for treating a disease, disorder or medical condition.

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