Methods and compositions for enhancing adjuvant activity of natural and semisynthetic saponins

Abstract

In one aspect, the disclosure relates to immunogenic compositions comprising a semisynthetic saponin adjuvant (VSA) including, but not limited to, VSA-1 and VSA-2, derived from a Momordica saponin and at least one antigen. In a further aspect, the immunogenic compositions can include other known adjuvants, emulsifiers, and / or surface-active agents and can be formulated as liposomes, micelles, emulsions, and the like. Also disclosed are methods of eliciting effective immune responses to viral, bacterial, protozoal, and neoplastic diseases using the disclosed immunogenic compositions.

Description

ATTORNEY DOCKET NO. 222120-2140METHODS AND COMPOSITIONS FOR ENHANCING ADJUVANT ACTIVITY OF NATURAL AND SEMISYNTHETIC SAPONINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 694,940, filed on September 16, 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under contract numbers 75N93022C00033 and 75N93022C00037 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Vaccination is a successful medical practice that has eliminated or limited infectious diseases such as smallpox, polio, typhus, rotavirus, and hepatitis A and B. Traditionally, vaccines were whole-organism-based, using live attenuated / inactivated viruses and bacteria, a strategy that is not applicable to many other severe infectious diseases, cancers, and autoimmune diseases. The emergence of new pathogens and inadequate protection conferred by some of the existing vaccines such as vaccines for tuberculosis, influenza, and pertussis (especially in certain age groups or immunocompromised individuals) highlights the need for more sophisticated and effective vaccines. Subunit vaccines that use well-characterized and highly purified antigens such as recombinant proteins and peptides have proven to be a promising new approach. However, with highly refined antigens (thus with limited or no pathogen-associated-molecular-patterns (PAMPs)), subunit vaccines are poorly immunogenic and need adjuvants to enhance antigenspecific immune responses. Vaccine adjuvants are non-immunogenic substances that can improve or modulate antigen-specific immune responses toward their co-administered antigens, constituting an indispensable element of modern vaccines. They can: (a) enhance the ability of a vaccine to elicit strong and durable immune responses (including in immunologically compromised individuals such as immunologically immature (neonates), aged, and immune suppressed individuals), (b) reduce antigen dose and the number of immunizations, and (c) modulate the nature of immune response (e.g., in favor of a humoral or cell-mediated response).ATTORNEY DOCKET NO. 222120-2140

[0004] The lack of safe and effective adjuvants for different application scenarios severely hinders the development of subunit vaccines. The Food and Drug Administration (FDA) has approved only a few adjuvants (i.e., alum, AS04, MF59 / AS03, CpG, and AS01b) with different working mechanisms and limitations for human use. For example, alum (various aluminum salts), the first and most commonly used adjuvant, had been the only human vaccine adjuvant for more than nine decades until 2009. It primarily enhances Th2 humoral responses, effective for neutralizing vaccines but ineffective for vaccines targeting intracellular pathogens or cancer cells when a Th1 or mixed Th1 / Th2 response is required. AS04, approved in 2009, is a combination of alum and monophosphoryl lipid A (MPL). However, MPL, along with MF59 / AS03, are unable to stimulate the production of a cytotoxic CD8+ T cell response which is crucial against malignant cells or intracellular pathogens that cause many devastating diseases. Most recently, AS01b was approved for use in Shingrix® against herpes zoster and Mosquirix® against malaria. It is a combination adjuvant containing MPL and saponin QS-21 in a liposomal formulation, inducing strong humoral and cellular immune responses. The unique saponin adjuvant QS-21 (FIG. 1) is also a key component of experimental combination adjuvants AS02 (an oil-in-water emulsion containing QS21 and MPL), AS05 (liposomes containing QS21 , MPL, and alum), and AS15 (liposome formulation containing QS-21, MPL, and CpG). It can potentiate a balanced Th1 / Th2 response with antigen-specific CTL production. Nevertheless, it has its own inherent drawbacks.

[0005] QS-21 is a natural product isolated from the tree bark of Quillaja saponaria Molina (QS), an evergreen tree native to temperate central Chile. Overexploitation of the natural source has already resulted in ecological damage and shortage of available supplies, leading to more strict environmental regulations and increased price. The supply shortage of QS saponins can be a serious issue because they will be in demand for other high-volume vaccination needs. In addition to Shingrix® and malaria vaccine Mosquirix®, QS-21 is in clinical trials for tuberculosis, hepatitis, HIV, and cancers such as melanoma and NSCLC. Preclinical data also support broad applicability across many other indications, including for anthrax, chlamydia, CMV, dengue, Ebola, group A Streptococcus, Helicobacter pylori, HPV, HSV, influenza, Lyme disease, Moraxella catarrhalis, rotavirus, RSV, Streptococcus pneumoniae, SARS, smallpox, Staphylococcus aureus, and tetanus. Despite its excellent immunostimulating capacity and approval of clinical use, QS-21 has other drawbacks. For example, QS-21 shows dose-dependent adjuvant activity; however, its toxicity limits doses to about 50 pg, with the exception of cancer at <200 pg. The chemical instability of QS-21 is another issue. Hydrolytic removal of the acyl chain fragment of the QS-21ATTORNEY DOCKET NO. 222120-2140 molecule can occur slowly in solution, resulting in the adjuvant’s loss of its valuable capability in stimulating a Th1 response.

[0006] The severely limited supply of QS-21 , along with its dose-limiting toxicity, laborious and low-yielding purification, and chemical instability, hinder its wider clinical use. There are ongoing efforts to address QS-21 shortage, e.g., to obtain QS-21 from novel and sustainable botanical products; to produce QS-21 by developing a cell-culture-based technology; or to synthesize semisynthetic QS-21 analogs such as TQL-1055. Despite these intense efforts, overcoming QS-21 shortage remains an unmet urgent need. There is an imperative need of new adjuvants (especially QS-21 -type potent saponin adjuvants) for creating new vaccines against a broad range of diseases including emerging / re-emerging devastating infectious diseases and cancers. These needs and other needs are satisfied by the present disclosure.SUMMARY

[0007] In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to immunogenic compositions comprising a semisynthetic saponin adjuvant (VSA) including, but not limited to, VSA-1 and VSA-2, derived from a Momordica saponin and at least one antigen. In a further aspect, the immunogenic compositions can include other known adjuvants, emulsifiers, and / or surface-active agents and can be formulated as liposomes, micelles, emulsions, and the like. Also disclosed are methods of eliciting effective immune responses to viral, bacterial, protozoal, and neoplastic diseases using the disclosed immunogenic compositions.

[0008] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Many aspects of the present disclosure can be better understood with reference to theATTORNEY DOCKET NO. 222120-2140 following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0010] FIG. 1 shows the structure of natural saponin adjuvant QS-21.

[0011] FIG. 2 shows serum lgG1 and lgG2a anti-OVA responses in mice immunized by the s.c. route with OVA (20.0 pg) alone (None) or with QS-21 (10.0 pg), VSA1 (50 pg), or VB4 containing 50 pg of VSA-1. Mice were immunized on days 0, 14 and 28. Serum samples were collected prior to each immunization and 6 weeks after the initial immunization.

[0012] FIG. 3 shows serum lgG1 and lgG2a anti-OVA responses in mice immunized by the s.c. route with OVA (20.0 pg) alone (None) or with AS01 b (10% of human dose), or V1S50M5C containing 50 pg of VSA-1 and 5 pg of MPL. Mice were immunized on days 0, 14 and 28. Serum samples were collected prior to each immunization and 6 weeks after the initial immunization.

[0013] FIG. 4 shows serum lgG1 and lgG2a anti-gE responses in mice immunized by the s.c. route with gE (5 pg) alone (None) or with QS-21 (10.0 pg), or VM5010B containing 50 pg of VSA- 1 and 10 pg of MPL. Mice were immunized on days 0, 14 and 28. Serum samples were collected prior to each immunization and 6 weeks after the initial immunization.

[0014] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0015] Disclosed herein are new saponin-based adjuvants that inherit the favorable adjuvant activity of QS-21 but are devoid of its aforementioned drawbacks. In one aspect, these new adjuvants include VSA-1 and VSA-2 (Scheme 1). In a further aspect, the VSA adjuvants can be obtained via one-step derivatization of natural Momordica saponin (MS) I and II that are isolated from commercially available and inexpensive seeds of Momordica cochinchinensis Spreng (MC) (Scheme 1). In a still further aspect, their structures only differ in the triterpenoid core, i.e. , MS I has a gypsogenin core (R = H) while MS II has a quillaic acid core (R = OH), identical to the coreATTORNEY DOCKET NO. 222120-2140 of QS-21 (FIG. 1). The structures of MS I and II strikingly resemble that of the de-acylated QS saponins.Scheme 1 : One-step preparation of VSA adjuvants from natural Momordica cochinchinensis

[0016] In one aspect, the adjuvant formulations and combinations of the present disclosure offer numerous advantages over known adjuvants. In one aspect, the plant Momordica cochinchinensis Spreng is a perennial vine, and easy to grow, which circumvents the drawback of limited supplies of QS saponins. In another aspect, the abundance of MS I and II in the MS seeds is high, and their isolation is more efficient and thus more cost-effective than QS saponins. In still another aspect, the processed seeds are widely available in the U.S. and a 500 g pack can provide purified MS I and II > 1.0 g. However, in another aspect, the saponin can be extracted from other parts of the plant including, but not limited to, roots, stems, flowers, leaves, and the like, or from another Momordica plant or variety. In one aspect, ready access to VSA adjuvantsATTORNEY DOCKET NO. 222120-2140 avoids the formidable obstacle in the research of developing unnatural saponin-based vaccine adjuvants. In a further aspect, the easy isolation of the synthetic precursors and subsequent one- step routine chemical synthesis will simplify large-scale cGMP production of VSA adjuvants for high-volume vaccination needs, including in a pandemic. In a still further aspect, the use of structurally defined and homogenous adjuvants can avoid regulation hurdles.Immunogenic Compositions

[0017] In one aspect, disclosed herein are immunogenic compositions including at least one semisynthetic saponin adjuvant (VSA) derived from a Momordica saponin and at least one antigen. In a further aspect, the Momordica saponin can be extracted from a Momordica cochinchinensis Spreng seed, another part of the Momordica cochinchinensis Spreng plant, such as, for example, a root, stem, flower, or leaf, or another Momordica species.

[0018] In an aspect, the disclosed compositions are useful as vaccines against a variety of disease including viral diseases, bacterial diseases, diseases caused by protozoans, cancers, and other diseases and conditions. Thus, in one aspect, the at least one antigen can be an anthrax antigen, a chlamydia antigen, a cytomegalovirus antigen, a dengue antigen, an Ebola antigen, a group A Streptococcus antigen, a Helicobacter pylori antigen, a human papillomavirus antigen, a herpes simplex virus I or II antigen, an influenza antigen, a Lyme disease antigen, a Moraxella catarrhalis antigen, a rotavirus antigen, a respiratory syncytial virus antigen, a Streptococcus pneumoniae antigen, a SARS CoV-1 or -2 antigen, a smallpox antigen, a Staphylococcus aureus antigen, a tetanus antigen, a herpes zoster antigen, a malaria antigen, a tuberculosis antigen, a hepatitis A or B antigen, a human immunodeficiency virus antigen, an antigen associated with a cancer, or any combination thereof. In a further aspect, the antigen associated with a cancer can be a melanoma antigen or a non-small cell lung cancer (NSCLC) antigen. In some aspects, the disclosed immunogenic compositions can be administered to subjects who already have a disease such as, for example, melanoma, and administration of the compositions can assist the subjects’ own immune systems in attacking the diseases.

[0019] In yet another aspect, the VSA can be VSA-1 , VSA-2, or a combination thereof. In a further aspect, the VSA can be present in an amount of from about 10 pg to about 100 pg per unit dose, or from about 10 pg to about 500 pg, from about 10 pg to about 250 pg, from about 100 pg to about 200 pg, or from about 500 pg to about 1000 pg. In some aspects, the immunogenic compositions include one or more additional adjuvants including, but not limited to, MPL, CpG, alum, or any combination thereof. Thus, in a further aspect, pairs of additional adjuvants (e.g.,ATTORNEY DOCKET NO. 222120-2140MPL / CpG, CpG / alum, and MPL / alum) are also contemplated and should be considered disclosed, as should the ternary combination of adjuvants (e.g., MPL / CpG / alum).

[0020] In one aspect, the immunogenic compositions further include one or more emulsifiers or surface-active agents such as, for example, a natural or semisynthetic saponin, a polysorbate, a monosaccharide, a disaccharide, a polysaccharide, or any combination thereof. In an aspect, the polysorbate can be polysorbate 20, polysorbate 40, polysorbate 80, or any combination thereof. In another aspect, the monosaccharide can be glucose and the disaccharide can be sucrose or trehalose. In still another aspect, the polysaccharide can be chitosan or a derivative thereof.

[0021] In one aspect, the disclosed immunogenic compositions can be formulated in any dosage form useful for administration of vaccines and can include liposomes, micelles, or the like. In a further aspect, the composition can further include AS03, MF59, or a combination thereof. Still further in this aspect, the composition may be formulated as an emulsion. In any of these aspects, the immunogenic composition can further include at least one pharmaceutically acceptable excipient or carrier including, but not limited to, a polyamine polymer, a polyethylene glycol amine, poly(ethyleneimine), a nanocarbon, an amine-containing biological molecule, or any combination thereof.Method for Eliciting an Effective Immune Response

[0022] In one aspect, disclosed herein is a method for eliciting an effective immune response to an antigen in a subject, the method including at least the step of administering one or more unit doses of a disclosed immunogenic composition to the subject. In a further aspect, the method enhances a response of at least one serum immunoglobulin to the antigen in the subject such as, for example, lgG1 , lgG2a, or both.

[0023] In one aspect, the subject is a mammal such as, for example, a pet, livestock animal, experimental animal, or a human, including, but not limited to, a cat, dog, cattle, swine, horse, sheep, goat, rabbit, mouse, rat, or guinea pig.

[0024] In a further aspect, administering can be accomplished by oral administration, intramuscular injection, subcutaneous injection, or intranasal administration. In a still further aspect, from one to four unit doses of the immunogenic composition can be administered to the subject. In one aspect, when more than one unit dose is administered, such as from two to four unit doses, the doses can be administered at intervals ranging from two weeks to twenty-four months.ATTORNEY DOCKET NO. 222120-2140

[0025] Immunogenic responses can be measured or assessed in the subject in various ways as known in the art. In one aspect, the immune response in the subject includes at least one antigenspecific antibody titer. In a further aspect, the at least one antigen-specific antibody titer in the subject can be at least 1.5-fold higher than the same antigen-specific antibody titer in a subject to whom an otherwise identical immunogenic composition lacking the VSA was administered. In an alternative or additional aspect, the immune response in the subject can be measured by at least one opsonization titer. Further in this aspect, the at least one opsonization titer can be at least 50% higher than the same opsonization titer in a subject to whom an otherwise identical immunogenic composition lacking the VSA was administered.

[0026] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0027] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0028] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0029] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.ATTORNEY DOCKET NO. 222120-2140

[0030] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0031] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0032] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0033] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.Definitions

[0034] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by,” “comprising,” “comprises,” “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.ATTORNEY DOCKET NO. 222120-2140

[0035] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a serum immunoglobulin,” “a pharmaceutically acceptable carrier,” or “an antigen” include, but are not limited to, mixtures or combinations of two or more such serum immunoglobulins, pharmaceutically acceptable carriers, or antigens, and the like.

[0036] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0037] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. 'about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x,’ about y,’ and ‘about z’ as well as the ranges of ‘greater than x,’ greater than y, ’ and ‘greater than z.’ In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0038] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1 % to 5%” should be interpreted to include not only the explicitly recited values of about 0.1 % to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% toATTORNEY DOCKET NO. 222120-2140 about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0039] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0040] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0041] The term “adjuvant molecule” as used herein refers to a molecule that enhances the immune response of a subject to a vaccine.

[0042] The terms “administering” and “administration” as used herein refer to introducing a composition (e.g., a vaccine, adjuvant, or immunogenic composition) of the present disclosure into a subject.

[0043] The term "antigen" as used herein refers to a molecule with one or more epitopes that stimulate a host's immune system to make a secretory, humoral, and / or cellular antigen-specific response, or to a DNA or RNA molecule that is capable of producing such an antigen in a vertebrate. The term is also used interchangeably with "immunogen." For example, a specific antigen can be complete protein, portions of a protein, peptides, fusion proteins, glycosylated proteins, or combinations thereof.ATTORNEY DOCKET NO. 222120-2140

[0044] The term "composition" as used herein refers to a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such a term in relation to a pharmaceutical composition is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the immunogenic compositions of the present disclosure encompass any composition made by admixing a compound of the present disclosure and a pharmaceutically acceptable carrier.

[0045] As used herein, “CpG” refers to oligodeoxynucleotides having unmethylated sequences of cytosine-phosphate-guanosine dinucleotide sequences. Several different CpG types are known.

[0046] As used herein, “MF59” refers to an oil-in-water adjuvant used in human influenza vaccines and known to induce Th2 immune responses, among others. In one aspect, MF59 includes squalene droplets stabilized with small amounts of polysorbate 80 and sorbitan trioleate.

[0047] As used herein, “AS03” refers to a squalene-based oil-in-water emulsion similar to MF59 but that also includes a-tocopherol.

[0048] Compounds of the disclosure can be prepared using reactions and methods generally known to the person of ordinary skill in the art, having regard to that knowledge and the disclosure of this application including the Examples. The reactions are performed in solvent appropriate to the reagents and materials used and suitable for the reactions being effected. It will be understood by those skilled in the art of organic synthesis that the functionality present on the compounds should be consistent with the proposed reaction steps. This will sometimes require modification of the order of the synthetic steps or selection of one particular process scheme over another in order to obtain a desired compound of the disclosure. It will also be recognized that another major consideration in the development of a synthetic route is the selection of the protecting group used for protection of the reactive functional groups present in the compounds described in this disclosure. An authoritative account describing the many alternatives to the skilled artisan is Greene and Wuts (Protective Groups In Organic Synthesis, Wiley and Sons, 1991).ATTORNEY DOCKET NO. 222120-2140

[0049] A compound of the disclosure of the disclosure may be formulated into a pharmaceutical composition for administration to a subject by appropriate methods known in the art. Pharmaceutical compositions of the present disclosure or fractions thereof comprise suitable pharmaceutically acceptable carriers, excipients, and vehicles selected based on the intended form of administration, and consistent with conventional pharmaceutical practices. Suitable pharmaceutical carriers, excipients, and vehicles are described in the standard text, Remington: The Science and Practice of Pharmacy (21. sup. st Edition. 2005, University of the Sciences in Philadelphia (Editor), Mack Publishing Company), and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. By way of example for oral administration in the form of a capsule or tablet, the active components can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, methyl cellulose, magnesium stearate, glucose, calcium sulfate, dicalcium phosphate, mannitol, sorbital, and the like. For oral administration in a liquid form, the chug components may be combined with any oral, non-toxic, pharmaceutically, acceptable inert carrier such as ethanol, glycerol, water, and the like. Suitable binders (e.g., gelatin, starch, corn sweeteners, natural sugars including glucose; natural and synthetic gums, and waxes), lubricants (e.g. sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride), disintegrating agents (e.g. starch, methyl cellulose, agar, bentonite, and xanthan gum), flavoring agents, and coloring agents may also be combined in the compositions or components thereof. Compositions as described herein can further comprise wetting or emulsifying agents, or pH buffering agents.

[0050] The term “immunogenic composition” as used herein are those which result in specific antibody production or in cellular immunity when injected into a host.

[0051] The immunogenic compositions and / or vaccines of the present disclosure may be formulated by any of the methods known in the art. They can be typically prepared as injectables or as formulations for intranasal administration, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection or other administration may also be prepared. The preparation may also, for example, be emulsified, or the protein(s) / peptide(s) encapsulated in liposomes.

[0052] The active immunogenic ingredients are often mixed with excipients or carriers, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. The concentration of the immunogenic polypeptide in injectable, aerosolATTORNEY DOCKET NO. 222120-2140 or nasal formulations is usually in the range of about 0.2 to 5 mg / mL. Similar dosages can be administered to other mucosal surfaces.

[0053] In addition, if desired, the vaccines may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or other agents, which enhance the effectiveness of the vaccine. Examples of agents which may be effective include, but are not limited to, aluminum hydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP); N-acetyl- nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N-acetylmuramyl-L- alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- ethylamine (CGP 19835A, referred to as MTP-PE); and RIBI, which contains three components extracted from bacteria: monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween 80 emulsion. The effectiveness of the auxiliary substances may be determined by measuring the amount of antibodies (especially IgG, IgM, or IgA) directed against the immunogen resulting from administration of the immunogen in vaccines which comprise the adjuvant in question. Additional formulations and modes of administration may also be used.

[0054] The immunogenic compositions and / or vaccines of the present disclosure can be administered in a manner compatible with the dosage formulation and in such amount and manner as will be prophylactically and / or therapeutically effective, according to what is known to the art. Precise amounts of the active ingredient required to be administered may depend on the judgment of the physician or veterinarian and may be peculiar to each individual, but such a determination is within the skill of such a practitioner.

[0055] The vaccine or immunogenic composition may be given in a single dose; two-dose schedule, for example, two to eight weeks apart; or a multi-dose schedule. A multi-dose schedule is one in which a primary course of vaccination may include 1 to 10 or more separate doses, followed by other doses administered at subsequent time intervals as required to maintain and / or reinforce the immune response (e.g., at 1 to 4 months for a second dose, and if needed, a subsequent dose(s) after several months).

[0056] The term “immunoglobulin” as used herein refers to a class of proteins that exhibit antibody activity and bind to other molecules (e.g., antigens and certain cell-surface receptors) with a high degree of specificity. Immunoglobulins can be divided into five classes: IgM, IgG, IgA, IgD, and IgE. IgG is the most abundant antibody class in the body and assumes a twisted "Y" shape configuration. With the exception of the IgMs, immunoglobulins are composed of four peptideATTORNEY DOCKET NO. 222120-2140 chains that are linked by intrachain and interchain disulfide bonds. IgGs are composed of two polypeptide heavy chains (H chains) and two polypeptide light chains (L chains) that are coupled by non-covalent disulfide bonds.

[0057] The term "immunological response" as used herein refers to the development in a subject of a humoral and / or a cellular immune response to an antigen present in the composition of interest. For purposes of the present disclosure, a "humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T-lymphocytes and / or other white blood cells.

[0058] The terms "subject", "individual", or "patient" as used herein are used interchangeably and refer to an animal preferably a warm-blooded animal such as a mammal. Mammal includes without limitation any members of the Mammalia. A mammal, as a subject or patient in the present disclosure, can be from the family of Primates, Carnivora, Proboscidea, Perissodactyla, Artiodactyla, Rodentia, and Lagomorpha. In a particular embodiment, the mammal is a human. In other embodiments, animals can be treated; the animals can be vertebrates, including both birds and mammals. In aspects of the disclosure, the terms include domestic animals bred for food or as pets, including horses, cattle, sheep, poultry, fish, swine, canines, felines, and zoo animals, goats, apes (e.g. gorilla or chimpanzee), humans, and rodents such as rats and mice.

[0059] The term "pharmaceutically acceptable carrier" as used herein refers to a diluent, adjuvant, excipient, or vehicle with which a probe of the disclosure is administered and which is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. When administered to a patient, the probe and pharmaceutically acceptable carriers can be sterile. Water is a useful carrier when the probe is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol, and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifyingATTORNEY DOCKET NO. 222120-2140 agents, or pH buffering agents. The present compositions advantageously may take the form of solutions, emulsion, sustained-release formulations, or any other form suitable for use.

[0060] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0061] The term “vaccine” as used herein refers to an immunogenic amount of one or more virosomes, fragment(s), or subunit(s) thereof. Such vaccines can include one or more viral surface envelope glycoproteins and portions thereof, and adjuvant molecule and portions thereof on the surfaces of the virosomes, or in combination with another protein or other immunogen, such as one or more additional virus components naturally associated with viral particles or an epitopic peptide derived therefrom.

[0062] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e., one atmosphere).Pharmaceutical Compositions

[0063] Pharmaceutical compositions of the present disclosure are suitable for injection, such as parenteral administration, such as intravenous, intramuscular, or subcutaneous administration. Pharmaceutical compositions for injection can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.

[0064] Pharmaceutical compositions of the present disclosure suitable for parenteral administration can include sterile aqueous or oleaginous solutions, suspensions, or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some aspects, the final injectable form is sterile and must be effectively fluid for use in a syringe. The pharmaceutical compositions should be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g.,ATTORNEY DOCKET NO. 222120-2140 glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0065] Injectable solutions, for example, can be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In some aspects, a disclosed parenteral formulation can comprise about 0.01-0.1 M, e.g. about 0.05 M, phosphate buffer. In a further aspect, a disclosed parenteral formulation can comprise about 0.9% saline.

[0066] In various aspects, a disclosed parenteral pharmaceutical composition can comprise pharmaceutically acceptable carriers such as aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include but not limited to water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles can include mannitol, normal serum albumin, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer’s, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like. In a further aspect, a disclosed parenteral pharmaceutical composition can comprise may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. Also contemplated for injectable pharmaceutical compositions are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the subject or patient.

[0067] Thus, the pharmaceutical compositions of the present disclosure can be presented as discrete units suitable for oral administration such as capsules, cachets, or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the present disclosure, and / or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and / or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, suchATTORNEY DOCKET NO. 222120-2140 methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

[0068] It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. That is, a “unit dosage form” is taken to mean a single dose wherein all active and inactive ingredients are combined in a suitable system, such that the patient or person administering the drug to the patient can open a single container or package with the entire dose contained therein, and does not have to mix any components together from two or more containers or packages. Typical examples of unit dosage forms are tablets (including scored or coated tablets), capsules or pills for oral administration; single dose vials for injectable solutions or suspension; suppositories for rectal administration; powder packets; wafers; and segregated multiples thereof. This list of unit dosage forms is not intended to be limiting in any way, but merely to represent typical examples of unit dosage forms.

[0069] Because of the ease in administration, oral administration can be a preferred dosage form, and tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed. However, other dosage forms may be suitable depending upon clinical population (e.g., age and severity of clinical condition), solubility properties of the specific disclosed compound used, and the like. Accordingly, the disclosed compounds can be used in oral dosage forms such as pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques.ATTORNEY DOCKET NO. 222120-2140

[0070] The disclosed pharmaceutical compositions in an oral dosage form can comprise one or more pharmaceutical excipient and / or additive. Non-limiting examples of suitable excipients and additives include gelatin, natural sugars such as raw sugar or lactose, lecithin, pectin, starches (for example corn starch or amylose), dextran, polyvinyl pyrrolidone, polyvinyl acetate, gum arabic, alginic acid, tylose, talcum, lycopodium, silica gel (for example colloidal), cellulose, cellulose derivatives (for example cellulose ethers in which the cellulose hydroxy groups are partially etherified with lower saturated aliphatic alcohols and / or lower saturated, aliphatic oxyalcohols, for example methyl oxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate), fatty acids as well as magnesium, calcium or aluminum salts of fatty acids with 12 to 22 carbon atoms, in particular saturated (for example stearates), emulsifiers, oils and fats, in particular vegetable (for example, peanut oil, castor oil, olive oil, sesame oil, cottonseed oil, corn oil, wheat germ oil, sunflower seed oil, cod liver oil, in each case also optionally hydrated); glycerol esters and polyglycerol esters of saturated fatty acids C12H24O2 to C18H36O2 and their mixtures, it being possible for the glycerol hydroxy groups to be totally or also only partly esterified (for example mono-, di- and triglycerides); pharmaceutically acceptable mono- or multivalent alcohols and polyglycols such as polyethylene glycol and derivatives thereof, esters of aliphatic saturated or unsaturated fatty acids (2 to 22 carbon atoms, in particular 10-18 carbon atoms) with monovalent aliphatic alcohols (1 to 20 carbon atoms) or multivalent alcohols such as glycols, glycerol, diethylene glycol, pentacrythritol, sorbitol, mannitol and the like, which may optionally also be etherified, esters of citric acid with primary alcohols, acetic acid, urea, benzyl benzoate, dioxolanes, glyceroformals, tetra hydrofurfuryl alcohol, polyglycol ethers with C1-C12-alcohols, dimethylacetamide, lactamides, lactates, ethylcarbonates, silicones (in particular medium-viscous polydimethyl siloxanes), calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, magnesium carbonate and the like.

[0071] Other auxiliary substances useful in preparing an oral dosage form are those which cause disintegration (so-called disintegrants), such as: cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, sodium carboxymethyl cellulose or microcrystalline cellulose. Conventional coating substances may also be used to produce the oral dosage form. Those that may for example be considered are: polymerizates as well as copolymerizates of acrylic acid and / or methacrylic acid and / or their esters; copolymerizates of acrylic and methacrylic acid esters with a lower ammonium group content (for example EudragitR RS), copolymerizates of acrylic and methacrylic acid esters and trimethyl ammonium methacrylate (for example EudragitR RL);ATTORNEY DOCKET NO. 222120-2140 polyvinyl acetate; fats, oils, waxes, fatty alcohols; hydroxypropyl methyl cellulose phthalate or acetate succinate; cellulose acetate phthalate, starch acetate phthalate as well as polyvinyl acetate phthalate, carboxy methyl cellulose; methyl cellulose phthalate, methyl cellulose succinate, -phthalate succinate as well as methyl cellulose phthalic acid half ester; zein; ethyl cellulose as well as ethyl cellulose succinate; shellac, gluten; ethylcarboxyethyl cellulose; ethacrylate-maleic acid anhydride copolymer; maleic acid anhydride-vinyl methyl ether copolymer; styrol-maleic acid copolymerizate; 2-ethyl-hexyl-acrylate maleic acid anhydride; crotonic acid-vinyl acetate copolymer; glutaminic acid / glutamic acid ester copolymer; carboxymethylethylcellulose glycerol monooctanoate; cellulose acetate succinate; polyarginine.

[0072] Plasticizing agents that may be considered as coating substances in the disclosed oral dosage forms are: citric and tartaric acid esters (acetyl-triethyl citrate, acetyl tributyl-, tributyl-, triethyl-citrate); glycerol and glycerol esters (glycerol diacetate, -triacetate, acetylated monoglycerides, castor oil); phthalic acid esters (dibutyl-, diamyl-, diethyl-, dimethyl-, dipropylphthalate), di-(2-methoxy- or 2-ethoxyethyl)-phthalate, ethylphthalyl glycolate, butylphthalylethyl glycolate and butylglycolate; alcohols (propylene glycol, polyethylene glycol of various chain lengths), adipates (diethyladipate, di-(2-methoxy- or 2-ethoxyethyl)-adipate; benzophenone; diethyl- and diburylsebacate, dibutylsuccinate, dibutyltartrate; diethylene glycol dipropionate; ethyleneglycol diacetate, -dibutyrate, -dipropionate; tributyl phosphate, tributyrin; polyethylene glycol sorbitan monooleate (polysorbates such as Polysorbar 50); sorbitan monooleate.

[0073] Moreover, suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents may be included as carriers. The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include, but are not limited to, lactose, terra alba, sucrose, glucose, methylcellulose, dicalcium phosphate, calcium sulfate, mannitol, sorbitol talc, starch, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0074] In various aspects, a binder can include, for example, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. In a further aspect, a disintegrator can include, for example, starch, methyl cellulose, agar, bentonite, xanthan gum,ATTORNEY DOCKET NO. 222120-2140 and the like.

[0075] In various aspects, an oral dosage form, such as a solid dosage form, can comprise a disclosed compound that is attached to polymers as targetable drug carriers or as a prodrug. Suitable biodegradable polymers useful in achieving controlled release of a drug include, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, caprolactones, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and hydrogels, preferably covalently crosslinked hydrogels.

[0076] Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

[0077] A tablet containing a disclosed compound can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0078] In various aspects, a solid oral dosage form, such as a tablet, can be coated with an enteric coating to prevent ready decomposition in the stomach. In various aspects, enteric coating agents include, but are not limited to, hydroxypropylmethylcellulose phthalate, methacrylic acidmethacrylic acid ester copolymer, polyvinyl acetate-phthalate, and cellulose acetate phthalate. Akihiko Hasegawa “Application of solid dispersions of Nifedipine with enteric coating agent to prepare a sustained-release dosage form” Chem. Pharm. Bull. 33:1615-1619 (1985). Various enteric coating materials may be selected on the basis of testing to achieve an enteric coated dosage form designed ab initio to have a preferable combination of dissolution time, coating thicknesses and diametral crushing strength (e.g., see S. C. Porter et al. “The Properties of Enteric Tablet Coatings Made From Polyvinyl Acetate-phthalate and Cellulose acetate Phthalate,” J. Pharm. Pharmacol. 22:42p (1970)). In a further aspect, the enteric coating may compriseATTORNEY DOCKET NO. 222120-2140 hydroxypropyl-methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymer, polyvinyl acetate-phthalate, and cellulose acetate phthalate.

[0079] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.EXAMPLES

[0080] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.Example 1 : Immune Responses to Adjuvants and Combinations of Adjuvants

[0081] Systematic studies of VSA adjuvants’ immunostimulating activities were conducted by varying specific VSA formulation and combining different VSAs. Results are presented in FIGs. 2-4. In these experiments it was shown that VSA adjuvants, when used in combination, can have increased immunostimulating activity even at reduced dosages.

[0082] Serum lgG1 and lgG2a anti-OVA responses were monitored in mice immunized by the s.c. route with OVA (20.0 pg) alone (None) or with QS-21 (10.0 pg), VSA1 (50 pg), or VB4 containing 50 pg of VSA-1. Mice were immunized on days 0, 14, and 28. Serum samples were collected prior to each immunization and 6 weeks after the initial immunization with responses for the VB4 containing 50 pg of VSA-1 being highest after 6 weeks (FIG. 2).

[0083] Serum lgG1 and lgG2a anti-OVA responses were monitored in mice immunized by the s.c. route with OVA (20.0 pg) alone (None) or with AS01b (10% of human dose), or V1S50M5C containing 50 pg of VSA-1 and 5 pg of MPL. Mice were immunized on days 0, 14, and 28. Serum samples were collected prior to each immunization and 6 weeks after the initial immunization withATTORNEY DOCKET NO. 222120-2140 responses for the V1S50M5C containing 50 pG of VSA-1 and 5 pg of MPL being highest after 6 weeks (FIG. 3).

[0084] Serum IgG 1 and lgG2a anti-gE responses were monitored in mice immunized by the s.c. route with gE (5 pg) alone (None) or with QS-21 (10.0 pg), or VM5010B containing 50 pg of VSA- 1 and 10 pg of MPL. Mice were immunized on days 0, 14, and 28. Serum samples were collected prior to each immunization and 6 weeks after the initial immunization with responses for the VM5010B containing 50 pg of VSA-1 and 10 pg of MPL being highest after 6 weeks (FIG. 4).

[0085] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the abovedescribed embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.REFERENCES1. Ballou WR. The development of the RTS,S malaria vaccine candidate: challenges and lessons. Parasite Immunol. 2009;31 (9):492-500. doi: 10.1111 / j.1365-3024.2009.01143.x.2. Banday AH, et al. Cancer vaccine adjuvants-- recent clinical progress and future perspectives. Immunopharmacol Immunotoxicol. 2015;37(1): 1 -11 . Epub 2014 / 10 / 17. doi: 10.3109 / 08923973.2014.971963.3. Bastola R, et al. Vaccine adjuvants: smart components to boost the immune system. Archives of pharmacal research. 2017;40(11):1238-48. Epub 2017 / 10 / 14. doi: 10.1007 / s12272-017- 0969-z.4. Bonam SR, et al. An overview of novel adjuvants designed for improving vaccine efficacy. Trends in Pharmacological Sciences. 2017;38(9):771-93.5. Brunner R, et al. The ABC of clinical and experimental adjuvants - a brief overview. Immunol Lett. 2010;128(1):29-35. doi: 10.1016 / j.imlet.2009.10.005.6. Coffman RL, et al. Vaccine adjuvants: putting innate immunity to work. Immunity. 2010;33(4):492-503. doi: 10.1016 / j.immuni.2010.10.002.7. Delrieu J, et al. 'Clinical trials in Alzheimer's disease': immunotherapy approaches. J Neurochem. 2012;120 Suppl 1 :186-93. doi: 10.1111 / j.1471-4159.2011.07458.x.ATTORNEY DOCKET NO. 222120-2140 Di Pasquale A, et al. Vaccine adjuvants: from 1920 to 2015 and beyond. Vaccines (Basel). 2015;3(2):320-43. Epub 2015 / 09 / 08. doi: 10.3390 / vaccines3020320. Didierlaurent AM, et al. Adjuvant system AS01 : helping to overcome the challenges of modern vaccines. Expert review of vaccines. 2017;16(1):55-63. Epub 2016 / 07 / 28. doi: 10.1080 / 14760584.2016.1213632. Garcon N, et al. From discovery to licensure, the Adjuvant System story. Hum Vaccin Immunother. 2017; 13(1): 19-33. Epub 2016 / 09 / 17. doi: 10.1080 / 21645515.2016.1225635. Gargon N, et al. Innate Immunity and Vaccine Adjuvants: From Concepts to the Development of a Unique Adjuvant System AS04 Used for the Formulation of a Human Papillomavirus (HPV) Vaccine. Curr Cancer Ther Rev. 2010;6:126-37. Garcon N, et al. Recent clinical experience with vaccines using MPL- and QS-21-containing adjuvant systems. Expert Rev Vaccines. 2011 ;10(4):471-86. doi: 10.1586 / erv.11.29. Gargon N, et al. Vaccine Adjuvants. In: Gargon N, Stern PL, Cunningham AL, editors. Understanding Modern Vaccines Perspectives in Vaccinology Amsterdam: Elsevier; 2011. p. 89-113. Giudice GD, et al. Correlates of adjuvanticity: A review on adjuvants in licensed vaccines. Semin Immunol. 2018;2018:14-21. Glenny AT, et al. Immunological notes. XVI1.-XXIV. J Pathol Bacteriol. 1926;29:31-40. HogenEsch H, et al. Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want, npj Vaccines. 2018;3(51). doi: 10.1038 / s41541-018-0089-x. James SF, et al. Shingrix: the new adjuvanted recombinant herpes zoster vaccine. Annals of Pharmacotherapy. 2018;52(7):673-80. Kensil CR, et al. Current vaccine adjuvants: An overview of a diverse class. Frontiers in Bioscience. 2004;9:2972-88. Kensil CR, et al. Effects of QS-21 on Innate and Adaptive Immune Responses. In: Hackett CJ, Harn DAJ, editors. Vaccine Adjuvants: Immunological and Clinical Principles. Totowa, NJ: Humana Press Inc.; 2005. p. 221-34. Kensil CR, et al. Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex. 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Claims

ATTORNEY DOCKET NO. 222120-2140CLAIMSWhat is claimed is:

1. An immunogenic composition comprising a semisynthetic saponin adjuvant (VSA) derived from a Momordica saponin and at least one antigen.

2. The immunogenic composition of claim 1 , wherein the Momordica saponin comprises a saponin extracted from Momordica cochinchinensis Spreng or another Momordica plant.

3. The immunogenic composition of claim 2, wherein the saponin is extracted from a seed, root, stem, flower, or leaf4. The immunogenic composition of claim 1 , wherein the at least one antigen comprises an anthrax antigen, a chlamydia antigen, a cytomegalovirus antigen, a dengue antigen, an Ebola antigen, a group A Streptococcus antigen, a Helicobacter pylori antigen, a human papillomavirus antigen, a herpes simplex virus I or II antigen, an influenza antigen, a Lyme disease antigen, a Moraxella catarrhalis antigen, a rotavirus antigen, a respiratory syncytial virus antigen, a Streptococcus pneumoniae antigen, a SARS CoV-1 or -2 antigen, a smallpox antigen, a Staphylococcus aureus antigen, a tetanus antigen, a herpes zoster antigen, a malaria antigen, a tuberculosis antigen, a hepatitis A or B antigen, a human immunodeficiency virus antigen, an antigen associated with a cancer, or any combination thereof.

5. The immunogenic composition of claim 4, wherein the antigen associated with a cancer comprises a melanoma antigen or a non-small cell lung cancer antigen.

6. The immunogenic composition of claim 1, wherein the VSA comprises VSA-1 , VSA-2, or any combination thereof.

7. The immunogenic composition of claim 1 , further comprising one or more additional adjuvants.

8. The immunogenic composition of claim 7, wherein the one or more additional adjuvants comprise MPL, CpG, alum, or any combination thereof.

9. The immunogenic composition of claim 7, wherein the one or more additional adjuvants are MPL and CpG.

10. The immunogenic composition of claim 7, wherein the one or more additional adjuvants are CpG and alum.

11. The immunogenic composition of claim 7, wherein the one or more additional adjuvants are MPL and alum.ATTORNEY DOCKET NO. 222120-214012. The immunogenic composition of claim 1 , further comprising one or more emulsifiers or surface-active agents.

13. The immunogenic composition of claim 12, wherein the one or more emulsifiers or surfaceactive agents comprise a natural or semisynthetic saponin, a polysorbate, a monosaccharide, a disaccharide, a polysaccharide, or any combination thereof.

14. The immunogenic composition of claim 13, wherein the polysorbate comprises polysorbate 20, polysorbate 40, polysorbate 80, or any combination thereof.

15. The immunogenic composition of claim 13, wherein the monosaccharide comprises glucose.

16. The immunogenic composition of claim 13, wherein the disaccharide comprises sucrose, trehalose, or any combination thereof.

17. The immunogenic composition of claim 13, wherein the polysaccharide comprises chitosan.

18. The immunogenic composition of claim 1 , wherein the composition is formulated as liposomes or micelles.

19. The immunogenic composition of claim 1 , wherein the composition further comprises AS03, MF59, or any combination thereof.

20. The immunogenic composition of claim 1 , wherein the composition is formulated as an emulsion.

21. The immunogenic composition of claim 1 , further comprising at least one pharmaceutically acceptable excipient or carrier.

22. The immunogenic composition of claim 21 , wherein the at least one pharmaceutically acceptable excipient or carrier comprises a polyamine polymer, a polyethylene glycol amine, poly(ethyleneimine), a nanocarbon, an amine-containing biological molecule, or any combination thereof.

23. The immunogenic composition of claim 1 , wherein the VSA is present in an amount of from about 10 pg to about 1000 pg per unit dose.

24. A method for eliciting an effective immune response to an antigen in a subject, the method comprising administering one or more unit doses of the immunogenic composition of any one of claims 1-23 to the subject.

25. The method of claim 24, wherein the method enhances a response of at least one serum immunoglobulin to the antigen in the subject.ATTORNEY DOCKET NO. 222120-214026. The method of claim 25, wherein the at least one serum immunoglobulin comprises lgG1 , lgG2a, or any combination thereof.

27. The method of claim 24, wherein the subject is a mammal.

28. The method of claim 27, wherein the mammal is a human, cat, dog, cattle, swine, horse, sheep, goat, rabbit, mouse, rat, or guinea pig.

29. The method of claim 24, wherein administering comprises oral administration, intramuscular injection, subcutaneous injection, or intranasal administration.

30. The method of claim 24, comprising administering from one to four unit doses of the immunogenic composition to the subject.

31. The method of claim 30, wherein from two to four unit doses are administered at two week to twenty-four month intervals.

32. The method of claim 24, wherein the immune response in the subject comprises at least one antigen-specific antibody titer.

33. The method of claim 32, wherein the at least one antigen-specific antibody titer in the subject is at least 1.5-fold higher than the same antigen-specific antibody titer in a subject to whom an otherwise identical immunogenic composition lacking the VSA was administered.

34. The method of claim 24, wherein the immune response in the subject can be measured by at least one opsonization titer.

35. The method of claim 34, wherein the at least one opsonization titer is at least 50% higher than the same opsonization titer in a subject to whom an otherwise identical immunogenic composition lacking the VSA was administered.

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