Immunologically active saponin fractions and adjuvant formulations

Abstract

Disclosed are immunologically active saponin fraction composition, its method of manufacture and use in vaccine adjuvants, and in adjuvanted vaccines. Methods of preparation, vaccine and treatment kits comprising the adjuvants, and uses of vaccines and vaccine kits comprising the adjuvants are also disclosed.

Description

Immunologically Active Saponin Fractions and Adjuvant FormulationsTechnical Field

[0001] The present disclosure relates to a novel immunostimulatory triterpenic saponin fraction of Quillaja saponaria Molina suitable as a component of vaccine adjuvant formulations. Additionally, the present disclosure discloses processes for the isolation of this new saponin fraction from an extract of Q. saponaria Molina by chromatographic methods. Furthermore, the present disclosure relates to immunologically active compositions comprising the new saponin fraction, adjuvant formulations comprising them, and kits of parts and vaccine compositions made using said compositions for the prevention and treatment of diseases.Background

[0002] There exists a range of potential adjuvant formulations for the effective provision of an antigen and stimulation of immune response. For example, saponins (including but not limited to QuilA or QS21 ) have stimulatory immune-modulator effects.

[0003] Saponins are steroid or triterpenoid glycosides widely spread in the plant kingdom. These compounds have a wide range of medicinal and commercial uses (Hostettmann and Marston, 1995). The Chilean tree Quillaja saponaria Molina is an abundant source of triterpenic saponins. The saponins of this tree are a complex family of more than 50 different glycosides, most of them consisting of quillaic acid aglycone substituted with oligosaccharides at C-3 and C-28 positions (Kite et al. 2004). Kensil and Marciani (1991 ) described the separation of 22 chromatographic fractions of saponins (QA-1 to QA-22) from an extract of Quillaja bark by reverse phase HPLC.

[0004] Although Quillaja saponins have been used as surfactants in the cosmetic, and food and beverage industries (Fleck etal., 2019), nowadays there is an increasing interest in using Quillaja saponins as components in pharmaceutical formulations, particularly as immunostimulant agents for the formulation of vaccine adjuvants (Kensil et al., 1991 ). Adjuvants are key vaccine components of modern vaccines that ensure the induction of a robust immune response to antigens or antigen-producing nucleic acids. In some instances, adjuvants ensure that a sufficient immune response is raised against otherwise poorly immunogenic vaccine antigens, such as some recombinant antigens. Additionally, by enabling optimization of the immune response, adjuvants have facilitated the reduction of antigen quantity required in a vaccine dose (i.e. “dose-sparing” strategies) of particular value wherean antigen is expensive or not available in otherwise sufficient quantities to fulfill the requirements of a vaccination program, thereby substantially improving global vaccine supply, in particular in pandemic situations. Moreover, adjuvants improve immune responses in populations with typically weakened immune responses, such as infants, elderly and immunocompromised individuals.

[0005] The saponin QS-21 is one of the most potent adjuvants known, which is currently used in both exploratory studies as well as in a few licensed vaccines (Lacaille-Dubois, 2019). Saponin QS-21 showed very low toxicity in mice (lethal dose, 500 pg) and, in animal immunization tests invoked high neutralizing antibody titers as well as cytotoxic T-lymphocyte (CTL) responses (Cleland et al., 1996).

[0006] However, the isolation of QS-21 is a very expensive procedure requiring careful selection of the raw material employed followed by complex process of isolation of the product (Baig et al., 2019; De Kessel et al., 2020) and the limited supply has increasingly become the major constraint on the widespread use of QS-21 (Ragupathi, etal., 201 1 ). Furthermore, there is high variability in the saponin composition between Q. saponaria trees, even in the same local environment, and QS-21 is just one of typically 22 saponin fractions extracted from this bark; in some trees, the content of QS-21 is very low compared to the most abundant saponins. It is unfortunate that the current high price of QS21 -containing adjuvant formulations is severely hindering the ability of low and middle income countries in particular to provide populations with effective and affordable vaccine programs. This inequality is exacerbated in times of global pandemic when high global demand for effective adjuvants places strain on the limited supply of currently available saponin fractions with immunostimulant properties.

[0007] Therefore, there is a need to provide new compositions of saponins with immunostimulant properties from selected and harvested Q. saponaria raw materials, which can be isolated and purified using an alternative method at lower cost and at higher yield compared to QS-21 and other existing saponin fractions. Additionally, for sustainability reasons, there is a desire to further improve the efficiency of production of saponin-based adjuvants from crops of Q. saponaria.

[0008] The present invention discloses a triterpenic saponin fraction denominated "saponin fraction DV", isolated by chromatographic means from an extract of Q. saponaria Molina, whose major components are saponins having a monoisotopic m / z for [M-H]- of 1726, 1856, 1988 and 2003 by negative electrospray ionization mass spectroscopy analysis. Saponin fraction DV is characterized by its significant immunostimulant activity, low cost of preparation, and increased yields per kg of Q. saponaria biomass. This disclosure of the present invention also comprises a method of production at large scale of saponin fraction DV, as well as avariety of compositions comprising saponin fraction DV suitable for use as adjuvants in vaccines, pharmaceutical and vaccine compositions comprising saponin fraction DV, and uses thereof.

[0009] As such, it is an object of the current disclosure to provide novel, efficacious and reduced-cost saponin fractions ("saponin fraction DV"), adjuvant formulations comprising said novel reduced-cost saponin fraction, and vaccine compositions comprising said novel low-cost saponin fraction suitable for use in vaccines. Adjuvant formulations comprising the novel saponin fraction DV are particularly useful when employing dose-sparing strategies of antigen use in vaccines, which together with the lower cost of production of saponin fraction DV, enables very low cost but very efficacious adjuvant formulations, which are particularly needed for use in vaccines intended predominantly for use in low and middle income countries. Accordingly, data presented in the Examples herein demonstrate the suitability of saponin fraction DV for use in vaccines for malaria, influenza, covid and tuberculosis, each of which represent a significant burden to the health services of low and middle income countries.Summary

[0010] Compositions of the present application comprising saponin fraction DV described herein are immunostimulatory, suitable for use as adjuvants, suitable for inclusion in immunostimulatory formulations, and suitable for use in compositions suitable for disease prevention or treatment, such as vaccines. Compositions of the present application comprising saponin fraction DV and an antigen described herein are able to activate immune responses to enhance the subsequent acquisition of immunity to the condition associated with the antigen of the composition, so are suitable for use as vaccines and / or immune-mediated methods of treatment. The current application teaches methods of producing saponin fraction DV, including methods suitable for production on an industrial scale capable of producing sufficient volumes of saponin fraction DV suitable for the production of a sufficient number of adjuvanted vaccine doses to prevent or help respond to outbreaks of infectious diseases, epidemics and pandemics. The current disclosure also provides a range of formulations and compositions for adjuvant and vaccine compositions comprising immunologically active saponin fraction(s), appropriate for the provision of various antigens for the stimulation of immune responses, such as in vaccination and immunization programs.Description of drawings and figuresFigure 1 : Process flow chart of the chromatographic purification of saponin fraction DV at small scale.Figure 2: Process flow chart of the chromatographic purification of saponin fraction DV at large scale.Figure 3: Representative example of the UHPLC chromatographic analysis of the product saponin fraction DV.Figure 4: Zoomed view of the UHPLC chromatogram of the product saponin fraction DV shown in Figure 3.Figure 5: Zoomed view of the QVI group peaks in the UHPLC chromatogram of the product saponin fraction DV shown in Figure 3.Figure 6: Mass spectra analysis of the DV saponin fraction.Figure 7: One proposed structure of a saponin compound detectable at 1726 m / z using mass spectroscopy in negative ion mode.Figure 8: Proposed structure of a saponin compound detectable at 1856 m / z using mass spectroscopy in negative ion mode.Figure 9: Proposed structure of a saponin compound detectable at 1988 m / z using mass spectroscopy in negative ion mode.Figure 10: Proposed structure of a saponin compound detectable at 2003 m / z using mass spectroscopy in negative ion mode.Figure 11 : Comparison of the lytic activity of saponin fraction DV on sheep red blood cells, as compared with a commercial GMP-grade saponin QS21 .Figure 12: Association of saponin fraction DV with cholesterol-containing liposomes and emulsions.Figures 13a-e: Accelerated stability of saponin fraction DV-containing DOPC / Cholesterol liposome-based adjuvant formulations compared to QS21 -containing DOPC / Cholesterol liposome-based adjuvant formulations examined for particle size (Fig.13a), polydispersity index (Fig.13b), DOPC recovery (Fig.13c), cholesterol recovery (Fig.13d) and saponin recovery using HPLC-CAD (Fig.13e).Figures 14a-e: Accelerated stability of saponin fraction DV-containing emulsion-based adjuvant formulations compared to QS21 -containing emulsion-based formulations examined for particle size (Fig.14a), polydispersity index (Fig.14b), squalene recovery (Fig.14c), cholesterol recovery (Fig.14d) and saponin recovery using HPLC-CAD (Fig.14e).Figure 15: Accelerated stability of DV-containing DOPC / Cholesterol / 3D-(6-acyl) PHAD® liposome-based adjuvant formulations compared to QS21 -containing DOPC / Cholesterol / 3D-(6-acyl) PHAD® liposome-based adjuvant formulations examined for particle size (Fig.15a), polydispersity index (Fig.15b), DOPC recovery (Fig.15c), cholesterol recovery (Fig.15d), saponin recovery using UPLC-MS (Fig.15e) and 3D6AP recovery (Fig.15f).Figure 16a: CD4+ T cell responses induced by saponin fraction DV-containing adjuvant formulations as compared with QS21 -containing adjuvant formulations using COVID-19 antigen in mice.Figure 16b: CD8+ T cell responses induced by saponin fraction DV-containing adjuvant formulations as compared with QS21 -containing adjuvant formulations using COVID-19 antigen in mice.Figure 17a: Antibody responses (total IgG EC50 tiers) induced by vaccines comprising saponin fraction DV-containing adjuvant formulations as compared with QS21 -containing adjuvant formulations using a SARS-CoV-2 spike antigen in mice.Figure 17b: Antibody responses (total lgG2c EC50 tiers) induced by vaccines comprising saponin fraction DV-containing adjuvant formulations as compared with QS21 -containing adjuvant formulations using a SARS-CoV-2 spike antigen in mice.Figure 18: T cell responses induced by saponin fraction DV-containing adjuvant formulations as compared with QS21 -containing adjuvant formulations using influenza antigens in mice.Figure 19: Antibody responses induced by saponin fraction DV-containing adjuvant formulations as compared with QS21 -containing adjuvant formulations using influenza antigens in mice.Figure 20: T cell responses induced by vaccines comprising saponin fraction DV-containing adjuvant formulations as compared with QS21 -containing adjuvant formulations using a MTB Ag85B antigen in mice.Figure 21a: Antibody responses (total IgG results - EC50 titers) induced by vaccines comprising saponin fraction DV-containing adjuvant formulations as compared with QS21 - containing adjuvant formulations using a MTB antigen in mice.Figure 21 b: Antibody responses (total lgG2c results - EC50 titers) induced by vaccines comprising saponin fraction DV-containing adjuvant formulations as compared with QS21 - containing adjuvant formulations using a MTB antigen in mice.Figure 22: T cell responses induced by vaccines comprising saponin fraction DV-containing adjuvant formulations as compared with QS21 -containing adjuvant formulations using Plasmodium falciparum MSP-1 antigen in mice.Figure 23a: Antibody responses in mice induced by vaccines comprising saponin fraction DV- containing adjuvant formulations compared with QS21 -containing adjuvant formulations using a malaria antigen.Figure 23b: Antibody responses in mice induced by vaccines comprising saponin fraction DV- containing adjuvant formulations compared with QS21 -containing adjuvant formulations using a malaria antigen.Figure 24: T cell responses induced by vaccines comprising saponin fraction DV-containing adjuvant formulations as compared with vaccines comprising QS21 -containing adjuvant formulations using varicella-zoster gE antigen in mice.Figure 25a: Antibody responses (total IgG EC50 titers) induced by vaccines comprising saponin fraction DV-containing adjuvant formulations as compared with QS21 -containing adjuvant formulations using a varicella-zoster antigen in mice.Figure 25b: Antibody responses (total lgG2c EC50 titers) induced by vaccines comprising saponin fraction DV-containing adjuvant formulations as compared with QS21 -containing adjuvant formulations using a varicella-zoster antigen in mice.Figure 26: CLS adjuvant formulation containing saponin fraction DV.Figure 27: CLS adjuvant formulation comprising pharmaceutical grade QS21 .Figure 28a: CD4+ T cell responses induced by saponin fraction DV-containing CLS adjuvant formulations as compared with QS21 -containing CLS adjuvant formulations using COVID-19 antigen in mice.Figure 28b: CD8+ T cell responses induced by saponin fraction DV-containing CLS adjuvant formulations as compared with QS21 -containing CLS adjuvant formulations using COVID-19 antigen in mice.Figure 29a: Antibody responses (total IgG EC50 titers) induced by saponin fraction DV- containing CLS adjuvant formulations as compared with QS21 -containing CLS adjuvant formulations using COVID-19 antigen in mice.Figure 29b: Antibody responses (total lgG2c EC50 titers) induced by saponin fraction DV- containing CLS adjuvant formulations as compared with QS21 -containing CLS adjuvant formulations using COVID-19 antigen in mice.Incorporation by reference

[0011] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.Detailed Description

[0012] For purposes of this application, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference.

[0013] All methods described herein can be performed in any suitable order of steps unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate specific embodiments and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the material presented herein.

[0014] All percentages, ratios and proportions herein are by weight, unless otherwise specified. A weight percent (weight %, also as wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the composition in which the component is included (e.g., on the total amount of the reaction mixture).

[0015] Terms used herein may be preceded and / or followed by a single dash, “ ”, or a double dash, “=”, to indicate the bond order of the bond between the named substituent and its parent moiety; a single dash indicates a single bond and a double dash indicates a double bond or a pair of single bonds in the case of a spiro-substituent. In the absence of a single or double dash it is understood that a single bond is formed between the substituent and its parent moiety; further, substituents are intended to be read “left to right” with reference to the chemical structure referred to unless a dash indicates otherwise. For example, arylalkyl, arylalkyl-, and alkylaryl indicate the same functionality.

[0016] For simplicity, chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, etc.). Nevertheless, such terms are also used to convey corresponding multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, while an “alkyl” moiety can refer to a monovalent radical (e.g. CH3-CH2-), in some circumstances a bivalent linking moiety can be “alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH2- CH2-), which is equivalent to the term “alkylene.” (Similarly, in circumstances in which adivalent moiety is required and is stated as being “aryl,” those skilled in the art will understand that the term “aryl” refers to the corresponding divalent moiety, arylene). All atoms are understood to have their normal number of valences for bond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 for S, depending on the oxidation state of the S). Nitrogens in the presently disclosed compounds can be hypervalent, e.g., an N-oxide or tetrasubstituted ammonium salt. On occasion a moiety may be defined, for example, as -B-(A)a, wherein a is 0 or 1 . In such instances, when a is 0 the moiety is -B and when a is 1 the moiety is -B-A.

[0017] As used herein, the term “alkyl” or “alkane” includes a saturated hydrocarbon having a designed number of carbon atoms, such as 1 to 40 carbons (i.e., inclusive of 1 and 40), 1 to 35 carbons, 1 to 25 carbons, 1 to 20 carbons, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18. Alkyl groups or alkanes may be straight or branched and depending on context, may be a monovalent radical or a divalent radical (i.e., an alkylene group). For example, the moiety “-(C1 C6 alkyl) O-” signifies connection of an oxygen through an alkylene bridge having from 1 to 6 carbons and C1 -C3 alkyl represents methyl, ethyl, and propyl moieties. Examples of “alkyl” include, for example, methyl, ethyl, propyl, isopropyl, butyl, iso, sec and tert butyl, pentyl, and hexyl. Examples of “alkane” include, for example, methane, ethane, propane, isopropane, butane, isobutane, sec-butane, tert-butane, pentane, hexane, heptane, and octane.

[0018] The term “substituted,” when used to modify a specified group or radical, means that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below, unless specified otherwise.

[0019] Many of the compositions described herein can be assimilated as a dilute solution, and as such, the person skilled in the art will appreciate that when describing the concentration ranges of the components, the “% wt / wt” and “mg / mL” can be considered to have the following conversions: 1% (wt / wt) = 10 mg / mL; 1 mg / mL = 0.1 % (wt / wt).

[0020] The terms "substantially" or "approximately" or “about”, as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.

[0021] The term “and / or” as used herein is intended to represent an inclusive “or”. Thewording X and / or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and / or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.

[0022] The term “non-naturally occurring” as used herein about a substance, refers to any substance that is not normally found in nature or natural biological systems. Non-naturally occurring compositions may include compositions completely or partially synthetized by human intervention and / or compositions prepared by human processes modifying a natural substance.

[0023] The term “comprise” and “include” as used throughout the specification and the accompanying claims as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0024] The articles "a" and "an" are used herein refers to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0025] Terms like “preferably”, “commonly”, “particularly”, and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0026] The term "in vivo”, as used herein refers to within a living cell or organism, including, for example an animal, a mammal, a primate or a human.

[0027] The term “in vitro”, as used herein refers to outside a living cell or organism, including, without limitation, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor and the like.

[0028] The term “isolated" as used herein about a composition, refers to any composition, which by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. “Isolated” or “purified” compositions include but are not limited to compositions of the invention for which the ratio of the compositions relative to other constituents with which they are associated in nature is increased. In an important embodiment the amount of the composition is increased relative to other constituents with which the composition is associated in nature (i.e. the novel saponin fraction DV of the current invention is at least partially purified from the harvested Q. saponaria raw materials). In an embodiment the composition of the invention may be isolated into a pure or substantially pure form. In this context a substantially pure compound means that the composition is separated from other extraneous or unwanted material present from the onsetof producing the compound or generated in the manufacturing process. Such a substantially pure composition of saponin fraction DV may contain less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the composition when found in nature. In an embodiment the isolated composition of saponin fraction DV is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100 % pure by weight.

[0029] As used herein and commonly used by those skilled in the art, the term “saponin” refers to triterpene glycosides that have a foaming quality when agitated in water. Natural sources of saponins include the Saponaria genus of flowering plants and the soapbark tree Quillaja Saponaria. QS21 , also known as QS-21 and QA21 , is a purified fraction from the Quillaja Saponaria Molina tree, one method of purification being disclosed (as QA21 ) in US patent No. 5,057,540. Quillaja saponin has also been disclosed as an adjuvant by Scott et al, Int. Archs. Allergy AppL Immun., 1985, 77, 409.

[0030] For the purpose of the disclosure, a “saponin fraction” means one or more saponin molecules grouped together by a degree of co-purification. In the case of the saponin fractions mentioned herein, the saponin fraction means a group of saponin molecules at least partially purified from a natural or synthetic mixture of saponins. Such a saponin fraction may be characterized by groups of peaks (including overlapping peaks) as seen using, for example, UV-HPLC. Saponin fractions may undergo further purification to generate one or more subtractions.

[0031] When referring to features of a saponin fraction, it is common practice in the art to refer to the presence or absence of compounds identified or detectable using mass spectroscopy in negative ion mode. By the term ‘m / z’ is meant the mass to charge ratio of the monoisotope peak. Unless otherwise specified, ‘m / z’ is determined by negative ion electrospray mass spectrometry. It is appreciated by those skilled in the art that one or more compounds identified by their presence at a specific m / z value in negative ion mode may interchangeably be referred to as the same value +1 when not using negative ion mode (i.e. when accounting for the mass of the associated ion as well as the compound itself). For example, a compound detectable using mass spectroscopy at 1856 m / z in negative ion mode may alternatively be referred to as compound 1857. As is understood by a person skilled in this art, the current precision of labelling a peak in a spectra obtained through typical methods of mass detection andidentification by software, may in some instances result in the labeling of a spectroscopic peak that is +1-2 m / z from the actual m / z of the compound. As used herein and exemplified here with the arbitrary reading “XYZ”, the term “no compound at XYZ m / z detectable using mass spectroscopy”, as is understood by those skilled in this art, means that there is no detectable compound at XYZ m / z in a mass spectroscopy analysis, i.e. the absorbance value at XYZ m / z is indistinguishable from background levels of absorbance in the sample.

[0032] In embodiments of the current invention, the novel saponin fraction (referred to herein as “saponin fraction DV”) is suitable for use in adjuvants and vaccines is an immunologically active saponin composition extracted from Quillaja saponaria Molina comprising a major peak, at between approximately 50% and approximately 80% of the total composition measured using UHPLC with UV absorbance at 214 nm, comprising one or more compounds detectable at 1988 m / z using mass spectroscopy in negative ion mode, and one or more compounds corresponding to a peak at 1856 m / z detectable using mass spectroscopy in negative ion mode. In some embodiments, saponin fraction DV further comprises one or more compounds corresponding to a peak at 1726 m / z, 1856 m / z, 2030 m / z, and / or 2003 m / z detected using mass spectroscopy in negative ion mode. In some embodiments, the saponin fraction DV has no compound at 2018 m / z and / or 2480 m / z detectable using mass spectroscopy in negative ion mode. UHPLC chromatographic analysis of samples of saponin fraction DV are provided in Figures 3, 4 and 5 herein.

[0033] The immunologically active compositions of the current invention may contain saponin fraction DV in substantially pure form, such that the saponin fraction DV is at least 80% pure, at least 85% pure, at least 90% pure, preferably at least 95% pure, more preferably at least 98% pure, and most preferably 100% pure. In some embodiments, only partially purified saponin fraction DV may be used. In other embodiments, the saponin fraction DV may be supplemented with additional saponins and / or with additional saponin fractions such as the Quil A extract of the bark of Quillaja Saponaria. Other non-limiting examples of immunologically active saponin fractions that may be added to saponin fraction DV and are useful in adjuvant or vaccine compositions include QS21 , QS7, QS18 (also known as QA18) and QS17 (also known as QA17). In further embodiments, the saponin fraction DV may be supplemented with saponins produced from recombinant microbes and / or triterpene saponin analogues such as those disclosed in WO17079582, WO18200645 or WO2023122801 A2, the contents of which are incorporated herein. In some adjuvants, saponin derivates that are immuno-stimulatory and surface active may be used. All saponins suitable for use in the compositions described herein are used in concentrations which are non-toxic to the patient or subject when appropriately formulated with one or more sterols. In some aspects thesaponin fraction DV, optionally with additional saponin and / or saponin fractions present, may be present in immunologically active adjuvants in the total saponin content range of 0.02 mg / mL to 0.4 mg / mL, 0.04 mg / mL to 0.2 mg / mL, 0.06 mg / mL to 0.2 mg / mL or 0.08 mg / mL to 0.1 mg / mL. Preferred concentrations of saponin fraction DV in bulk production of adjuvant formulation are between 2.0 mg / mL and 0.02mg / mL. Preferred concentrations of saponin fraction DV in diluted adjuvant formulations suitable for doses of medicament (such as vaccines) for administration to humans are between 0.4 mg / mL and 0.025 mg / mL, preferably between 0.05 and 0.2 mg / mL.

[0034] Saponin fraction DV is immunostimulatory, meaning it helps to activate or enhance an immune response and is therefore capable of acting as an adjuvant. As used herein, the term “adjuvant” means one or more substances (such as a saponin fraction comprising several saponin molecules) capable of increasing or modulating the immune response (i.e. is an “immunomodulator”) and is frequently used to increase the efficacy or potency of a vaccine comprising one or more antigens by accelerating, prolonging or enhancing the immune response to a specific antigen which may be administered before, after or simultaneously with the adjuvant. Without wishing to be bound by theory, some adjuvants are able to mimic specific pathogen-associated molecular patterns that immune systems have evolved to recognize as indicative of an infection, or otherwise to activate the physiological means of recognizing such pathogen-associated molecular patterns. In the case of the immuno-stimulant properties of saponin, innate immunity is stimulated following destabilization of the endosome and triggering of pro-inflammatory pathways leading to increases T and B cell responses. Thus, the presence of an adjuvant, when used in conjunction with one or more antigens of interest in a vaccine, can greatly increase the innate immune response of the subject or patient to the antigen by augmenting the activities of innate cells such as of dendritic cells and macrophages and promote the induction of potent cellular and humoral responses. Thus the compositions provided herein comprising saponin fraction DV are useful as an immunological adjuvant, and may be co-administered with antigens, such as in vaccines against bacterial, viral, fungal and parasitic diseases, or for immunotherapy treatment of cancer or allergies or other non- infectious diseases such as auto-immune diseases (e.g. multiple sclerosis, Lupus or Alzheimers disease). For example, the saponin fraction DV may be used in combination with any suitable antigen or vaccine component, e.g., an antigenic agent selected from the group consisting of antigens from pathogenic and non-pathogenic organisms, viruses, bacteria, parasites and fungi. As a further example, the saponin fraction DV may be used in combination with proteins, peptides, antigen-coding mRNA, antigens and vaccines which are pharmacologically active for disease states and conditions such as influenza, SARS-CoV-2,SARS-CoV-1 , rhinovirus, malaria, tuberculosis, HIV / AIDS, leishmaniasis, Group A streptococcus, respiratory syncytial virus (RSV), Middle East Respiratory Syndrome virus (MERS), hepatitis B, varicella zoster, coronavirus or human papilloma virus infection, or any combination thereof. In certain embodiments, the saponin fraction DV and the antigen are each present in an amount effective to elicit an immune response when administered to an animal, mammal, domesticated animal, farm animal, human, embryo, or ovum vaccinated therewith. In some aspects, the saponin fraction DV may be present in adjuvant compositions of the current invention suitable for administration to humans or large animals in the range of 0.001 mg / mL to 1.0 mg / mL, 0.005 mg / mL to 0.09 mg / mL, 0.09 mg / mL to 0.1 mg / mL, 0.04 mg / mL to 0.4 mg / mL, or 0.08 mg / mL to 0.8 mg / mL. In some aspects, the saponin fraction DV may be present in a composition formulated with antigen suitable for administration to humans at a concentration range of between 0.04 to 0.4 mg / mL. In some aspects, the saponin fraction DV may be present in a composition formulated with antigen suitable for administration to humans such that the saponin fraction DV is present in the range of between 10 to 100 pg, more preferably between 25 to 50 pg of saponin fraction DV per human dose.

[0035] As used herein, the term “adjuvant formulation” used interchangeably with “adjuvant formulated as”, refers to a composition comprising saponin fraction DV, buffers, and optionally other molecules including but not limited to any of additional adjuvants, excipients, stabilizers, antigens and antigen display vectors. Categories of adjuvant formulations are typically classified by the physico-chemical properties of the composition comprising the adjuvant, which are determined by the chemical properties of all components of the composition and the manner and order in which they are added to the developing adjuvant formulation during manufacture. By way of non-limiting examples, in some embodiments, the adjuvant composition comprising saponin fraction DV is formulated as a colloid suspension comprising saponin fraction DV, such as an emulsion (an emulsion-based adjuvant formulation), a micelle (a micelle-based adjuvant formulation), a liposome (a liposome-based adjuvant formulation), or any mixture thereof. Different antigens (and therefore different vaccines) are best formulated as liposomal or emulsion-based adjuvant formulations. In some embodiments, the adjuvant compositions comprising saponin fraction DV may be present in (i.e. formulated as) an oil-in-water emulsion, a suspension of liposomes, Immune simulating complexes (ISCOMs), polymer particles, polymersomes, inorganic particles, cage-like structures (CLS). In some embodiments, the adjuvant composition comprising saponin fraction DV is formulated as an emulsion (an emulsion-based adjuvant formulation). In other embodiments, the adjuvant compositions comprising saponin fraction DV is formulated as a liposome (a liposomal adjuvant formulation). In other embodiments, the adjuvant composition comprising saponinfraction DV is formulated as a micelle (a micellular adjuvant formulation). In other embodiments, the adjuvant composition comprising saponin fraction DV is formulated as a cage-like structure (CLS). In other embodiments, the adjuvant composition comprising saponin fraction DV comprises an aluminium salt. In other embodiments, the adjuvant composition comprising saponin fraction DV comprises a polymer nanoparticle.

[0036] As used herein with reference to emulsion-based adjuvants comprising saponin fraction DV, the term “emulsion” means a mixture of two or more liquids, at least two of which are immiscible owing to liquid-liquid phase separation, at least one present under the form of droplets, stabilized by at least one surfactant, dispersed within a continuous phase of one of the liquids. The emulsion can also contain a variety of additives, including but not limiting to stabilizers, antioxidants, sugars, salts or preservatives. In some embodiments, the adjuvant formulations comprising saponin fraction DV are oil in water emulsions, i.e. oil / lipid phase dispersed in an aqueous phase. In other embodiments, the emulsion-based adjuvant formulations comprising saponin fraction DV comprises one or more oils (such as squalene), saponin fraction DV, a sterol (such as cholesterol) and optionally or more additional immunomodulators, typically pathogen-associated molecular patterns (PAMPs) that are recognized by pattern recognition receptors (PRRs), including but not limited to Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-l-like receptors (RLRs), C-type lectin receptors (CLRs) and cytosolic dsDNA sensors (CDSs). In some aspects, the one or more additional immunomodulators to be formulated or otherwise used with saponin fraction DV may be a TLR ligand (2, 3, 4, 5, 6, 7, 8, 9), a NOD ligand, an RLR ligand, a CLR ligand, an inflammasome inducer, a STING ligand, a MINCLE agonist a TLR4 agonist, distributed within an aqueous phase (such as a buffer solution). In some aspects the lipid, such as an oil, may be present in the immunologically active oil in water emulsion (o / w emulsion-based adjuvant formulation) comprising saponin fraction DV in the range of 0.05 - 5% wt / wt (0.5 - 50 mg / mL), or 2 - 4% wt / wt (20 - 40 mg / mL). In some aspects the lipid (oil) is squalene present in the immunologically active oil in water emulsion (o / w emulsion-based adjuvant formulation) comprising saponin fraction DV in the range of 0.05 - 5% wt / wt (0.5 - 50 mg / mL), or 2 - 4% wt / wt (20 - 40 mg / mL). In some aspects, the lipid is solid at room temperature and therefore the emulsion may be referred to as solid lipid nanoparticles (SLN).

[0037] In some embodiments, the saponin fraction DV may be present in an oil-in-water emulsion (i.e. as an emulsion-based adjuvant). In some aspects, the saponin fraction DV may be present in a composition formulated with antigen suitable for administration to humans at a concentration range of between 0.04 to 0.4 mg / mL. In some aspects, the saponin fraction DV may be present in a composition formulated with antigen suitable for administration tohumans such that the saponin fraction DV is present in the range of between 10 to 100 pg, more preferably between 25 to 50 pg of saponin fraction DV per human dose.

[0038] As used herein, the term “micelle” means an immunologically active adjuvant formulation comprising an aggregate of surfactant molecules suspended in an aqueous solution, such that the hydrophilic regions of the surfactant are in contact with the aqueous solution and the hydrophobic regions of the surfactant are sequestered (clustered) towards the center of the micelle. Micelles comprising saponin fraction DV may be approximately spherical, cylindrical or ellipsoid in shape, with populations of micelles often comprising examples of each structure but having a distinct bias towards one type as a result of their precise composition and method of manufacture.

[0039] As used herein, the term “liposome” or “liposome-based adjuvant formulation” means an immunologically active adjuvant formulation comprising at least one lipid bilayer. In some aspects of the current disclosure relating to adjuvant formulations comprising saponin fraction DV, the liposomes may be unilamellar (comprised of one lipid bilayer), multilamellar (comprised of several lamellar phase lipid bilayers), cochleate or mixtures of any of these. The liposomal adjuvant formulations comprising saponin fraction DV may contain an amphipathic lipid which is a neutral lipid, for example phosphatidylcholine, which is preferably noncrystalline at room temperature, for example egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine or dilauryl phosphatidylcholine. In preferred embodiments, the liposomes comprising saponin fraction DV may also contain a charged lipid which increases the stability of the liposome-saponin structure for liposomes composed of saturated lipids. In these cases the amount of charged lipid is preferably 1 -20% w / w, most preferably 5-10% w / w. In some embodiments, the liposomes comprising saponin fraction DV may also contain a PEGylated lipid (a lipid comprising repetitive PEG moieties), a targeting lipid (a lipid comprising a moiety that allows a targeted delivery to a specific cell type and / or tissues) or any other lipid commonly used to prepare liposome formulations. In some embodiments, the liposomes comprising saponin fraction DV may also contain a sterol. In some embodiments, the ratio of sterol to phospholipid is 1 -50% (mol / mol), most preferably 20-40% (mol / mol). In some aspects of liposomal-based adjuvants disclosed herein comprising saponin fraction DV, the lipid may be present in the immunologically active liposome in water composition comprising saponin fraction DV may be in the range of 0.004 - 1 % wt / wt (0.04 - 10 mg / mL), or 0.1 - 0.8% wt / wt (1 - 8 mg / mL). In some aspects of liposomal-based adjuvants disclosed herein comprising saponin fraction DV, the lipid is DOPC (dioleoyl phosphatidylcholine) present in the immunologically active liposome in water composition comprising saponin fraction DV in the range of 0.004 - 1 % wt / wt (0.04 - 10 mg / mL), or 0.1 - 0.8% wt / wt (1 - 8 mg / mL).

[0040] In some embodiments, the saponin fraction DV may be present in a liposome (i.e. as a liposome-based adjuvant). In some aspects, the saponin fraction DV may be present in a liposomal composition formulated with antigen suitable for administration to humans or animals at a concentration range of 0.04 to 0.4 mg / mL. In some aspects, the saponin fraction DV may be present in a liposomal composition formulated with antigen suitable for administration to humans such that the saponin fraction DV is present in the range of 1 to 100 pg of saponin fraction DV per dose, preferably 10 to 100 pg of saponin fraction DV per dose, more preferably between 25 to 50 pg of saponin fraction DV per human dose. Additional components of the liposomal adjuvants (also referred to interchangeably as “liposome-based adjuvants”) disclosed herein containing the saponin fraction DV include: charged lipid, PEGylated lipid, targeting lipid or any other lipid commonly used to prepare liposome formulations. The liposomes of the liposome-based adjuvants disclosed herein may have an average diameter of less than 200nm or from 20 to 180 nm. The average (mean) diameter of lipid-based nanoparticles can be determined with dynamic light scattering (DLS), which is a technique commonly used by those skilled in art (such as taught in: Falke S., Betzel C; (2019) Dynamic Light Scattering (DLS); Pereira A., Tavares P., Limao-Vieira P. (eds) Radiation in Bioanalysis. Bioanalysis (Advanced Materials, Methods, and Devices), vol 8. Springer, Cham.; Chu B, Laser light scattering: Basic principles and practice. Academic Press, Boston, Second Edition, 1991 , 343 pp; and Brown W., ed. Dynamic light scattering, the method and some applications. Clarendon, Oxford, 1993. 735 pp.). The saponin fraction DV may be present in an aqueous solution. Thus, the formulations of the adjuvants or vaccine compositions can comprise both hydrophilic and hydrophobic components.

[0041] In some embodiments, the saponin fraction DV may be present in a cage-like structure (i.e. as a “CLS” adjuvant formulation) otherwise known as cage-like particles. As used herein, the term “cage-like structure” (CLS) or “cage-like particle” adjuvant formulation means an adjuvant formulation in the form of open cage-like structures comprising one or more sterol, one or more phospholipid, and a saponin or saponin fraction (such as saponin fraction DV). The term “immune stimulating complex” (“ISCOM” from ImmuniStimuatingCOMplex) is a CLS with immunostimulating properties. The immunological activity of saponin fraction DV means that a CLS comprising saponin fraction DV may also be referred to as an ISCOM. The typical size of a CLS is 30-40nm, but may vary from 10nm to 100nm in diameter. In some aspects, the CLS or ISCOM comprising saponin fraction DV comprises saponin fraction DV in a concentration range of 0.001 mg / mL to 1 .0 mg / mL, 0.005 mg / mL to 0.09 mg / mL, 0.09 mg / mL to 0.1 mg / mL, 0.04 mg / mL to 0.4 mg / mL, or 0.08 mg / mL to 0.8 mg / mL. In some aspects, the CLS or ISCOM comprising saponin fraction DV comprises a sterol, such as cholesterol, [3-sitosterol, stigmasterol, ergosterol, ergocalciferol, and / or campesterol, in a concentration range of 0.0002 mg / mL to 0.2 mg / mL, 0.001 mg / mL to 0.018 mg / mL, 0.018 mg / mL to 0.02 mg / mL, 0.008 mg / mL to 0.08 mg / mL or 0.016 mg / mL to 0.16 mg / mL. In some aspects, the CLS or ISCOM comprising saponin fraction DV comprises a phospholipid, such as phosphatidylcholine (PE), DPPC, DLPC, DMPC, DPPC, DSPC, DOPC, DEPE, DMPE, DPPE, DOPE, DMPE. Na, DPP.Na, DOPA.Na, DMPG.Na, DOPG.Na, DMPS.Na, DAPC, DEPC, LPC, LPE, PC, PE, and / or other phospholipids listed Table 2, in a concentration range of 0.04 to 10 mg / mL, 0.0002 mg / mL to 0.2 mg / mL, 0.001 mg / mL to 0.018 mg / mL, 0.018 mg / mL to 0.02 mg / mL, 0.008 mg / mL to 0.08 mg / mL or 0.016 mg / mL to 0.16 mg / mL. In preferred embodiments, the saponin (such as saponinf fraction DV) to sterol (such as cholesterol) ratio is approximately 1 :1 .

[0042] Further adjuvant composition classes suitable for inclusion of saponin fraction DV include ISCOMs, polymer particles, polymersomes, inorganic particles and virus like particles.

[0043] In some embodiments, the adjuvant compositions comprising saponin fraction DV may comprise one or more oils. In some aspects the one or more oils are preferably present in bulk emulsion adjuvant compositions comprising saponin fraction DV in the range of between 10mg / mL and 100mg / mL. In some embodiments, the adjuvants or vaccine compositions may include oil produced from an animal, fish, vegetable, tree, and / or microbe (including recombinant bacteria, yeast or fungi engineered to produce such oils). For example, nuts, beans, seeds and grains are typically lipid-rich sources, and give rise to such common oils as safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, peanut oil, soybean oil, coconut oil, olive oil, jojoba oil, corn oil, wheat oil, oat oil, rye oil, rice oil, teff oil, triticale oil and the like may also be used. Further non-limiting examples of oils (lipids) suitable for use in adjuvants comprising saponin fraction DV of the current disclosure are provided in Table 1 .Table 1 : oils (lipids) suitable for use in adjuvants, including but not limited to those based on emulsions and liposomes comprising saponin fraction DV of the current disclosure.

[0044] Alternative oils suitable for inclusion in adjuvants and adjuvant formulations comprising saponin fraction DV, include Long Chain Triglycerides (LCT).

[0045] Fats and oils from mammalian milk (actually biocompatible lipids) may be used in the practice of aspects of this disclosure. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art. Most fish, whales and sharks contain oils which may be readily recovered and suitable for use herein, including but not limited to cod liver oils, shark liver oils (such as squalene), and whale oils such as spermaceti. In some aspects of the current disclosure, terpenoids (branched chain oils composed of 5-carbon isoprene units), such as squalene, are the oil. Squalene is a colorless triterpene with formula (C5H8)6, originally isolated from shark liver oil, though all plants and animals produce squalene as a biochemical intermediate, being a precursor of such materials as sterols and steroids.Squalene.

[0046] Squalene and squalane are available from commercial sources at high purity levels suitable for pharmaceutical use. As a non-limiting example, one suitable method of preparing squalene by purification distillation is taught in US20160051670A1 . Environmental concerns over shark hunting for squalene harvesting have motivated the production of squalene fromrecombinant microbes, non-limiting methods and biosynthetic pathways of which are taught in for example Spanova and Gunther, 201 1 (“Squalene - biochemistry, molecular biology, process biotechnology, and applications”. European Journal of Lipid Science and Technology. 1 13 (11 ): 1299-1320. Doi:10.1002 / ejlt.201 100203) and Pan et al. 2015 (“Biosynthesis of Squalene from Farnesyl Diphosphate in Bacteria: Three Steps Catalysed by Three Enzymes”. ACS Central Science. 1 (2): 77-82. Doi:10.1021 / acscentsci.5b001 15. PMID 26258173). Squalane, the saturated analog to squalene, can also be used. It should be clear to one skilled in the art that synthetic derivatives of such lipids may also be suitable, such as the 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol, which although they do not naturally occur in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate nut and seed oils. The adjuvants comprising saponin fraction DV, and the resulting formulated vaccines of the current disclosure may comprise a mixture or blend of the lipids described herein.

[0047] As used herein with reference to adjuvant formulations comprising saponin fraction DV and as understood by a person skilled in the art, the term “surfactant” (a well-recognized abbreviation of “surface-active agent”) relates to compounds that are able to lower the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. This ability of surfactants makes them useful as emulsifiers, foaming agents, detergents, wetting agents and dispersants. Surfactants are typically able to do this by virtue of their amphiphilic nature, i.e. at least part of the surfactant molecule is hydrophilic, permitting interaction with polar solvents such as water, and at least part of the surfactant is hydrophobic, permitting interaction with non-polar solvents. Surfactants especially suitable for use in the adjuvant formulations comprising saponin fraction DV are biocompatible with the patient or subject into which the adjuvant or vaccine formulation will be introduced. Surfactants may be classified by their relative interactions with the hydrophilic and hydrophobic phases, which is described by those skilled in the art by the hydrophile / lipophile balance (“HLB”) value of the surfactant. In some embodiments, the adjuvant or vaccine compositions comprising saponin fraction DV are formulated as emulsions comprising lipid droplets coated with at least one surfactant or a combination of surfactants.

[0048] As used herein with reference to adjuvant formulations comprising saponin fraction DV, the term “hydrophilic surfactant” refers to an aqueous phase surfactant, including but not limited to those with an HLB value between 8 and 18. Hydrophilic surfactants suitable for use in some embodiments of adjuvant formulations comprising saponin fraction DV have an HLB of at least 10, preferably at least 15, or at least 16. Hydrophilic surfactants suitable for use in some embodiments of adjuvant formulations comprising saponin fraction DV include, but arenot limited to, one or more of polyoxyethylene sorbitan esters (commonly referred to as the Tweens), alkyl sulfate (e.g., sodium lauryl sulfate), polyoxyethylene lauryl ether, polyoxyethylene monostearate, fatty acid polyethyoxylate, ethoxylated cetyl / stearyl alcohol, polyoxyl lauryl ether, glycolipids or ‘green surfactants’ including but not limited to mono-, di-, tri- or poly-lipidated derivatives of sugar alcohols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, or blends comprising one or more thereof. Further examples of Tweens suitable for use in adjuvant formulations comprising saponin fraction DV include polyoxyethylenesorbitan monooleate (e.g., polysorbate 80, TWEEN 80), polyoxyethylenesorbitan monolaurate (polysorbate 20, TWEEN 20). In some aspects, the hydrophilic surfactant may be present in adjuvant formulations comprising saponin fraction DV in the range of 0.05% to 10% wt / wt (0.5 - 100 mg / mL), 0.1% to 8.5% wt / wt (1 - 85 mg / mL), 0.5% to 7% wt / wt (5 - 70 mg / mL) or 0.5% to 5% wt / wt (5 - 50 mg / mL).

[0049] As used herein with reference to adjuvant formulations comprising saponin fraction DV, the term “hydrophobic surfactant” (otherwise known as lipophilic surfactants) refers to a lipid phase surfactant, including but not limited to those with an HLB value between 3 and 6. Hydrophobic surfactants suitable for use in some embodiments of the adjuvant or vaccine compositions comprising saponin fraction DV have an HLB of less than 6, preferably of less than 4, and more preferably less than 3. Hydrophobic surfactants suitable for use in some embodiments of the adjuvant formulations comprising saponin fraction DV are one or more sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (commonly known as Span 85), sorbitan monolaurate (e.g., Span 60), or blends comprising one or more thereof. Nonionic surfactants suitable for use in some embodiments of the adjuvant formulations comprising saponin fraction DV include ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear or branched EO / PO block copolymers sold under the trade name TERGITOL™. Nonionic surfactants suitable for use in some embodiments of the adjuvant formulations comprising saponin fraction DV include glycolipids or ‘green surfactants’ including but not limited to mono-, di-, tri- or poly- lipidated derivatives of sugar alcohols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Nonionic surfactants suitable for use in some embodiments of the adjuvant formulations comprising saponin fraction DV include octoxynols, which can vary in the number of repeating ethoxy (oxy-1 ,2-ethanediyl) groups, such as octoxynol-9 (Triton X- 100, or t-octylphenoxypolyethoxyethanol). In some aspects the hydrophobic surfactant may be present in the adjuvant formulations comprising saponin fraction DV in the range of 0.05% to 10% wt / wt (0.5 - 100 mg / mL), 0.1 % to 8.5% wt / wt (1 - 85 mg / mL), 0.5% to 7% wt / wt (5 - 70 mg / mL) or 0.5% to 5% wt / wt (5 - 50 mg / mL).

[0050] In some embodiments, the adjuvant formulations comprising saponin fraction DV may contain a phospholipid. As used herein, the term “phospholipid” refers to any lipid comprising a phosphate group, including but not limited to phospholipids, glycerophospholipids or a mixture thereof. Phospholipids suitable for use in some embodiments of the adjuvant formulations comprising saponin fraction DV include unmodified phospholipids and glycerophospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and blends thereof such as commercially available lecithins, such as those derived from soybean oil, sunflower oil, etc.

[0051] In some aspects the one or more phospholipids (such as a phospholipid or glycerophospholipid) are preferably present in bulk liposomal adjuvant formulations comprising saponin fraction DV in the range of between 1 mg / mL and 100mg / mL, and in bulk liposomal adjuvant compositions comprising saponin fraction DV in the range of between 10g / mL and 40mg / mL. In some aspects the one or more phospholipids or amphipathic lipids (such as a phospholipid or glycerophospholipid) may be present in the adjuvant comprising saponin fraction DV in the range of 0.004 - 1 % wt / wt (0.04 - 10 mg / mL), or 0.1 - 0.8% wt / wt (1 - 8 mg / mL). In some aspects the phospholipid is DOPC present in the adjuvant comprising saponin fraction DV in the range of 0.004 - 1 % wt / wt (0.04 - 10 mg / mL), or 0.1 - 0.8% wt / wt (1 - 8 mg / mL).

[0052] Non-limiting examples of phospholipids suitable for use in adjuvant formulations, such as emulsions or liposomes, comprising saponin fraction DV, are provided in Table 2.Table 2: - phospholipids (also amphipathic lipids) suitable for use in aspects of the current disclosure, including but not limited to use in liposome or CLS adjuvant formulations comprising saponin fraction DV.

[0053] Mixtures of surfactants may be used in aspects of the current invention, including but not limited to Tween 80 / Span 85 mixtures, or Tween 80 / Triton-X100 mixtures. For example, in some aspects a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is suitable for use in some embodiments of the adjuvant formulations comprising saponin fraction DV. In another aspect, the surfactants include Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. In another aspect, a mixture of surfactants may comprise laureth 9 plus a polyoxyethylenesorbitan ester and / or an octoxynol. Possible amounts of surfactants (% by weight) suitable for use in some embodiments of the adjuvant formulations comprising saponin fraction DV are: 0.05% to 10% wt / wt (0.5 - 100 mg / mL), 0.1 % to 8.5% wt / wt (1 - 85 mg / mL), 0.5% to 7% wt / wt (5 - 70 mg / mL) or 0.5% to 5% wt / wt (5 - 50 mg / mL).

[0054] In preferred embodiments, the adjuvant formulations comprising saponin fraction DV further comprise a sterol. As used herein with reference to adjuvant formulations comprising saponin fraction DV, and as commonly used by those skilled in the art, the term “sterol” refers to an organic alcohol that contains at least a sterane moiety. The sterol serves to diminish the cellular lytic activity of saponins, therefore reducing the reactogenicity of a saponin-containing adjuvant formulation, such as those disclosed herein comprising DV saponin fraction. In some aspects, the sterol may be present in the adjuvant formulation in the range of 0.02 mg / mL - 50 mg / mL, 0.04 mg / mL - 25 mg / mL, 0.08 mg / mL - 50 mg / mL, or 0.1 mg / mL to 1 mg / mL. The sterol may be present in the adjuvant formulation comprising saponin fraction DV to have a sterol / saponin ratio (weight / weight) at least s 1. Sterols can be purified and isolated from most eukaryotes, including plants (phytosterols, such as campesterol, sitosterol, stigmasterol), animals (zoosterols, such as cholesterol), fungi (such as ergosterol), and some bacteria. Suitable sterols for use in some embodiments of adjuvant formulations comprising saponin fraction DV include but are not limited to p-sitosterol, stigmasterol, ergosterol, ergocalciferol, cholesterol, anionic cholesterols (such as deoxycholate and other bile acids) and cationic cholesterols (such as DC-cholesterol and DOPC-cationics). Further non-limiting examples of sterols suitable for use in some embodiments of adjuvant formulations comprising saponin fraction DV are provided in Table 3.Table 3: sterols suitable for use in adjuvant formulations based on emulsions and liposomes of the current disclosure comprising saponin fraction DV.

[0055] Sterols are well characterized. For example, cholesterol is disclosed in the Merck Index, 1 1th Edn., page 341 , as a naturally occurring sterol found in animal fat, but is also available from plant-derived synthetic production (e.g. not limiting Phytochol®, Botanichol®,Synthechol®). In some embodiments of the adjuvant formulations comprising saponin fraction DV, cholesterol or sitosterol is the sterol. Cholesterol is the principal sterol synthesized by all animals and has formula C27H46O.Cholesterol

[0056] As used herein, adjuvants comprising saponin fraction DV are formulated using water or a water-based solution (typically an aqueous buffer). In aspects of the current disclosure, the aqueous component can be distilled water or can include further components e.g. solutes. In some embodiments, the water-based solution includes salts to form an aqueous buffer e.g. citrate or phosphate salts, such as sodium salts. Suitable aqueous solutions for use in the current disclosure include pharmaceutically acceptable buffers with a pH between 4 and 9 including, but not limited to phosphate, citrate, acetate, Tris, borate, histidine, lactate, tromethamine, gluconate, aspartate, tartrate, succinate, maleate, and / or fumarate buffers. In some embodiments, suitable pharmaceutically acceptable aqueous buffers include one or more of: a phosphate buffer (e.g., PBS buffer); a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. In some embodiments, pharmaceutically acceptable aqueous buffer strengths are within the 5-20 mM range, the 8 to 17mM range or the 10 to 15mM range. In some embodiments, the one or more pharmaceutically acceptable aqueous buffer has a typical pH within the pH 4-9 range, the 5-8 range, or the 6-7 range. In some embodiments, the pH range of the aqueous buffer in the composition comprising saponin fraction DV is pH 6.5-7 range, pH 5.5-7 range, pH 6-6.5 range or pH 5.5-6. The composition may include a preservative such as thiomersal or 2-phenoxyethanoL

[0057] In some embodiments, the adjuvant formulations or vaccine compositions described herein comprising saponin fraction DV may contain antioxidant vitamins and / or minerals such as B carotene, vitamin A, vitamin C, vitamin D, vitamin E, co-enzyme Q10, sulfur based antioxidants like glutathione, lipoic acid, metal chelators like citrate, EDTA, EGTA, selenium or combinations thereof.

[0058] The adjuvant formulations or vaccine compositions described herein comprisingsaponin fraction DV may additionally comprise excipients, including but not limited to one or more of, a sugar, such as sucrose, sorbitol, salts, such as sodium chloride, and / or proteins such as gelatin. In some embodiments an excipient such as gelatin, may be added to adjust the viscosity or stability, such as protection against temperature fluctuations during transport and / or storage, of the final adjuvant or vaccine.

[0059] The adjuvant formulations or vaccine compositions described herein comprising saponin fraction DV can be used to treat a patient or subject suffering from a variety of diseases. The term “treatment” refers to medical treatments including but not limited to preventative treatments and prophylactic treatments to reduce the severity or duration of a medical condition (such as an infectious disease) in an individual subject, patient, patient group, cohort or population. The term “disease” as used interchangeably with “medical indication” herein refers to a medical condition resulting from a pathogenic infection, a transmissible disease, an acquired disease, an infectious disease, a foodborne disease, an airborne disease, or an internal dysfunction such as a cancer, allergies, autoimmune diseases and neurodegenerative diseases. For the avoidance of doubt, “disease” includes pre-disease states or subclinical conditions, such as pre-cancer identified by abnormal structure or function of cellular tissues. Non-limiting examples of target diseases in some aspects of the current disclosure include diseases such as influenza, SARS-CoV-2, SARS-CoV-1 , rhinovirus, malaria, tuberculosis, HIV / AIDS, leishmaniasis, Group A streptococcus, respiratory syncytial virus (RSV), Middle East Respiratory Syndrome virus (MERS), hepatitis B, varicella zoster, coronavirus or human papilloma virus infection. In some aspects of the current disclosure, use of a vaccine comprising the adjuvant formulations disclosed herein comprising saponin fraction DV may be performed as a prophylactic treatment, a vaccination program, as a means of reducing the ability of the patient or subject to act as a vector for spread of a transmissible disease, or as a therapeutic treatment means of reducing the severity of prognosis or symptoms of an existing disease.

[0060] The term “patient” or “subject” as used herein may refer to a target group (i.e. a plurality of patients or subjects) of vertebrates (animals, such as a domesticated animal, farm animal, mammal, primate, fowl or fish), species (such as a human, pig, cow, horse, dog, cat) and / or a specific vaccine recipient, patient, subject, population or sub-population or population cohort considered to be at high risk of contracting or transmitting a disease or having been diagnosed with having the disease or suffering from a high disease burden measured by mortality, morbidity, financial cost, days of work lost, and so on. In some aspects of the current disclosure, the patient or subject may be a human considered to be at risk of contracting a transmissible disease, such as influenza, SARS-CoV-2, SARS-CoV-1 , rhinovirus, malaria,tuberculosis, HIV / AIDS, leishmaniasis, Group A streptococcus, respiratory syncytial virus (RSV), Middle East Respiratory Syndrome virus (MERS), hepatitis B, varicella zoster, coronavirus or human papilloma virus infection.

[0061] As used herein, the term “immunologically active” refers to the ability of a molecule to activate or enhance an immune response. For example, an antigen or molecule comprising an antigenic component may be described herein as immunologically active because it stimulates a patient or subject immune system and activates an immune response (i.e. is “immunogenic”). The adjuvant or vaccine compositions described herein may contain an antigen. As used herein, the term “antigen” is used interchangeably with “molecule comprising an antigen”, and is recognized by one skilled in the art to mean a molecule or molecular structure associated with an infection or disease condition, such as an antigen natively present on or in a pathogen, that can be bound by an antigen-specific antibody, antigen-binding antibody fragment or B-cell antigen receptor. Antigens may be proteins, peptides and / or polysaccharides, of which a great many are known in the art. The presence of non-self (“foreign”) antigens in the body of a patient or subject vertebrate, such as a mammal or human, normally triggers an immune response. However, “self” antigens associated with auto-immune diseases (such as multiple sclerosis, Lupus or Alzheimer’s) and cancers, may be employed in some methods of treatment of those diseases and often require a large adjuvant effect. Antigens may also be present on antigen carriers, such as virosomes or virus like particles (VLPs). In some embodiments, the adjuvant formulation comprising saponin fraction DV may be used to formulate an antigen and preferably increase one or more desirable features of a vaccine composition, wherein the vaccine composition is an inactivated vaccine, a live attenuated vaccine, a toxoid, a subunit vaccine, a recombinant vaccine, a polysaccharide vaccine, a conjugate vaccine, or a recombinant vector vaccine. In other embodiments of the current disclosure, the adjuvant formulation comprising saponin fraction DV may comprise a carrier for a subunit antigen, a toxoid antigen, a polysaccharide antigen, a soluble antigen or a recombinantly produced antigen.

[0062] The adjuvant formulations and vaccine compositions described herein comprising saponin fraction DV may contain an “antigen carrier” (otherwise known as a “platform”) such as nanoparticles, keyhole limpet hemocyanin (KLH), CRM197 a genetically detoxified form of diphtheria toxin, rotavirus VP6 or combinations thereof, influenza virus derived virosomes, hepatitis B surface antigen virus like particles, or other platforms presenting a repetitive array of the same or diverse antigens (multivalent antigens) on their surface.

[0063] Inactivated vaccines are composed of micro-organisms that have been killed with chemicals and / or heat and are no longer infectious. Examples are vaccines against influenza,cholera, plague, and hepatitis A. Most vaccines of this type are likely to require booster shots, i.e. subsequent or repeated inoculations.

[0064] Live, attenuated vaccines are composed of micro-organisms that have been cultivated under conditions which disable their ability to induce disease. These responses are more durable, however, they may require booster shots. Examples include yellow fever, measles, rubella, and mumps.

[0065] Toxoids are inactivated toxic compounds from micro-organisms in cases where these (rather than the micro-organism itself) cause illness, used prior to an encounter with the toxin of the micro-organism. Examples of toxoid-based vaccines include tetanus and diphtheria.

[0066] Subunit, recombinant, polysaccharide, and conjugate vaccines are composed of fragments or sequences from a pathogenic (disease-causing) organism. A characteristic example is the subunit vaccine against Hepatitis B virus.

[0067] Current work in this field is pioneering treatment (including pretreatment) technologies such as polynucleotide vaccines, which comprise polynucleotides (such as mRNA / DNA / tRNA / XNA) encoding antigens associated with the pathogen or disease. These vaccines are inexpensive and generate a strong immunity. A related group of vaccines are recombinant vector vaccines (platform-based vaccines) that are harmless recombinant viruses able to encode one or more antigens from a pathogenic organism. They are used widely in veterinary and human medicine.

[0068] The term "immune response” and “immunity” as used interchangeably herein refers to the adaptive immune system of vertebrates. In some aspects of the current disclosure, adjuvant formulations comprising saponin fraction DV and antigens derived from a disease (a vaccine) may be intentionally administered to a recipient patient or subject to induce the memory function of the adaptive immune system towards antigens derived from the disease. Such “artificial” (man-mediated through the use of a vaccine) active inducement of the adaptive immune system of a vertebrate patient or subject is frequently referred to as immunization, and if performed on a population of patient or subjects, as an immunization campaign or program. Immunization confers a degree of protection, seen as mild or inapparent disease symptoms when the immunized patient or subject comes into contact with pathogens causing the disease. This resistance to the disease following immunization is therefore a function of the immunological memory and is the goal of most vaccinations. With reference to the antigen being delivered and forming the basis of immune recognition (and therefore optimally immunity), there are general classifications of vaccines broadly characterized by the manner in which antigens are present in the vaccine: inactivated vaccines, live attenuated vaccines, toxoids, a fourth group comprising subunit, recombinant, polysaccharide,polypeptide, conjugate vaccines, and recombinant vector vaccines.

[0069] Saponins (including but not limited to fraction DV, QuilA or QS21 ) also have stimulatory immune-modulator effects so may be described herein as “immunologically active”. In some aspects, adjuvant formulations comprising the saponin fraction DV may further comprise one or more additional immunomodulating (such as immune-stimulating) compounds. Non-limiting examples of immune-modulating compounds (’’immunomodulators”) suitable for use in adjuvant formulations comprising saponin fraction DV include pathogen-associated molecular patterns (PAMPs) that are recognized by pattern recognition receptors (PRRs): Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-l-like receptors (RLRs), C-type lectin receptors (CLRs) and cytosolic dsDNA sensors (CDSs). In some aspects, the one or more additional immunomodulators is selected from a TLR ligand (1 , 2, 3, 4, 5, 6, 7, 8, 9), a NOD ligand (NOD-1 and / or NOD-2 agonists), an RLR ligand, a CLR ligand, an inflammasome inducer, a STING ligand, a MINCLE agonist, Th17 agonists, and NKT cell activators.

[0070] It is an object of the current disclosure to provide adjuvant formulations enabling a substantial immune response (and thus effectiveness of vaccination) when used to deliver an antigen in an inoculation. It should be noted that some TLR agonists, such as TLR-4 agonists, when administered in combination with saponins as immune-stimulators, activate different pathways of immune-modulation and therefore have a synergistic rather than additive immune-stimulation effect. Therefore, some possible embodiments of the adjuvant formulations and vaccines comprising saponin fraction DV comprise an oil, a TLR4 agonist, a saponin and a sterol, dispersed in an aqueous buffer solution.Methods of producing saponin fraction DV

[0071] Saponin fraction DV may be extracted from any tissue of Quillaja saponaria selected from one or more of leaves, roots, buds, flowers, seeds, shoots, twigs, stems and trunks. In some preferred aspects, the tissue of Quillaja saponaria used as a raw material for saponin fraction DV extraction is the bark.

[0072] In some aspects of the current disclosure the saponin fraction DV is manufactured at small scale (up to 1 g) using a method comprising the steps of: (i) production of a concentrated extract of partially purified crude saponin extract by removal of non-saponin impurities from a highly purified saponin extract (content of Quillaja saponins equal or higher than 85% w / w on dry solids, preferably between 90 and 95% w / w) by adsorption on organic and / or inorganic adsorbents, by filtration, by selective precipitation with organic solvents, or by a combination thereof (preferably by treatment with an aliphatic alcohol followed by filtration, recovery of thefiltrate and concentration by solvent evaporation); (ii) first chromatographic fractionation wherein the concentrated extract of partially purified crude saponin resulting from step a) is fractionated by liquid chromatography (LC) using water, organic solvents or a combination thereof as eluents, either in isocratic or gradient elution mode. In addition, the mobile phase eluents might be adjusted with pH modifiers such as organic acids or organic bases, inorganic acids or inorganic bases or a combination thereof. The stationary phase employed in LC might be either hydrophobic or hydrophilic and column packed. LC separation is preferably performed by reverse phase High Performance Liquid Chromatography (HPLC) in isocratic elution mode using a mixture of acidified organic solvent and acidified water as eluant - preferably a mixture of methanol or acetonitrile and water, containing 0.01 to 0.5% v / v of an organic acid (0.05 to 0.3% of formic acid). The eluate is recovered as discrete fractions and selected eluates containing a major component peak of DV Fraction are then pooled together forming “Crude Saponin DV Fraction” and concentrated (such as by vacuum) and then freeze- dried to obtain a “Dry Saponin DV Fraction”, and; (iii) a second chromatographic fractionation wherein such intermediate “Dry Saponin DV Fraction” is dissolved in an aqueous solution of acetonitrile 10 to 50% v / v (preferably 20-40% v / v) and sub-fractionated by liquid chromatography (LC) using water and / or organic solvents as eluents, either in isocratic or gradient mode. In addition, the mobile phase eluents might be adjusted with pH modifiers such as organic and / or inorganic acids or bases. The stationary phase employed in LC may be either hydrophobic or hydrophilic and column packed. In some aspects, LC separation is preferably performed by reverse phase High Performance Liquid Chromatography (HPLC) in gradient elution mode using acidified organic solvent and acidified water as eluants - preferably methanol or acetonitrile and water, both containing 0.01 to 0.5% v / v of an organic acid (such as 0.05 to 0.3% of formic acid). The second eluate is recovered as discrete fractions and selected eluates containing a major component peak of DV Fraction are then pooled together forming the “Pool Saponin DV Fraction”, concentrated (such as by vacuum evaporation) to “Saponin Fraction DV Precursor” and then optionally freeze-dried to obtaining a “Saponin Fraction DV”.

[0073] In some aspects of the current disclosure the saponin fraction DV is manufactured at large scale (higher than 1 g) using a method comprising the steps of: (i) production of a partially purified saponin extract by removal of non-saponin impurities from a crude liquid extract (content of Quillaja saponins equal or higher than 85% w / w, but preferably between 90 and 95% w / w) by adsorption on organic and / or inorganic adsorbents, by filtration, by selective precipitation with organic solvents, or by a combination thereof (preferably by treatment with insoluble polysaccharide and an aliphatic alcohol followed by filtration and recovery of thefiltrate); (ii) first chromatographic fractionation wherein the concentrated extract of partially purified crude saponin extract resulting from step a) is fractionated by liquid chromatography (either high or low pressure LC) using water, organic solvents or a combination thereof as eluents, either in isocratic or gradient elution mode. In addition, the mobile phase eluents might be adjusted with pH modifiers such as organic acids or organic bases, inorganic acids or inorganic bases or a combination thereof. The stationary phase employed in LC might be either hydrophobic or hydrophilic and column packed. LC separation is preferably performed by normal phase purification in isocratic elution mode using a mixture of organic solvents and water acidified with an organic acid (preferably onto silica stationary phase eluted with a mixture of dichloromethane, methanol, water and acetic acid). The eluate is recovered as discrete fractions and selected eluates containing a major component peak of DV Fraction are then pooled together and dried forming “Enriched Saponin Fraction DV”; (iii) an adsorption step to remove impurities where such “Enriched Saponin Fraction DV” is dissolved in an aqueous solution of acetonitrile 10 to 50% v / v (preferably 20-40% v / v) acidified with 0.05- 0.40% v / v of acetic acid (preferably 0.1 -0.3% v / v), and cleaned by filtration chromatography using water and / or organic solvents as eluents rendering a “Filtered enriched Saponin Fraction DV” (preferably onto silica stationary phase eluted with an aqueous solution of acetonitrile 10 to 50% v / v (preferably 20-40% v / v) acidified with an organic acid (preferably acetic acid 0.05- 0.25% v / v), and; (iii) a final chromatographic fractionation wherein such intermediate Filtered enriched Saponin Fraction DV is sub-fractionated by liquid chromatography (LC) using water and / or organic solvents as eluents, either in isocratic or gradient mode. In addition, the mobile phase eluents might be adjusted with pH modifiers such as organic and / or inorganic acids or bases. The stationary phase employed in LC might be either hydrophobic or hydrophilic and column packed. LC separation is preferably performed by reverse phase low pressure Liquid Chromatography in gradient elution mode using acidified organic solvent and acidified water as eluants - preferably methanol or acetonitrile and water, both containing 0.01 to 0.5% v / v of an organic acid (0.1 to 0.3% of acetic acid). The eluate is recovered as discrete fractions and selected eluates containing a major component peak of DV Fraction are then pooled together forming the “Pooled further enriched Saponin Fraction DV” and concentrated by vacuum and then freeze-dried to obtain a “Saponin Fraction DV”.

[0074] In some aspects of the current invention, the novel saponin fraction (referred to herein as “saponin fraction DV”) is suitable for use in adjuvants and vaccines and is an immunologically active saponin composition extracted from Quillaja saponaria Molina comprising a major peak, at between approximately 50% and approximately 80% purity of the total composition measured using UHPLC with UV absorbance at 214 nm, comprising one ormore compounds detectable at 1988 m / z using mass spectroscopy in negative ion mode, and one or more compounds corresponding to a peak at 1856 m / z detectable using mass spectroscopy in negative ion mode. In some embodiments, saponin fraction DV further comprises one or more compounds corresponding to a peak at 1726 m / z, 1856 m / z, 2030 m / z, and / or 2003 m / z detected using mass spectroscopy in negative ion mode (preferably at most 80% of the saponin content represented by the combined amount of 1988 m / z and 1856 m / z compounds; at most 40% of the saponin content represented by 1856 m / z compound; at most 10% of the saponin content represented by 2003 m / z compound). In some embodiments, the saponin fraction DV has no compound at 2018 m / z and / or 2480 m / z detectable using mass spectroscopy in negative ion mode.Methods of producing the adjuvant formulations or vaccine compositions.

[0075] In some aspects of the current disclosure in which the adjuvant formulation comprises a saponin fraction DV stable oil-in-water emulsion manufactured using a method comprising the steps of: (i) preparation through rapid agitation (optionally by homogenization and / or microfluidization) or auto-emulsification of a first emulsion, also known as a preliminary emulsion or a pre-emulsion, having a first average lipid droplet size (optionally less than 5pm); (ii) further agitation (such as by fluidization, microfluidization, shaking, stirring, homogenizing, or exposure to power ultrasound / sonication) of the first emulsion to form a second emulsion having a second average lipid droplet size which is less than the first average lipid droplet size (preferably less than 220 nm); and (iii) nanofiltration, preferably by filter sterilization, of the second emulsion.

[0076] The pH of the adjuvant formulations and vaccine compositions disclosed herein comprising saponin fraction DV have a typical pH within the pH 4-9 range, generally between 5.0 and 8.1 , and more typically between 6.0 and 8.0 e.g. between 6.5 and 7.5. Methods of producing compositions as disclosed herein may therefore include a step of adjusting the pH of the adjuvant formulation or vaccine composition prior to packaging.

[0077] In some aspects of the current disclosure the adjuvant formulation comprises a saponin fraction DV stable liposome manufactured using any methods known to the skilled in this art suitable for production of liposomal adjuvant formulations, including but not limited to high shear homogenization, film hydration method, ethanol injection method, microfluidic mixing method, crossflow mixing method, impingement jet mixing method, reverse phase evaporation method, detergent removal method, membrane extrusion method.

[0078] In some embodiments, incorporation of an antigen may be performed either during or immediately after production of the adjuvant formulation comprising saponin fraction DV. Inother embodiments of the invention, larger volumes of adjuvant formulation comprising saponin fraction DV may be produced and stored (often referred to as “bulk adjuvant”), for subsequent incorporation of antigen into the formulation at a time of need, optionally followed by aliquoting into dosage volumes suitable for injection. In other embodiments, the adjuvant formulation comprising saponin fraction DV may be stored in separate receptacles to the antigen, permitting supply of a kit of parts comprising adjuvant formulation comprising saponin fraction DV and antigen, which may be extemporaneously mixed into the final injectable formulation (such as an injectable vaccine), for example in a local medical clinic or at the location of a subject to be treated or vaccinated. In certain aspects, such a kit of parts permits transportation of vaccine components that would otherwise be less stable if instead formulated as a single vaccine formulation.

[0079] In some embodiments, the antigen can be added to the adjuvant formulations disclosed herein comprising saponin fraction DV extemporaneously (at the time of administration of the vaccine compositions) or directly after the formation of the adjuvant formulations comprising saponin fraction DV, thereby forming a pre-mixed vaccine composition.

[0080] In some embodiments, a vaccine composition comprising saponin fraction DV may be prepared by combining antigen (present in some embodiments as a liquid solution or suspension of antigen in an aqueous buffer) with bulk adjuvant in a 4:1 to a 1 :4 ratio by volume. In some embodiments, a vaccine composition comprising saponin fraction DV may be prepared by combining antigen (present in some embodiments as a liquid solution or suspension of antigen in buffer) with bulk adjuvant in 1 :1 ratio by volume. For example, a typical human dose of 0.5mL may comprise 0.25mL adjuvant (often referred to as “bulk adjuvant”) and 0.25mL antigen. Dosage volumes for small animals are typically 0.05mL, comprising 0.025mL adjuvant and 0.025mL antigen. The skilled person appreciates that whilst the smaller volumes must be used in pre-clinical testing, the larger dosages would likely be more appropriate for human subjects.

[0081] Suitable antigens for use with adjuvant formulations comprising saponin fraction DV may be involved in, or derived from, for example, an infectious disease, cancer, autoimmune disease, allergy, asthma, or any other condition where stimulation of an antigen-specific immune response would be desirable or beneficial to the patient or subject administered to. Various antigens can be used with adjuvant formulations comprising saponin fraction DV, including but not limited to: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and / or saccharide antigens; fungal antigens; parasite antigens; and soluble antigens such as virosomes and tumor antigens. The disclosure is particularly useful forantigens derived from a coronavirus, a rhinovirus, an influenza virus, a Plasmodium parasite, a mycobacterium, Leishmania parasite, streptococcus bacteria, mycobacteria such as Mycobacterium tuberculosis, respiratory syncytial virus (RSV), a human papilloma virus, a human immunodeficiency (HIV) virus, hepatitis B virus, varicella zoster virus, or any combination thereof.Packaging and kits comprising adjuvant formulations or vaccine compositions

[0082] The adjuvant formulations comprising the saponin fraction DV may be diluted with a buffer prior to packaging into a vial or a syringe. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Dilution can reduce the concentration of the adjuvant's components while retaining their relative proportions e.g. to provide a “half-strength” adjuvant.

[0083] Vaccines (formulations comprising one or more adjuvants and antigens) are typically administered to human patients or subjects in a dosage volume of, for example, 0.2 mL (200 piL), 0.25 mL, 0.3 mL, 0.5 mL, 1 .0 mL, 2.0 mL, although a half dose (i.e. about 0.25 mL) may be administered to children. Containers enclosing compositions of the current application may therefore be marked to show a half-dose volume e.g. to facilitate delivery to children. For instance, a syringe containing a 0.5 mL dose may have a mark showing a 0.25 mL volume.

[0084] Where a glass container (e.g. a syringe or a vial) is used, the container can be made from a borosilicate glass or a soda lime glass. The composition may include material for a single immunization, or may include material for multiple immunizations (i.e. a ‘multidose’ kit). The composition may also include a preservative.

[0085] One aspect of the application provides kits and compositions prepared using the methods presented herein comprising a vaccine or adjuvant formulation comprising saponin fraction DV. The compositions prepared according to the methods of the application are suitable for administration to vertebrates, such as human subjects or patients, and the disclosure provides a method of raising an immune response in a subject or patient, comprising the step of administering such a composition to the subject or patient. The disclosure also provides these kits and compositions comprising saponin fraction DV for use as medicaments.

[0086] The packing may enclose sufficient composition comprising saponin fraction DV according to the current disclosure for a single immunization, or may include sufficient volume for multiple immunizations (i.e. a ‘multidose’ kit), and in such multidose arrangement aspects of the current disclosure, the composition can include a preservative (such as for examplethiomersal or 2-phenoxyethanol).

[0087] Where antigen and adjuvant formulation comprising saponin fraction DV are presented as separate components within a kit, they are physically separate from each other within the kit, and this separation can be achieved in various ways. For instance, the components may be in separate containers, such as vials, the contents of which can then be mixed when needed e.g. by removing the contents of one vial and adding them to the other vial, or by separately removing the contents of both vials and mixing them in a third container.

[0088] In another arrangement, one of the kit components is in a syringe and the other is in a container such as a vial. The syringe can be used (e.g. with a needle) to insert its contents into the vial for mixing, and the mixture can then be withdrawn into the syringe. The mixed contents of the syringe can then be administered to a patient or subject, typically through a new sterile needle. Packing one component in a syringe eliminates the need for using a separate syringe for administration to the patient or subject.

[0089] In another possible arrangement, the two kit components are held together but separately in the same syringe e.g. a dual-chamber syringe, such that when the syringe is actuated (e.g. during administration to a patient) the contents of the two chambers are mixed. This arrangement avoids the need for a separate mixing step at time of use.

[0090] The contents of the various kit components will generally all be in liquid form. In some arrangements, a component (typically the antigen component) is in dry form (e.g. in a lyophilized form), with the other component being in liquid form. The two components can be mixed in order to reactivate the dry component and give a liquid composition for administration to a patient or subject. A lyophilized component will typically be located within a vial rather than a syringe. Dried components may include stabilizers such as lactose, sucrose, trehalose, sorbitol or mannitol, as well as mixtures thereof e.g. lactose / sucrose mixtures, sucrose / mannitol mixtures, etc. A non-limiting example of one possible arrangement is a liquid emulsion or liposome -based adjuvant formulation comprising saponin fraction DV in a prefilled syringe and an antigen component in a vial or in a second compartment of the pre-filled syringe.

[0091] If vaccines contain components in addition to the saponin fraction DV -containing adjuvant formulation and one or more antigens, then these further components may be included in one of these two-kit components or may be part of a third kit component.

[0092] Suitable containers for such “mixed vaccines”, or for individual kit components, include sterile vials and disposable syringes. Where a composition / component is located in a container, such as a vial, the container can be made of a glass or plastic material. The vial can also be sterilized before the composition is added to it. To avoid problems with latex -sensitive patients, vials can be sealed with a latex-free stopper and the vial can be packaged in the absence of latex packing materials. In one embodiment, the vial has a butyl rubber stopper.

[0093] In some aspects, the vial may include a single dose of vaccine / component, or it may include more than one dose (a ‘multidose’ vial) e.g. 5, 6, or 10 doses. For example, with a single dose volume of 0.5 mL, a vial may include 10x0.5 mL composition for 10 doses , or 2.5 mL for 5 doses, and the container may be marked to show a single dose volume. In certain aspects, a container may contain multiple dosage volumes (e.g., a multiple dose vial). In some embodiments, the vaccines comprising saponin fraction DV are to be diluted with sterile solution (e.g., sterile 0.9% sodium chloride Injection, USP) before administration to subjects or patients.

[0094] The vials can be made of glass, such as colorless glass. A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filled syringe can be inserted into the cap, the contents of the syringe can be expelled into the vial (e.g. to reconstitute lyophilized material therein), and the contents of the vial can be removed back into the syringe. After removal of the syringe from the vial, a needle can then be attached and the composition comprising saponin fraction DV can be administered to a patient or subject. The cap is preferably located inside a seal or cover, such that the seal or cover has to be removed before the cap can be accessed.

[0095] Where a composition / component is packaged into a syringe, the syringe will not normally have a needle attached to it, although a separate needle may be supplied with the syringe for assembly and use. Safety needles can be used. 1 -inch 23-gauge, 1 -inch 25-gauge and %-inch 25-gauge needles are typical. Syringes may be provided with peel-off labels on which the lot number, infection season (such as influenza season) and expiration date of the contents may be printed, to facilitate record keeping. The plunger in the syringe preferably has a stopper to prevent the plunger from being accidentally removed during aspiration. The syringes may have a latex rubber cap and / or plunger. Disposable syringes contain a single dose of vaccine comprising saponin fraction DV. The syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap is preferably made of a butyl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber or hard plastic shield.

[0096] In some embodiments, the composition comprising saponin fraction DV is packaged in microneedle patch which administers the composition when adhered to the surface of a subject or patient to be treated or vaccinated.Methods of use of the adjuvant and vaccine compositions, methods of vaccination andtreatment

[0097] This disclosure also provides the use of: (i) an aqueous preparation of an antigen; and (ii) an adjuvant formulation comprising saponin fraction DV prepared according to the disclosure, in the manufacture of a medicament for raising an immune response in a subject or patient.

[0098] In some aspects, the recipient of an adjuvant formulation comprising saponin fraction DV or a vaccine composition comprising saponin fraction DV is a patient or subject undergoing therapeutic treatment. Non-limiting examples include cancer treatment, allergy treatment, autoimmune disease treatment, chronic hepatitis, cyctomegalovirus (CMV) infection, or tuberculosis infection.

[0099] In some aspects, the recipient of an adjuvant formulation comprising saponin fraction DV or a vaccine composition comprising saponin fraction DV is a patient or subject undergoing prophylactic intervention. Non-limiting examples include influenza, SARS-CoV-2, SARS-CoV- 1 , rhinovirus, malaria, tuberculosis, HIV / AIDS, leishmaniasis, Group A streptococcus (GAS), respiratory syncytial virus (RSV), Middle East Respiratory Syndrome virus (MERS), hepatitis B, varicella zoster, herpes simplex virus (HSV), coronavirus or human papilloma virus (HPV) infection.

[0100] Various antigens can be used with the adjuvant formulation comprising saponin fraction DV, including but not limited to: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and / or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens. The application is particularly useful for vaccines against, for example, influenza, SARS-CoV-2, SARS-CoV-1 , MERS (Middle Eastern Respiratory syndrome) rhinovirus, malaria, tuberculosis, HIV / AIDS, leishmaniasis, Group A streptococcus, respiratory syncytial virus (RSV), Middle East Respiratory Syndrome virus (MERS), hepatitis B, varicella zoster, coronavirus or human papilloma virus infection.

[0101] The compositions comprising saponin fraction DV can be administered in various ways. Possible methods of administration (including typical methods of immunization) include intramuscular injection or subcutaneous injection, though alternative methods include intranasal, oral, intradermal, transcutaneous, and transdermal administration of an adjuvant formulation comprising saponin fraction DV or a vaccine composition comprising saponin fraction DV. Most vaccines are given by hypodermic or intramuscular injection; however, live attenuated polio and some typhoid and cholera vaccines are given orally in order to produce immunity based in the bowel. In many aspects of the current disclosure, the method of administration of an adjuvant formulation comprising saponin fraction DV or a vaccine composition comprising saponin fraction DV is intramuscular or subcutaneous injection.

[0102] Vaccines prepared according to the disclosure may be used to inoculate both children and adults. The patient or subject may be less than 1 year old, 1 -5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. The patient or subject may be elderly (e.g. at least 50 years old, preferably at least 65 years), the young (e.g. less than 5 years old), hospitalized patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, immunodeficient patients and subjects, and people travelling abroad. The vaccines are not suitable solely for these groups, however, and may be used more generally in a population.

[0103] An adjuvant formulation comprising saponin fraction DV or a vaccine composition comprising saponin fraction DV may be administered to patients or subjects at substantially the same time as (e.g., during the same medical consultation or visit to a healthcare professional) other vaccines.ExamplesMaterials and methods

[0104] Chemicals used in the examples herein, e.g. for buffers and substrates, are commercial products of at least reagent grade. Water utilized in the examples was sterile water for injection (sterile nonpyrogenic solute-free preparation of distilled water suitable for injection).Example 1 : Analysis of saponin fractions by UHPLC liquid chromatography.

[0105] The chemical profiles of the saponins present in the crude saponin extract employed as raw material, intermediate fractions isolated during chromatographic purification and the purified saponin fraction DV, were determined by ultrahigh performance liquid chromatography (UHPLC) in a Waters Acquity H-Class UPLC chromatography system coupled to a Waters PDA Acquity diode array absorbance detector. The analytical column used was an Acquity UPLC® Peptide BEH C-18 300 A (particle size 1 .7 pm, inner diameter 2.1 mm; length 100 mm, Waters, MA, USA). The column was maintained at 50°C during each chromatographic run. The injected samples were eluted at constant flow rate (0.41 ml / min) in gradient elution according to Table 4 (the eluted peaks were detected by monitoring of the absorbance at 210 nm).Table 4: Solvent gradient employed in the analysis of saponin fraction DV by UHPLC / UV.Example 2: Characterization of saponin fraction DV by liquid chromatography coupled to mass spectrometry.

[0106] The product, an example of saponin fraction DV, was characterized by UHPLC / MS in a Waters Acquity Premier with Waters SQD2 mass detector. The column employed for chromatographic analysis was an Xselect PREMIER Peptide HSS T3 100 A 2.5 pm, 100 x 2.1 mm (Waters, MA, USA). The column was maintained at 40°C during each chromatographic run. The injected samples (10 pL, DMSO solution of DV at 1 mg / mL) were eluted at constant flow rate (0.3 ml / min) in gradient elution according to the Table 5. Table 5: Solvent gradient employed in the analysis of saponin fraction DV by UHPLC / MS.Example 3: Analysis of normal phase chromatography fractions of saponin fraction DV by thin-layer chromatography (TLC).

[0107] In addition to liquid UHPLC chromatography, thin-layer chromatography (TLC) was employed to characterize the chromatographic fractions eluted during the normal phase chromatography step of the saponin fraction DV purification. To this end 2-4 microliters of sample (containing 15-25 g / L of saponins) were loaded onto aluminum TLC plates covered with Silicagel 60. The plate was developed with a mixture 15:9:2:1 of chloroform / methanol / water / acetic acid. After this procedure, the plate was air-dried and sprayed with a visualization reagent (prepared by addition of 1 gram vanillin to 100 ml absolute ethanol with 1 .5 ml concentrated sulfuric acid). The sprayed plate was heated 10 min at 105°C to visualize the bands.Example 4: Analysis of reverse phase chromatography fractions of saponin fraction DV by thin-layer chromatography (TLC).

[0108] In addition to liquid UHPLC chromatography, thin-layer chromatography (TLC) was employed to characterize the chromatographic fractions eluted during the reverse phase chromatography step of the saponin fraction DV purification. To this end 2-4 microliters of sample (containing 15-25 g / L of saponins) were loaded onto aluminum TLC plates covered with Silicagel 60. The plate was developed with a mixture 1 :1 of water / acetonitrile containing 0.16% v / v of acetic acid. After this procedure, the plate was air-dried and sprayed with Pancaldi’s reagent - (NH4)6Mo04 42 g, Ce(SC>4)2 2 g, H2SO4 62 mL, H2O 1 L. The sprayed plate was heated 10 min at 105°C to visualize the bands.Example 5: Small scale chromatographic purification of saponin fraction DV.

[0109] An example of saponin fraction DV was manufactured at small scale by chromatographic purification from a highly purified saponin powder prepared from the bark of Quillaja saponaria as described in the Example 4 of Padilla- Iglesias and Valencia-Michaud (2020). In total, the purification process has six steps summarized in Figure 1 , which are described as follows:Step 1 : Extraction of saponins with methanol:

[0110] Sixty (60) g of highly purified saponin powder is suspended in 600 ml of methanol. The solids are extracted with stirring at 30°C for 15 min. Undissolved solids were removed by centrifugation or, preferably, filtration through Whatman N°1 paper. The methanol extract was concentrated by vacuum evaporation to provide a “Concentrated Methanol Extract” containing approximately 100-250 g / L. Total saponin concentration in the concentrated methanol extract was determined by UHPLC assay (as described in the Example 1 )-Step 2: Fractionation by preparative chromatography[0011 1] Aliquots of the Concentrated Methanol Extract resulting from Step 1 , containing approximately 100-300 mg of saponins, were separated through RP-HPLC in an octodecylsilane column fitted to a guard octodecylsilane column using isocratic elution with a mixture of water and methanol containing 0.15% formic acid, as shown in Table 6.

[0112] The eluate was recovered as discrete fractions; the saponin profile of the collected fractions was determined off-line by UHPLC assay (Example 1 ). Fractions containing the major peaks in saponin fraction DV were selected for further recovery of saponin fraction DV. The selected fractions of several preparative runs were mixed together, rendering the “Crude Saponin DV Fraction”.Table 6: Solvent employed in the fractionation of the concentrated methanol extract by preparative HPLC.Step 3: Solvent removal of the liquid “Crude saponin DV Fraction”:

[0113] The methanol and formic acid in the Crude Saponin DV Fraction resulting from Step 2 were removed by vacuum evaporation in a rotary-evaporator. The residual aqueous solution thus obtained was frozen and lyophilized, rendering “Dry Saponin DV Fraction”.Step 4: Fractionation by preparative chromatography

[0114] The “Dry saponin DV Fraction” resulting from Step 3 was dissolved in a mixture of water and acetonitrile to a concentration of 100 g / L. Aliquots of the solution of Dry Saponin DV Fraction” containing 14 mg of saponins were separated through RP-HPLC in in an octodecylsilane column fitted to a guard octodecylsilane column using the solvent gradient of water and acetonitrile (containing formic acid 0.15%) as shown in Table 7.Table 7: Solvent gradient employed in the production of DV by preparative HPLC.

[0115] The eluate was recovered as discrete fractions and the saponin profile of the collected fractions was determined off-line by UHPLC assay (Example 1 ). Fractions containing the major peak of saponin fraction DV were selected for further recovery of the product. The selected fractions of several runs were mixed together, rendering the “Pool Saponin DV Fraction”.Step 5: Solvent removal by rotary evaporation:

[0116] The acetonitrile and formic acid in the Pool Saponin DV Fraction” resulting from Step 4 were removed by vacuum evaporation, rendering the “Saponin Fraction DV Precursor”. The integrity of the saponin profile in the Saponin Fraction DV Precursor was then verified by the UHPLC assay as described in Example 1 .Step 6: Final lyophilization:

[0117] The Saponin Fraction DV Precursor resulting from Step 5 was aliquoted, frozen and lyophilized, rendering the final product “saponin fraction DV”. The integrity of the saponin profile in DV was verified by UHPLC assay. To this end, a solution containing 1 g / L saponin fraction DV was analyzed according to the method described in Example 1 .Example 6: Chromatographic purification of saponin fraction DV at large scale.

[0118] A further method to produce saponin fraction DV at reduced cost and large scale is described in this example. Highly purified saponin powder was prepared from bark of Quillaja saponaria as described in the Example 1 of Padilla- Iglesias and Valencia-Michaud (2020). In total, the large-scale process for purification of saponin fraction DV has five steps summarized in Figure 1 and as described in the following example:Step 1 : Treatment of highly purified saponin powder with cellulose powder and methanol:

[0119] Ten (10) g of highly purified saponin powder was placed in a round bottom flask and suspended in methanol (250 ml) in the presence of highly pure cellulose powder (Arbocel BWW40; 5 g). The resultant suspension was stirred at room temperature for at least 2 hours and filtered on a sintered glass funnel, the cake was washed twice with methanol. The resulting mother liquors were pooled together and concentrated under vacuum in a rotary evaporator down to approximately 40 ml of “Concentrated Methanol Extract”. The total saponin concentration in the concentrated methanol extract was determined by the UHPLC assay as described in the Example 1 .Step 2: Silica gel normal phase purification:

[0120] The Concentrated Methanol Extract resulting from Step 1 was diluted with 60 ml of methanol. Dichloromethane (90 ml) was added dropwise to the solution. The mixture was loaded onto a column containing 2,000 g of Silica gel 60, and purified according to the following conditions: a) Eluent mixture: Dichloromethane:methanol:water:acetic acid at 20:9:2:1 v / v / v / v. b) Flow rate: 80ml / cm2 / h. c) Fractioning: Fractions of approximately 360-400 ml; d) Monitoring: i) TLC according to the procedure described in Example 3, and; ii) UHPLC according to the procedure described in Example 1 .

[0121] The fractions enriched with saponin fraction DV (containing more than 40% major saponin fraction QVI group of peaks on total chromatographic area basis - see Figures 3-5) were pooled together and concentrated by drying under vacuum in a rotary evaporator at 50°C, to provide the “Enriched Saponin Fraction DV”.Step 3: Silica gel filtration:

[0122] The Enriched Saponin Fraction DV resulting from Step 2 was dissolved in a mixture of purified water / acetonitrile 7:3 v / v containing 0.16% v / v acetic acid (20 ml per g of Enriched Saponin Fraction DV). The resulting solution was filtrated on silica gel according to the following parameters: a) Stationary phase: Silica gel. b) Eluent mixture: Water / acetonitrile 7:3 v / v containing 0.16% v / v acetic acid. c) Flow rate: 45 ml / cm2 / h. d) Volume of washing: 10 ml per ml of loaded solution.

[0123] The combined filtrates were pooled together to provide the “Filtered Enriched Saponin Fraction DV”.Step 4: Reverse phase C18 chromatography:

[0124] The Filtered Enriched Saponin Fraction DV was fractionated by a low-pressure reverse phase chromatographic column according to the following parameters: a) Stationary phase: Octadecylsilane b) Eluent mixture: in gradient: Water / ACN 7:3 v / v containing 0.16% v / v of acetic acid. c) Water / ACN 5:5 v / v containing 0.16% v / v of acetic acid. d) Flow rate: 25 ml / cm2 / h. e) Fractioning: Fractions of equivalent to 1.5-1.7 volumes of the RP-18 packed as stationary phase in the column. f) Monitoring: TLC according to the procedure described in Example 4.

[0125] The resulting further enriched saponin fraction DV eluted from the column were pooled together to provide the “Pooled Further Enriched Saponin Fraction DV”.Step 5: Freeze drying:

[0126] The Pooled Saponin Fraction DV obtained in Step 4 was frozen and progressively freeze dried (40 h, vacuum <1mbar), resulting in the final product, the ’’Saponin fraction DV” as a white fluffy powder.

[0127] The integrity of the saponin profile in DV was verified by UHPLC assay, according to the method described in the Example 1 .Example 7: Chromatographic and spectroscopic characterization of saponin fraction DV.

[0128] To this end, a solution containing 1 g / L saponin fraction DV was analyzed according to the method described in Example 1 (Figure 3). Saponin fraction DV was characterized by the presence of two major peaks eluting approximately to 8.65 and 9.0 min, corresponding to a small preceding peak and a large multi-component peak (referred to herein as the "QVI group of peaks”), respectively (Figure 4). Within the QVI group of peaks, the largest peak, referred to herein as the “Major Peak”, has a shoulder peak, referred to herein as the “Shoulder of the major peak” and a preceding peak, referred to herein as the “Preceding peak”, as shown in Figure 5. The QVI group of peaks of saponin fraction DV therefore collectively comprise a tight group of overlapping peaks that signify a range of closely related compounds, which for convenience are collectively referred to herein as the “QVI compounds” of peaks. In addition to those components, some additional peaks are observed before and after the QVI group of peaks; the first saponin group and the second saponin group, respectively (Figure 4).

[0129] Saponin fraction DV was additionally characterized by LC / MS according to the method described in the Example 2 (Figure 6). According to the MS analysis the major components are saponins having a monoisotopic m / z for [M-H]- of 1726, 1856, 1988 and 2003, hereinafter referred as Components 1726, 1856, 1988 and 2003, respectively. Table 8 summarizes the relative elution of these compounds according to the chromatogram in Figure. Without being bound by theory, the inventors have disclosed the most likely structures of Components 1726, 1856, 1988 and 2003 in Figures 7 to 10.Table 8: Relative elution of Components 1726, 1856, 1988 and 2003 in the chromatogram inFigure 5.

[0130] According to the characterization of saponin fraction DV, the ratio between chromatographic areas of minor and major peaks should be in the range of 1 1 :87-40:60. The content of the major peak as percentage of total area in the chromatogram, should be in the range 50-87%.Example 8: Lytic activity of the saponin fraction DV

[0131] In this example, the lytic activity of saponin fraction DV was compared with the lytic activity of a GMP-grade QS21 saponin benchmark adjuvant. Solutions of each saponin were prepared in buffered saline at concentrations from 0 to 50 pg / mL of saponins. Each sample was then mixed with purified sheep red blood and incubated at 25°C for 30 minutes. After incubation, samples were centrifuged and supernatants were collected. Supernatants were subsequently diluted with water for injection and absorbance was monitored at 412 nm to detect release of hemoglobin. Negative control was obtained using buffered saline without saponin and positive control was obtained using a Triton-X100 solution at 1% (w / v). Results are reported in Figure 1 1 . It was observed that saponin fraction DV had the same hemolytic effect as that of the GMP-grade QS21 saponin.Example 9: Association of the saponin fraction DV with cholesterol-containing liposomes and emulsions

[0132] In this example, the ability of saponin fraction DV to associate with cholesterol- containing liposomes and emulsions was assessed by measuring the lytic activity of formulations prepared with various ratios of cholesterol / saponin fraction DV (w / w). These were compared with the same formulations prepared with GMP-grade QS-21 . Solutions of saponin fraction DV or QS-21 in buffered saline were mixed with neutral liposomes (DOPC 4 mg / mL, cholesterol 1 mg / mL, buffered saline), neutral liposomes containing the TLR4 agonist 3D-(6- acyl) PHAD® (DOPC 4 mg / mL, cholesterol 1 mg / mL, 3D-(6-acyl) PHAD® 0.04 mg / mL, buffered saline), and squalene-in-water emulsion (squalene 42 mg / mL, cholesterol 2 mg / mL). Each sample was then mixed with purified sheep red blood and incubated at 25°C for 30 minutes. After incubation, samples were centrifuged and supernatants were collected. Supernatants were subsequently diluted with water for injection and absorbance was monitored at 412 nm to detect release of hemoglobin. Negative control was obtained using buffered saline without saponin and positive control was obtained using a T rito n -X 100 solution at 1% (w / v). Results are reported in Figure 12. The lytic activity of the saponin fraction DV wasquenched for all formulations when the cholesterol / saponin fraction DV ratio was above 1 .0 (w / w) similar to that observed with GMP-grade saponin QS21 (see also Patent US6846489B1 , column 5, paragraph 4, quote: “If the QS21 is incubated with liposomes containing cholesterol prior to adding it to erythrocytes, the lysis of the erythrocytes is diminished depending on the ratio of cholesterol to QS21 . If a 1 :1 ratio is used no lytic activity is detected. If the liposomes do not contain cholesterol, then inhibition of lysis requires a one thousand fold excess of phospholipid over QS21 .”)Example 10: Manufacturing of saponin fraction DV-containing adjuvant formulations

[0133] Precursor DOPC / Cholesterol liposomes and 3D-(6-acyl) PHADO-loaded liposomes were produced by ethanol injection. DOPC and cholesterol with or without 3D-(6- acyl) PHAD® were dissolved in ethanol, then injected rapidly under stirring in an aqueous phase made of buffered saline, resulting in a homogenous suspension of liposomes. The ethanol was then removed by tangential flow filtration using a membrane with a molecular cutoff of 100 kDa. The saponin fraction DV-containing liposome adjuvants were prepared by mixing a volume of each precursor liposome with a concentrated saponin fraction DV solution in buffered saline. The resulting adjuvants were then diluted to working concentrations and sterile filtered through a 0.22 pm membrane under aseptic conditions. The sterile adjuvants were then stored in sterile polypropylene containers and kept at 2-8°C until use.

[0134] Precursor squalene in water emulsion containing cholesterol was produced by microfluidization. Squalene, polyoxyethylene sorbitan monooleate, sorbitan trioleate, cholesterol and buffer saline were mixed by high-speed homogenization and the mixture submitted to microfluidization at 20 000 psi to yield the squalene in water emulsion containing cholesterol. The saponin fraction DV emulsion adjuvant was prepared by mixing a volume of the precursor emulsion with a concentrated DV solution in buffered saline. The resulting emulsion-based adjuvants comprising saponin fraction DV were then diluted to working concentrations and sterile filtered through a 0.22 pm membrane under aseptic conditions. The sterile adjuvants were then stored in sterile polypropylene containers and kept at 2-8°C until use.Example 11 : Stability of saponin fraction DV-containing adjuvant formulations.

[0135] Adjuvant formulations based on liposomes and emulsions were manufactured with either GMP-grade QS21 or saponin fraction DV and stored at 40°C. After one month, a quality control of each batch was made to assess and compare the stability of saponin fraction DV with GMP-grade QS21 , as well as the influence of the saponin on the overall stability ofthe adjuvants, in particular physicochemical properties and component quantitation. For the DOPC / Cholesterol liposomes, results are presented in Figures 13a-e. For the squalene in water emulsion, results are presented in Figure 14a-e. For the DOPC / Cholesterol / 3D-(6-acyl) PHAD® liposomes, results are presented in Figure 15a-e. The colloidal stability and chemical stability of all adjuvants were identical for the monitored period, independently of the saponin used in the compositions, QS21 or saponin fraction DV. In the case of the DOPC / Cholesterol / 3D-(6-acyl) PHAD® liposomes, for which the degradation of 3D-(6-acyl) PHAD® at 40°C is a known feature, the type of saponin used in the adjuvant, QS21 or saponin fraction DV, did not have any influence on the degradation rate. Overall, this indicated that saponin fraction DV and QS21 have the same physicochemical properties when formulated in liposomes or emulsions.Example 12: Pre-clinical evaluation of saponin fraction DV -containing adjuvant formulations for a COVID-19 vaccine in miceExample 12 a: cellular response

[0136] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 2 pg per dose (50 pL) of HexaPro (S6P), a stabilized Spike protein from SARS-CoV-2, either (i) non adjuvanted, (ii) adjuvanted with DOPC / Cholesterol liposomes without saponins, (iii) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD liposomes without saponins, (iv) adjuvanted with squalene-in-water emulsion containing cholesterol (v) adjuvanted with DOPC / Cholesterol / QS21 liposomes (5 pg of QS21 saponins), (vi) adjuvanted with DOPC / Cholesterol / saponin fraction DV liposomes (5 pg of saponin fraction DV saponins), (vii) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / QS21 liposomes (1 pg of 3D(6-acyl)- PHAD and 5 pg of QS21 saponins), (viii) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)- PHAD / saponin fraction DV liposomes (1 pg of 3D(6-acyl)-PHAD and 5 pg of saponin fraction DV), (ix) adjuvanted with squalene-in-water emulsion containing cholesterol and QS21 (5 pg of QS21 saponins) or (x) adjuvanted with squalene-in-water emulsion containing cholesterol and saponin fraction DV (5 pg of saponin fraction DV), all adjuvant formulations being manufactured as in Example 9. On day 28, spleens from mice were collected. Splenocytes were isolated by crushing spleens through a 70 pm cell strainer, then washed with incomplete RPMI medium (1% penicillin / streptomycin, 20 mM Hepes + 1X MEM NEAA) followed by red blood cell removal using Lympholyte®-M cell separation medium. One million cells were stimulated in round-bottom 96-well plates in complete RPMI medium (incomplete RPMI + 6% FCS + 50 pM p-mercaptoethanol) with 50 pL / well of peptide medium at a final concentration of 1.5 pg / mL and incubated for 2 h at 37°C, 5% CO2. Cytokine secretion was blocked usingGolgiPlug™ (available from BD Biosciences) protein transport inhibitor (50 pL / well, 1 :1000 final dilution) and cells were further incubated overnight at 37°C, 5% CO2. Cells were then transferred to V-bottom 96-well plates and stained for 15 minutes at 4°C using the LIVE / DEAD™ Kit. Afterwards, cell surface staining was performed with purified anti- CD16 / CD32 (BD Bioscience), anti-CD3-FITC (eBioscience), anti-CD8a-PerCP-Cy5.5 (BioLegend®) and anti-CD4-Pacific Blue (BioLegend®) for 15 min on ice. After washing, fixation and permeabilization, cells were incubated for 30 min at 4°C with anti-IL-2-APC (BD Pharmingen™) and anti-IFNy-PE (BD Pharmingen™) for intracellular staining. Cells were washed 3 times with Perm / Wash buffer and were finally suspended in 300 pL PBS prior to acquisition on an Attune™ NxT flow cytometer (Invitrogen). Compensation was performed using the Arc™ Amine Reactive Compensation Bead Kit and AbC™ Total Antibody compensation bead kit (both from Invitrogen), and compensation matrices were calculated with the Attune™ NxT software (Invitrogen). FACS data were analyzed using FlowJo software (FlowJo LLC, USA). Results are shown as means of cumulated frequencies of cells expressing IL-2 and / or IFNy. Results are presented in Figures 16a and b. Results show that non- adjuvanted formulation and adjuvants without saponin did not induce any / or barely detectable T cell responses. In contrast, all adjuvants comprising saponin fraction DV induced detectable CD4+ T cell responses and CD8+ T cell responses similar to the QS21 -containing counterpart formulations. We conclude from these data that the saponin fraction DV, and associated saponin fraction DV-containing adjuvant formulations, of the current disclosure have comparable adjuvanticity to GMP-grade QS21 and benchmark QS21 -containing adjuvant formulations. We further conclude that saponin fraction DV is a very suitable alternative to QS21 for use as an adjuvant.Example 12 b: antibody response

[0137] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 2 pg per dose (50 pL) of HexaPro (S6P), a stabilized Spike protein from SARS-CoV-2, either (i) non adjuvanted, (ii) adjuvanted with DOPC / Cholesterol liposomes without saponins, (iii) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD liposomes without saponins, (iv) adjuvanted with squalene-in-water emulsion containing cholesterol (v) adjuvanted with DOPC / Cholesterol / QS21 liposomes (5 pg of QS21 saponins), (vi) adjuvanted with DOPC / Cholesterol / saponin fraction DV liposomes (5 pg of saponin fraction DV saponins), (vii) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / QS21 liposomes (1 pg of 3D(6-acyl)- PHAD and 5 pg of QS21 saponins), (viii) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)- PHAD / saponin fraction DV liposomes (1 pg of 3D(6-acyl)-PHAD and 5 pg of saponin fractionDV), (ix) adjuvanted with squalene-in-water emulsion containing cholesterol and QS21 (5 pg of QS21 saponins) or (x) adjuvanted with squalene-in-water emulsion containing cholesterol and saponin fraction DV (5 pg of saponin fraction DV), all adjuvant formulations being manufactured as in Example 9. Mice were bled on day 42 (3 weeks after the second injection). Anti-spike Ig responses were measured in mouse sera using enzyme linked immunosorbent assays (ELISA). Nunc Maxisorb™ plates (Thermo Fisher Scientific) were coated directly with S6P protein at 10 pg / mL in PBS 1 X overnight at 4°C, blocked for 1 h with PBS-t, PBS containing 0.05% (v / v) Tween 20 (Applichem), and 2% (w / v) bovine serum albumin (BSA) (Sigma). The plates were washed with PBS-t and incubated with 4x serial dilutions of mouse serum samples diluted 1 / 50 in PBS containing 0.05% (w / v) BSA. Plates were incubated for 1 h at 37°C, washed and then incubated for 1 h at 37°C with anti-mouse antibodies coupled to HRP (Southern Biotech) including goat anti-mouse total IgG or lgG2c at 1 :6000 dilution. Plates were then washed before addition of OPD (Sigma, P9187) substrate solution, the reaction was stopped at 30 minutes by adding 2M sulfuric acid. Absorbance was directly measured at 492 nm using a microplate reader (Biotek, Switzerland). Results are presented in Figure 17. Results show that 3 weeks after the second injection of vaccine formulation, all vaccines comprising saponin fraction DV-containing adjuvant formulations induced high total IgG antibody titers and high lgG2c antibody titers compared to the non-adjuvanted formulation or to the adjuvanted formulations without saponins. Furthermore, the vaccines comprising saponin fraction DV-containing adjuvant formulations induced similar titers to the QS21 - containing counterpart formulations. We conclude from these data that the vaccines comprising saponin fraction DV, and associated saponin fraction DV-containing adjuvant formulations, of the current disclosure has equivalent adjuvanticity compared to clinical-grade QS21 and represent a highly suitable alternative to QS21 for use in adjuvanted vaccines.Example 13: Pre-clinical evaluation in mice of saponin fraction DV-containing adjuvant formulations for an influenza vaccineExample 13a: cellular response

[0138] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 3 pg per dose (50 pL) of hemagglutinin proteins (0.75 pg for each strain under the form of inactivated split virions: H1 N1 , H3N2, B Victoria and B Yamagata; Fluarix® Tetra 2022-2023) using the Fluarix® Tetra 2022-2023 commercial vaccine (i) non adjuvanted, (ii) adjuvanted with DOPC / Cholesterol / QS21 liposomes (5 pg of QS21 saponins), (iii) adjuvanted withDOPC / Cholesterol / saponin fraction DV liposomes (5 pg of saponin fraction DV saponins), (iv) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / QS21 liposomes (1 pg of 3D(6-acyl)- PHAD and 5 pg of QS21 saponins), (v) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)- PHAD / saponin fraction DV liposomes (1 pg of 3D(6-acyl)-PHAD and 5 pg of saponin fraction DV), (vi) adjuvanted with squalene-in-water emulsion containing cholesterol and QS21 (5 pg of QS21 saponins) or (vii) adjuvanted with squalene-in-water emulsion containing cholesterol and saponin fraction DV (5 pg of saponin fraction DV), all adjuvant formulations being manufactured as in Example 9. On day 28, spleens from mice were collected. Splenocytes were isolated by crushing spleens through a 70 pm cell strainer, then washed with incomplete RPMI medium (1% penicillin / streptomycin, 20 mM Hepes + 1X MEM NEAA) followed by red blood cell removal using Lympholyte®-M cell separation medium. One million cells were stimulated in round-bottom 96-well plates in complete RPMI medium (incomplete RPMI + 6% FCS + 50 pM p-mercaptoethanol) with 50 pL / well of peptide medium at a final concentration of 1.5 pg / mL and incubated for 2 h at 37°C, 5% CO2. Cytokine secretion was blocked using GolgiPlug™ (available from BD Biosciences) protein transport inhibitor (50 pL / well, 1 :1000 final dilution) and cells were further incubated overnight at 37°C, 5% CO2. Cells were then transferred to V-bottom 96-well plates and stained for 15 minutes at 4°C using the LIVE / DEAD™ Kit. Afterwards, cell surface staining was performed with purified anti- CD16 / CD32 (BD Bioscience), anti-CD3-FITC (eBioscience), anti-CD8a-PerCP-Cy5.5 (BioLegend®) and anti-CD4-Pacific Blue (BioLegend®) for 15 min on ice. After washing, fixation and permeabilization, cells were incubated for 30 min at 4°C with anti-IL-2-APC (BD Pharmingen™) and anti-IFNy-PE (BD Pharmingen™) for intracellular staining. Cells were washed 3 times with Perm / Wash buffer and were finally suspended in 300 pL PBS prior to acquisition on an Attune™ NxT flow cytometer (Invitrogen). Compensation was performed using the Arc™ Amine Reactive Compensation Bead Kit and AbC™ Total Antibody compensation bead kit (both from Invitrogen), and compensation matrices were calculated with the Attune™ NxT software (Invitrogen). FACS data were analyzed using FlowJo software (FlowJo LLC, USA). Results are shown as means of cumulated frequencies of cells expressing IL-2 and / or IFNy. Results are presented in Figure 18. Results show that all adjuvants comprising saponin fraction DV induced CD4+ T cell responses while non-adjuvanted formulation did not. We conclude from these data that the saponin fraction DV, and associated saponin fraction DV-containing adjuvant formulations, of the current disclosure have comparable adjuvanticity to GMP-grade QS21 and benchmark QS21 -containing adjuvant formulations. We conclude that saponin fraction DV is a very suitable alternative to QS21 for use as an adjuvant.Example 13b: antibody response

[0139] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 3 ng per dose (50 pL) of hemagglutinin proteins (0.75 pg for each strain under the form of inactivated split virions: H1 N1 , H3N2, B Victoria and B Yamagata; Fluarix® Tetra 2022-2023) using the Fluarix® Tetra 2022-2023 commercial vaccine (i) non adjuvanted, (ii) adjuvanted with DOPC / Cholesterol / QS21 liposomes (5 pg of QS21 saponins), (iii) adjuvanted with DOPC / Cholesterol / saponin fraction DV liposomes (5 pg of saponin fraction DV), (iv) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / QS21 liposomes (1 pg of 3D(6-acyl)- PHAD and 5 pg of QS21 saponins), (v) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)- PHAD / saponin fraction DV liposomes (1 pg of 3D(6-acyl)-PHAD and 5 pg of saponin fraction DV), (vi) adjuvanted with squalene-in-water emulsion containing cholesterol and QS21 (5 pg of QS21 saponins) or (vii) adjuvanted with squalene-in-water emulsion containing cholesterol and saponin fraction DV (5 pg of saponin fraction DV), all adjuvant formulations being manufactured as in Example 9. Mice were bled on day 42 (3 weeks after the second injection). Serum antibody titers against HA were determined using hemagglutination inhibition assay (HAI), performed according to the WHO manual for laboratory diagnosis and virological surveillance of influenza (World Health Organization, 2011 ). All serum samples were pretreated overnight at 37°C with receptor-destroying enzyme and further diluted in 96-well roundbottom plates to a final serum dilution of 1 / 10. Individual sera were serially 2-fold diluted in V- bottom microtiter plates and mixed with whole inactivated virus adjusted to 4 hemagglutination units for 30 min at room temperature. Chicken red blood cells (RBC) were then added. Once control RBCs were settled (30-40 min), the plate was tilted, and the assay imaged for further analysis. HAI titers were calculated as the reciprocal of the last serum dilution that contained non-agglutinated RBCs (tearing). Samples with a result below the lowest dilution (1 :10) were assigned an HAI titer of 1 . Results are presented in Figure 19. Results show that all saponin fraction DV-containing adjuvants induced high hemagglutination inhibition titers compared to the non-adjuvanted formulation. Furthermore, the saponin fraction DV-containing formulations induced similar titers compared to the QS21 -containing counterpart formulations. We conclude from these data that the saponin fraction DV, and associated saponin fraction DV- containing adjuvants, of the current disclosure has equivalent adjuvanticity compared to benchmark GMP-grade QS21 and represent a very suitable alternative to QS21 .Example 14: Pre-clinical evaluation in mice of saponin fraction DV-containing adjuvant formulations for a tuberculosis vaccineExample 14a: cellular response

[0140] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 10 pg per dose (50 pL) of recombinant Mycobacterium tuberculosis (MTB) Ag85B protein either (i) non adjuvanted, (ii) adjuvanted with DOPC / Cholesterol / QS21 liposomes (5 pg of QS21 saponins), (iii) adjuvanted with DOPC / Cholesterol / saponin fraction DV liposomes (5 pg of saponin fraction DV), (iv) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / QS21 liposomes (1 pg of 3D(6-acyl)-PHAD and 5 pg of QS21 saponins), (v) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / saponin fraction DV liposomes (1 pg of 3D(6-acyl)- PHAD and 5 pg of saponin fraction DV), (vi) adjuvanted with squalene-in-water emulsion containing cholesterol and QS21 (5 pg of QS21 saponins) or (vii) adjuvanted with squalene- in-water emulsion containing cholesterol and saponin fraction DV (5 pg of saponin fraction DV), all adjuvant formulations being manufactured as in Example 9. On day 28, spleens from mice were collected. Splenocytes were isolated by crushing spleens through a 70 pm cell strainer, then washed with incomplete RPMI medium (1% penicillin / streptomycin, 20 mM Hepes + 1 X MEM NEAA) followed by red blood cell removal using Lympholyte®-M cell separation medium. One million cells were stimulated in round-bottom 96-well plates in complete RPMI medium (incomplete RPMI + 6% FCS + 50 pM p-mercaptoethanol) with 50 pL / well of protein medium at a final concentration of 10 pg / mL and incubated for 2 h at 37°C, 5% CO2. Cytokine secretion was blocked using GolgiPlug™ (available from BD Biosciences) protein transport inhibitor (50 pL / well, 1 :1000 final dilution) and cells were further incubated overnight at 37°C, 5% CO2. Cells were then transferred to V-bottom 96-well plates and stained for 15 minutes at 4°C using the LIVE / DEAD™ Kit. Afterwards, cell surface staining was performed with purified anti-CD16 / CD32 (BD Bioscience), anti-CD3-FITC (eBioscience), anti- CD8a-PerCP-Cy5.5 (BioLegend®) and anti-CD4-Pacific Blue (BioLegend®) for 15 min on ice. After washing, fixation and permeabilization, cells were incubated for 30 min at 4°C with anti- IL-2-APC (BD Pharmingen™) and anti-IFNy-PE (BD Pharmingen™) for intracellular staining. Cells were washed 3 times with Perm / Wash buffer and were finally suspended in 300 pL PBS prior to acquisition on an Attune™ NxT flow cytometer (Invitrogen). Compensation was performed using the Arc™ Amine Reactive Compensation Bead Kit and AbC™ Total Antibody compensation bead kit (both from Invitrogen), and compensation matrices were calculated with the Attune™ NxT software (Invitrogen). FACS data were analyzed using FlowJo software (FlowJo LLC, USA). Results are shown as means of cumulated frequencies of cells expressingIL-2 and / or IFNy. Results are presented in Figure 20. Results show that all vaccine formulations comprising saponin fraction DV adjuvant formulations induced high CD4+ T cell responses when compared to the non-adjuvanted formulation. Furthermore, the vaccines comprising saponin fraction DV-containing formulations induced similar CD4+ T cell responses to the QS21 -containing counterpart formulations. We conclude from these data that the saponin fraction DV, and associated saponin fraction DV-containing adjuvant formulations, of the current disclosure has equivalent adjuvanticity compared to GMP-grade QS21 and represent a very suitable alternative to QS21 for use in vaccines.Example 14b: antibody response

[0141] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 10 pg per dose (50 pL) of recombinant Mycobacterium tuberculosis (MTB) Ag85B protein either (i) non adjuvanted, (ii) adjuvanted with DOPC / Cholesterol / QS21 liposomes (5 pg of QS21 saponins), (iii) adjuvanted with DOPC / Cholesterol / saponin fraction DV liposomes (5 pg of saponin fraction DV), (iv) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / QS21 liposomes (1 pg of 3D(6-acyl)-PHAD and 5 pg of QS21 saponins), (v) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / saponin fraction DV liposomes (1 pg of 3D(6-acyl)- PHAD and 5 pg of saponin fraction DV), (vi) adjuvanted with squalene-in-water emulsion containing cholesterol and QS21 (5 pg of QS21 saponins) or (vii) adjuvanted with squalene- in-water emulsion containing cholesterol and saponin fraction DV (5 pg of saponin fraction DV), all adjuvant formulations being manufactured as in Example 9. Mice were bled on day 42 (3 weeks after the second injection). Anti-Ag85B Ig responses were measured in mouse sera using enzyme linked immunosorbent assays (ELISA). Nunc Maxisorb™ plates (Thermo Fisher Scientific) were coated directly with Ag85B protein at 10 pg / mL in PBS 1X overnight at 4°C, blocked for 1 h with PBS-t, PBS containing 0.05% (v / v) Tween 20 (Applichem), and 2% (w / v) bovine serum albumin (BSA) (Sigma). The plates were washed with PBS-t and incubated with 4x serial dilutions of mouse serum samples diluted 1 / 50 in PBS containing 0.05% (w / v) BSA. Plates were incubated for 1 h at 37°C, washed and then incubated for 1 h at 37°C with antimouse antibodies coupled to HRP (Southern Biotech) including goat anti-mouse total IgG or lgG2c at 1 :6000 dilution. Plates were then washed before addition of OPD (Sigma, P9187) substrate solution, the reaction was stopped at 30 minutes by adding 2M sulfuric acid. Absorbance was directly measured at 492 nm using a microplate reader (Biotek, Switzerland). Results presented in Figure 21 show that 3 weeks after the second injection of vaccine formulation, all vaccines comprising saponin fraction DV-containing adjuvant formulations induced high total IgG antibody titers and high lgG2c antibody titers compared to the non-adjuvanted formulation. Furthermore, the vaccines comprising saponin fraction DV-containing adjuvant formulations induced similar titers to the QS21 -containing counterpart formulations. We conclude from these data that the vaccines comprising saponin fraction DV, and associated saponin fraction DV-containing adjuvant formulations, of the current disclosure has equivalent adjuvanticity compared to GMP-grade QS21 and represent a highly suitable alternative to QS21 for use in adjuvanted vaccines.Example 15: Pre-clinical evaluation in mice of saponin fraction DV-containing adjuvant formulations for a malaria vaccineExample 15a: cellular responses

[0142] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 10 pg per dose (50 pL) of recombinant Plasmodium falciparum MSP1 protein either (i) non adjuvanted, (ii) adjuvanted with DOPC / Cholesterol / QS21 liposomes (5 pg of QS21 saponins), (iii) adjuvanted with DOPC / Cholesterol / saponin fraction DV liposomes (5 pg of saponin fraction DV), (iv) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / QS21 liposomes (1 pg of 3D(6-acyl)-PHAD and 5 pg of QS21 saponins), (v) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / saponin fraction DV liposomes (1 pg of 3D(6-acyl)- PHAD and 5 pg of saponin fraction DV), (vi) adjuvanted with squalene-in-water emulsion containing cholesterol and QS21 (5 pg of QS21 saponins) or (vii) adjuvanted with squalene- in-water emulsion containing cholesterol and saponin fraction DV (5 pg of saponin fraction DV), all adjuvant formulations being manufactured as in Example 9. On day 28, spleens from mice were collected. Splenocytes were isolated by crushing spleens through a 70 pm cell strainer, then washed with incomplete RPMI medium (1% penicillin / streptomycin, 20 mM Hepes + 1 X MEM NEAA) followed by red blood cell removal using Lympholyte®-M cell separation medium. One million cells were stimulated in round-bottom 96-well plates in complete RPMI medium (incomplete RPMI + 6% FCS + 50 pM p-mercaptoethanol) with 50 pL / well of protein medium at a final concentration of 10 pg / mL and incubated for 2 h at 37°C, 5% CO2. Cytokine secretion was blocked using GolgiPlug™ (available from BD Biosciences) protein transport inhibitor (50 pL / well, 1 :1000 final dilution) and cells were further incubated overnight at 37°C, 5% CO2. Cells were then transferred to V-bottom 96-well plates and stained for 15 minutes at 4°C using the LIVE / DEAD™ Kit. Afterwards, cell surface staining was performed with purified anti-CD16 / CD32 (BD Bioscience), anti-CD3-FITC (eBioscience), anti- CD8a-PerCP-Cy5.5 (BioLegend®) and anti-CD4-Pacific Blue (BioLegend®) for 15 min on ice. After washing, fixation and permeabilization, cells were incubated for 30 min at 4°C with anti-IL-2-APC (BD Pharmingen™) and anti-IFNy-PE (BD Pharmingen™) for intracellular staining. Cells were washed 3 times with Perm / Wash buffer and were finally suspended in 300 pL PBS prior to acquisition on an Attune™ NxT flow cytometer (Invitrogen). Compensation was performed using the Arc™ Amine Reactive Compensation Bead Kit and AbC™ Total Antibody compensation bead kit (both from Invitrogen), and compensation matrices were calculated with the Attune™ NxT software (Invitrogen). FACS data were analyzed using FlowJo software (FlowJo LLC, USA). Results are shown as means of cumulated frequencies of cells expressing IL-2 and / or IFNy. Results are presented in Figure 22. Results show that all vaccines comprising saponin fraction DV-containing adjuvant formulations induced high CD4+ T cell responses when compared to the non-adjuvanted formulation. Furthermore, the vaccines comprising saponin fraction DV-containing adjuvant formulations induced similar CD4+ T cell responses than the QS21 -containing counterpart formulations. We conclude from these data that the vaccines comprising the saponin fraction DV, and associated saponin fraction DV- containing adjuvant formulations, of the current disclosure have equivalent adjuvanticity compared to GMP-grade QS21 and represent a suitable alternative to QS21 .Example 15b: antibody response

[0143] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 10 pg per dose (50 pL) of recombinant Plasmodium falciparum MSP1 protein either (i) non adjuvanted, (ii) adjuvanted with DOPC / Cholesterol / QS21 liposomes (5 pg of QS21 saponins), (iii) adjuvanted with DOPC / Cholesterol / saponin fraction DV liposomes (5 pg of saponin fraction DV), (iv) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / QS21 liposomes (1 pg of 3D(6-acyl)-PHAD and 5 pg of QS21 saponins), (v) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / saponin fraction DV liposomes (1 pg of 3D(6-acyl)- PHAD and 5 pg of saponin fraction DV), (vi) adjuvanted with squalene-in-water emulsion containing cholesterol and QS21 (5 pg of QS21 saponins) or (vii) adjuvanted with squalene- in-water emulsion containing cholesterol and saponin fraction DV (5 pg of saponin fraction DV), all adjuvant formulations being manufactured as in Example 9. Mice were bled on day 42 (3 weeks after the second injection). Anti-MSP-1 Ig responses were measured in mouse sera using enzyme linked immunosorbent assays (ELISA). Nunc Maxisorb™ plates (Thermo Fisher Scientific) were coated directly with MSP-1 protein at 1 pg / mL in PBS 1X overnight at 4°C, blocked for 1 h with PBS-t, PBS containing 0.05% (v / v) Tween 20 (Applichem), and 2% (w / v) bovine serum albumin (BSA) (Sigma). The plates were washed with PBS-t and incubated with 4x serial dilutions of mouse serum samples diluted 1 / 50 in PBS containing 0.05% (w / v) BSA. Plates were incubated for 1 h at 37°C, washed and then incubated for 1 h at 37°C with anti-mouse antibodies coupled to HRP (Southern Biotech) including goat anti-mouse total IgG or lgG2c at 1 :6000 dilution. Plates were then washed before addition of OPD (Sigma, P9187) substrate solution, the reaction was stopped at 30 minutes by adding 2M sulfuric acid. Absorbance was directly measured at 492 nm using a microplate reader (Biotek, Switzerland). Results are presented in Figure 23. Results show that 3 weeks after the second injection of vaccine, all saponin fraction DV-containing adjuvant formulations induced high total IgG antibody titers and high lgG2c antibody titers compared to the non-adjuvanted formulation. Furthermore, the saponin fraction DV-containing adjuvant formulations induced similar titers to the QS21 -containing counterpart formulations. We conclude from these data that the saponin fraction DV, and associated saponin fraction DV-containing adjuvant formulations, of the current disclosure have equivalent adjuvanticity compared to GMP-grade QS21 and represent a suitable alternative to QS21 for use in adjuvanted vaccines.Example 16: Pre-clinical evaluation in mice of saponin fraction DV-containing adjuvant formulations for a varicella zoster vaccineExample 16a: cellular response

[0144] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 2 pg per dose (50 pL) of Varicella-zoster recombinant glycoprotein E (g E) either (i) non adjuvanted, (ii) adjuvanted with DOPC / Cholesterol / QS21 liposomes (5 pg of QS21 saponins), (iii) adjuvanted with DOPC / Cholesterol / saponin fraction DV liposomes (5 pg of saponin fraction DV), (iv) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / QS21 liposomes (1 pg of 3D(6- acyl)-PHAD and 5 pg of QS21 saponins), (v) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)- PHAD / saponin fraction DV liposomes (1 pg of 3D(6-acyl)-PHAD and 5 pg of saponin fraction DV), (vi) adjuvanted with squalene-in-water emulsion containing cholesterol and QS21 (5 pg of QS21 saponins) or (vii) adjuvanted with squalene-in-water emulsion containing cholesterol and saponin fraction DV (5 pg of saponin fraction DV), all adjuvant formulations being manufactured as in Example 9. On day 28, spleens from mice were collected. Splenocytes were isolated by crushing spleens through a 70 pm cell strainer, then washed with incomplete RPMI medium (1% penicillin / streptomycin, 20 mM Hepes + 1X MEM NEAA) followed by red blood cell removal using Lympholyte®-M cell separation medium. One million cells were stimulated in round-bottom 96-well plates in complete RPMI medium (incomplete RPMI + 6% FCS + 50 pM p-mercaptoethanol) with 50 pL / well of gE protein medium at a final concentration of 10 pg / mL and incubated for 2 h at 37°C, 5% CO2. Cytokine secretion was blocked using GolgiPlug™ (available from BD Biosciences) protein transport inhibitor (50 pL / well, 1 :1000final dilution) and cells were further incubated overnight at 37°C, 5% CO2. Cells were then transferred to V-bottom 96-well plates and stained for 15 minutes at 4°C using the LIVE / DEAD™ Kit. Afterwards, cell surface staining was performed with purified anti- CD16 / CD32 (BD Bioscience), anti-CD3-FITC (eBioscience), anti-CD8a-PerCP-Cy5.5 (BioLegend®) and anti-CD4-Pacific Blue (BioLegend®) for 15 min on ice. After washing, fixation and permeabilization, cells were incubated for 30 min at 4°C with anti-IL-2-APC (BD Pharmingen™) and anti-IFNy-PE (BD Pharmingen™) for intracellular staining. Cells were washed 3 times with Perm / Wash buffer and were finally suspended in 300 pL PBS prior to acquisition on an Attune™ NxT flow cytometer (Invitrogen). Compensation was performed using the Arc™ Amine Reactive Compensation Bead Kit and AbC™ Total Antibody compensation bead kit (both from Invitrogen), and compensation matrices were calculated with the Attune™ NxT software (Invitrogen). FACS data were analyzed using FlowJo software (FlowJo LLC, USA). Results are shown as means of cumulated frequencies of cells expressing IL-2 and / or IFNy. Results are presented in Figure 24. Results show that all saponin fraction DV-containing adjuvant formulations induced CD4+ T cell responses higher than the non- adjuvanted formulations. Furthermore, the saponin fraction DV-containing formulations induced similar CD4+ T cell responses to the QS21 -containing counterpart formulations. We conclude from these data that the vaccines comprising saponin fraction DV, and associated saponin fraction DV-containing adjuvant formulations of the current disclosure, have equivalent adjuvanticity compared to vaccines comprising GMP-grade QS21 , and saponin fraction DV represents a highly suitable alternative to QS21 .Example 16b: antibody response

[0145] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 2 pg per dose (50 pL) of Varicella-zoster recombinant glycoprotein E (g E) either (i) non adjuvanted, (ii) adjuvanted with DOPC / Cholesterol / QS21 liposomes (5 pg of QS21 saponins), (iii) adjuvanted with DOPC / Cholesterol / saponin fraction DV liposomes (5 pg of saponin fraction DV), (iv) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)-PHAD / QS21 liposomes (1 pg of 3D(6- acyl)-PHAD and 5 pg of QS21 saponins), (v) adjuvanted with DOPC / Cholesterol / 3D(6-acyl)- PHAD / saponin fraction DV liposomes (1 pg of 3D(6-acyl)-PHAD and 5 pg of saponin fraction DV), (vi) adjuvanted with squalene-in-water emulsion containing cholesterol and QS21 (5 pg of QS21 saponins) or (vii) adjuvanted with squalene-in-water emulsion containing cholesterol and saponin fraction DV (5 pg of saponin fraction DV), all adjuvant formulations being manufactured as in Example 9. Mice were bled on day 42 (3 weeks after the second injection). Anti-gE Ig responses were measured in mouse sera using enzyme linked immunosorbentassays (ELISA). Nunc Maxisorb™ plates (Thermo Fisher Scientific) were coated directly with gE protein at 0.5 pg / mL in PBS 1 X overnight at 4°C, blocked for 1 h with PBS-t, PBS containing 0.05% (v / v) Tween 20 (Applichem), and 2% (w / v) bovine serum albumin (BSA) (Sigma). The plates were washed with PBS-t and incubated with 4x serial dilutions of mouse serum samples diluted 1 / 50 in PBS containing 0.05% (w / v) BSA. Plates were incubated for 1 h at 37°C, washed and then incubated for 1 h at 37°C with anti-mouse antibodies coupled to HRP (Southern Biotech) including goat anti-mouse total IgG or lgG2c at 1 :6000 dilution. Plates were then washed before addition of OPD (Sigma, P9187) substrate solution, the reaction was stopped at 30 minutes by adding 2M sulfuric acid. Absorbance was directly measured at 492 nm using a microplate reader (Biotek, Switzerland). Results presented in Figures 25a and b. Results show that 3 weeks after the second injection, all saponin fraction DV-containing adjuvant formulations induced high total IgG antibody titers and high lgG2c antibody titers compared to the non-adjuvanted formulation. Furthermore, vaccines comprising the saponin fraction DV- containing formulations induced similar titers to the QS21 -containing counterpart formulations. We conclude from these data that the saponin fraction DV, and associated saponin fraction DV-containing adjuvant formulations, of the current disclosure have equivalent adjuvanticity compared to GMP-grade QS21 , and represent a highly suitable alternative to QS21 for use in adjuvants and adjuvanted vaccines.Example 17: Pre-clinical evaluation of saponin fraction DV-containing cage-like structured adjuvant formulation for a COVID-19 vaccine in miceExample 17a: ManufacturingCage-like structures containing saponin fraction DV (CLS-DV) were produced by detergent dilution. Egg phosphatidylcholine and cholesterol were first dissolved at 10 mg / mL in a 20% solution of MEGA10 detergent (N-decanoyl-N-methylglucamine). This lipid solution was diluted with PBS and mixed with concentrated saponin fraction DV solution at 10 mg / mL in PBS. The final mixture had a molar ratio of saponin / cholesterol / lipid of 2:2:1 and a cholesterol final concentration of 0.8 mg / mL. The detergent was removed by consecutive steps of dialysis overnight with a molecular weight cut-off of 10 kDa. The resulting adjuvant was then diluted to working concentrations and sterile filtered through a 0.22 pm membrane under aseptic conditions. The sterile adjuvants were stored in sterile polypropylene containers and kept at 2-8°C until use. The same protocol was applied to produce cage-like structures comprising pharmaceutical grade QS21 (CLS-QS21 ) rather than saponin fraction DV. Transmissionelectron microscopy (TEM) confirmed both of the resulting formulations possessed cage-like structures. Representative TEM images of the CLS adjuvant formulations containing saponin fraction DV or pharmaceutical grade QS21 are provided in Figure 26 and Figure 27 respectively.Example 17b: cellular response

[0146] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 2 pg per dose (50 pL) of HexaPro (S6P), a stabilized Spike protein from SARS-CoV-2, either (i) non adjuvanted, (ii) adjuvanted with cage-like structured adjuvant containing saponin fraction DV (5 pg of saponin fraction DV) or (iii) adjuvanted with cage-like structured adjuvant containing QS21 saponin (5 pg of QS21 saponin). Spleens from vaccinated mice were collected on day 28. Splenocytes were isolated by crushing spleens through a 70 pm cell strainer, then washed with incomplete RPMI medium (1% penicillin / streptomycin, 20 mM Hepes + 1 X MEM NEAA) followed by red blood cell removal using Lympholyte®-M cell separation medium. One million cells were stimulated in round-bottom 96-well plates in complete RPMI medium (incomplete RPMI + 6% FCS + 50 pM p-mercaptoethanol) with 50 pL / well of peptide medium at a final concentration of 1 .5 pg / mL and incubated for 2 h at 37°C, 5% CO2. Cytokine secretion was blocked using GolgiPlug™ (available from BD Biosciences) protein transport inhibitor (50 pL / well, 1 :1000 final dilution) and cells were further incubated overnight at 37°C, 5% CO2. Cells were then transferred to V-bottom 96-well plates and stained for 15 minutes at 4°C using the LIVE / DEAD™ Kit. Afterwards, cell surface staining was performed with purified anti-CD16 / CD32 (BD Bioscience), anti-CD3-FITC (eBioscience), anti- CD8a-PerCP-Cy5.5 (BioLegend®) and anti-CD4-Pacific Blue (BioLegend®) for 15 min on ice. After washing, fixation and permeabilization, cells were incubated for 30 min at 4°C with anti- IL-2-APC (BD Pharmingen™) and anti-IFNy-PE (BD Pharmingen™) for intracellular staining. Cells were washed 3 times with Perm / Wash buffer and were suspended in 300 pL PBS prior to acquisition on an Attune™ NxT flow cytometer (Invitrogen). Compensation was performed using the Arc™ Amine Reactive Compensation Bead Kit and AbC™ Total Antibody compensation bead kit (both from Invitrogen), and compensation matrices were calculated with the Attune™ NxT software (Invitrogen). FACS data were analyzed using FlowJo software (FlowJo LLC, USA). Results are presented in Figures 28a and 28b as means of cumulated frequencies of cells expressing IL-2 and / or IFNy. The results show that the non-adjuvanted formulations produced at best barely detectable T cell responses. In contrast, the CLS adjuvant comprising saponin fraction DV induced detectable CD4+ T cell responses and CD8+ T cell responses similar to the QS21 -containing counterpart formulation. We conclude fromthese data that the saponin fraction DV, and associated cage-like structured saponin fraction DV-containing adjuvant formulation of the current disclosure, has comparable adjuvanticity to GMP-grade QS21 and benchmark QS21 -containing cage-like structured adjuvant formulation. We further conclude that saponin fraction DV is a very suitable alternative to QS21 for use as an adjuvant.Example 12c: antibody response

[0147] C57BL / 6J mice were vaccinated intramuscularly on day 0 and day 21 with 2 pg per dose (50 pL) of HexaPro (S6P), a stabilized Spike protein from SARS-CoV-2, either (i) non adjuvanted, (ii) adjuvanted with cage-like structured adjuvant formulation comprising saponin fraction DV (5 pg of saponin fraction DV) or (iii) adjuvanted with cage-like structured adjuvant formujlation comprising QS21 saponin (5 pg of QS21 saponin). Mice were bled on day 42 (3 weeks after the second injection). Anti-spike Ig responses were measured in mouse sera using enzyme linked immunosorbent assays (ELISA). Nunc Maxisorb™ plates (Thermo Fisher Scientific) were coated directly with S6P protein at 10 pg / mL in PBS 1 X overnight at 4°C, blocked for 1 h with PBS-t, PBS containing 0.05% (v / v) Tween 20 (Applichem), and 2% (w / v) bovine serum albumin (BSA) (Sigma). The plates were washed with PBS-t and incubated with 4x serial dilutions of mouse serum samples diluted 1 / 50 in PBS containing 0.05% (w / v) BSA. Plates were incubated for 1 h at 37°C, washed and then incubated for 1 h at 37°C with anti-mouse antibodies coupled to HRP (Southern Biotech) including goat anti-mouse total IgG or lgG2c at 1 :6000 dilution. Plates were then washed before addition of OPD (Sigma, P9187) substrate solution, the reaction was stopped at 30 minutes by adding 2M sulfuric acid. Absorbance was directly measured at 492 nm using a microplate reader (Biotek, Switzerland). Results are presented in Figure 29a and Figure 29b. Results show that 3 weeks after the second injection of vaccine formulation, vaccine comprising saponin fraction DV-containing CLS adjuvant formulation induced high total IgG antibody titers and high lgG2c antibody titers compared to the non-adjuvanted formulation. Furthermore, the vaccines comprising saponin fraction DV-containing CLS adjuvant formulation induced similar titers to the QS21 -containing counterpart CLS formulation. We conclude from these data that the vaccine comprising saponin fraction DV, and associated saponin fraction DV-containing CLS adjuvant formulations of the current disclosure have equivalent adjuvanticity compared to clinical-grade QS21 and represent a highly suitable alternative to QS21 for use in adjuvanted vaccines.REFERENCES1. Baig, A.T., Denet, F.G.C., Diaz-Garcia, J.J., Farrenburg, C.A., Lawrence, L.L., Myers, K.R., Sandvick, J.K., Vandenburg, J.Y. 2020. “Saponin purification”. WO 2019 / 106192 A1 Patent Application.2. Cleland, J.L., Kensil, C.R., Lim, A., Jacobsen, N.E., Basa, L., Spellman, M., Wheeler, D.A., Wu, J-Y., Powell, M.F. 1996. “Isomerization and formulation stability of the vaccine adjuvant QS-21”. Journal of Pharmaceutical Sciences. 85: 22-28.3. De Kesel, C.B.G., Farrenburg, C.A., Sandvick, J.K., Vandenburg, J.Y. 2020. “Saponin purification”. WO 2020 / 245207 A1 Patent Application.4. Fleck JD, Betti AH, da Silva FP, Troian EA, Olivaro C, Ferreira F, Verza SG. 2019. “Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular chemical characteristics and biological activities”. Molecules. 24: 171.5. Hostettmann, K., Marston, A. 1995. "Saponins". Cambridge University Press. Cambridge.6. Kensil, C.A., Marciani, D.J. 1991 . “Saponin adjuvant”. US Patent 5,057,540.7. Kensil, C.R., Patel, U., Lennick, M., Marciani, D. 1991. “Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex”. Journal of Immunology. 146: 431-437.8. Kite, G.C., Howes, M-J.R., Simmonds, M.S. J. 2004. “Metabolomic analysis of saponins in crude extracts of Quillaja saponaria by liquid chromatography / mass spectrometry for product authentication”. Rapid Communications in Mass Spectrometry. 18: 2859- 2870.9. Lacaille-Dubois, M. A. 2019. “Updated insights into the mechanism of action and clinical profile of the immunoadjuvant QS-21 : A review”. Phytomedicine. 60:1529059.10. Padilla-Iglesias, L., Valencia-Michaud, A. 2020. “Method for obtaining saponins from plants”. US Patent 10,660,924 B2.1 1. Ragupathi, G., Gardner, J.R., Livingston, P.O., Gin, D.Y. 2011. “Natural and synthetic saponin adjuvant QS-21 for vaccines against cancer”. Expert Review of Vaccines. 10: 463-470.Listed EmbodimentsItems of the invention:Item 1 . An immunologically active saponin composition extracted from Quillaja saponaria Molina comprising: a) at between approximately 50% wt / wt and approximately 80% wt / wt of the total composition, a major peak measured using UHPLC with UV absorbance at 214 nm,comprising a compound detectable at 1988 m / z using mass spectroscopy in negative ion mode, and b) one or more compounds corresponding to a peak at 1856 m / z detected using mass spectroscopy in negative ion mode.Item 2. The immunologically active saponin composition of item 1 , wherein the active saponin composition has one or more compounds corresponding to a peak at 1726 m / z detected using mass spectroscopy in negative ion mode.Item 3. The immunologically active saponin composition of item 1 or 2, wherein the active saponin composition has no compound at 2018 m / z detectable using mass spectroscopy in negative ion mode.Item 4. The immunologically active saponin composition of any of items 1 to 3, wherein the active saponin composition has no compound at 2480 m / z detectable using mass spectroscopy in negative ion mode.Item 5. The immunologically active saponin composition of any of items 1 to 4, wherein the active saponin composition has one or more of: a) a compound at 1726 m / z detectable using mass spectroscopy in negative ion mode, b) a compound at 1856 m / z detectable using mass spectroscopy in negative ion mode, c) a compound at 2031 m / z detectable using mass spectroscopy in negative ion mode, or d) a compound at 2003 m / z detectable using mass spectroscopy in negative ion mode. Item 6. A method of extracting and at least partially purifying the immunologically active saponin compositions of any of items 1 to 5, comprising: a) analysis of samples of raw material biomass from Quillaja saponaria Molina using UHPLC with UV absorbance at 214 nm, wherein the biomass is sampled from bark, trunk, leaves, stems, roots, seeds, flowers, fruits or any combination of thereof, and selection of biomass comprising at least 1 .5% by mass saponins comprising at least 50% a compound detectable at 1988 m / z and one or more compounds corresponding to a peak at 1856 m / z as determined using mass spectroscopy in negative ion mode, b) Harvesting biomass from Quillaja saponaria Molina, c) Extraction of saponins from the selected and harvested biomass by a multi-step process comprising: i) Contacting the harvested biomass with water at a temperature between 40°C to 90°C to create a liquid extract, ii) removal of non-saponin components by one or more of adsorption on organic or inorganic adsorbents, precipitation with an inorganic salt, and precipitation with anorganic solvent, followed by filtration, iii) further purification of the extract by nanofiltration, ultrafiltration and / or diafiltration to provide a saponin-rich liquid precursor, iv) drying of the saponin-rich liquid precursor to provide a solid extract comprising approximately 70% wt / wt to 99% wt / wt saponins, d) solubilization of the solid saponin extract of step (c) in an aliphatic alcohol followed by filtration and recovery of the filtrate to produce a concentrated extract, e) Performing a first fractionation using liquid chromatography with water and / or an organic solvent, followed by selection and pooling of eluate fractions using UHPLC with UV absorbance at 214 nm, comprising a compound detectable at 1988 m / z using mass spectroscopy in negative ion mode, and one or more compounds corresponding to a peak at 1856 m / z detected using mass spectroscopy in negative ion mode, to provide an enriched saponin fraction extract, f) Dissolving the enriched saponin fraction of step (e) in acetonitrile or aqueous methanol 10 to 50% v / v and passing through an inorganic filter to provide a filtered enriched saponin fraction, g) Performing a second fractionation using liquid chromatography with water and / or an organic solvent, followed by selection and pooling of eluate fractions comprising approximately using UHPLC with UV absorbance at 214 nm, comprising a compound detectable at 1988 m / z using mass spectroscopy in negative ion mode, and one or more compounds corresponding to a peak at 1856 m / z detected using mass spectroscopy in negative ion mode, to provide a pooled further enriched saponin subtraction, h) Concentration and drying of the pooled further enriched saponin sub-fraction of step(g) to provide a solid saponin sub-fraction comprising 85% to 99.7% wt / wt saponin, of which between approximately 50% and approximately 80% w / w of saponin measured using UHPLC with UV absorbance at 214 nm is a compound detectable at 1988 m / z using mass spectroscopy in negative ion mode, and further comprises one or more compounds detectable at 1856 m / z using mass spectroscopy in negative ion mode, wherein step (a) may be performed before or after step (b) to provide selected harvested biomass for use in step (c).Item 7. The method of item 6, wherein the aliphatic alcohol is methanol and the organic solvent is selected from dichloromethane, acetonitrile, acetic acid or a mixture thereof.Item 8. The method of item 6 or 7, wherein the harvested biomass is at least 70% bark.Item 9. The method of any of items 6, 7 or 8, wherein the inorganic filter is silica orsilica gel.Item 10. The method of any of items 6 to 9, wherein the drying is performed by vacuum drying, freeze drying or spray drying.Item 1 1. A saponin composition produced by the method of any of items 6 to 8.Item 12. An adjuvant composition comprising the immunologically active saponin composition of any of items 1 to 5, or 10.Item 13. The adjuvant composition of item 12, wherein the adjuvant composition comprising the immunologically active saponin composition is formulated as a liposome-based adjuvant formulation or an ISCOM-based adjuvant formulation.Item 14. The adjuvant composition of item 12 or 13, wherein the adjuvant composition is a liposome-based adjuvant comprising: a) 0.04 to 10 mg / mL total of one or more lipids, b) 0.1 to 5 mg / mL total of one or more sterols, and c) 0.04 to 0.4 mg / mL of the saponin fraction of any of items 1 to 5, formulated in an aqueous buffer.Item 15. The adjuvant composition of item 12 or 13, wherein the adjuvant composition is an ISCOM-based adjuvant comprising a) 0.002 to 0.8 mg / mL total of one or more lipids, b) 0.008 to 0.08 mg / mL total of one or more sterols, and c) 0.04 to 0.4 mg / mL of the saponin fraction of any items 1 to 5, formulated in a aqueous buffer.Item 16. The adjuvant composition of item 14 or 15, wherein the lipid is one or more phospholipid, DOPC, DSPC, DLPC, DMPC, DMPG, DPPC, DSPC, DLPG, DOPS, DOPE, DEPE, DMPE, DPPE, DMPS or salts thereof, the sterol is cholesterol or sitosterol, and the aqueous buffer is selected from phosphate buffer, succinate buffer, acetate buffer, citrate buffer or TRIS buffer.Item 17. The adjuvant composition of item 12, wherein the adjuvant composition is formulated as an emulsion-based adjuvant formulation.Item 18. The adjuvant composition of item 17, wherein the adjuvant composition comprises: a) 5 to 50 mg / mL total of one or more oils, b) 5 to 50 mg / mL total of one or more surfactants, c) 0.04 to 0.4 mg / mL of the saponin fraction of any of items 1 to 5 d) 0.1 to 5 mg / mL total of one or more sterols, dispersed in an aqueous buffer.Item 19. The adjuvant composition of item 18, wherein the oil is selected from squalane,squalene, soybean oil or a combination thereof; the one or more surfactants are selected from polyoxyethylene sorbitan esters, polysorbate, polyoxyethylene monostearate, sorbitan esters, or linear or branched EO / PO block copolymers; the sterol is selected from cholesterol, sitosterol or a combination thereof; and the aqueous buffer is selected from phosphate buffer, succinate buffer, acetate buffer, citrate buffer or TRIS buffer.Item 20. The adjuvant composition of any of items 12 to 19, further comprising one or more additional immunomodulators, adjuvants, agonists, antagonists, excipients, preservatives, or antioxidants.Item 21. The adjuvant composition of item 20, wherein the one or more additional immunomodulators is selected from one or more TLR agonist, MINCLE agonist, STING ligand, NKT cell activator, NOD ligand, NLR ligand, RLR ligand, CLR ligand or CDS ligand.Item 22. The adjuvant composition of item 21 , wherein the one or more additional immunomodulators is 3D(6-acyl)-PHAD, PHAD, 3D-PHAD, MPL, or MPLA.Item 23. The adjuvant composition of item 20, wherein the one or more excipients is selected from a sugar, gelatin or a salt, and the one or more preservatives or antioxidants is selected from B carotene, vitamin A, vitamin C, vitamin D, vitamin E, co-enzyme Q10, selenium or combinations thereof.Item 24. Use of the immunologically active saponin composition of any of items 1 to 5 or adjuvant composition of any of items 12 to 23 in the preparation of a medicament.Item 25. A medicament comprising the immunologically active saponin composition of any of items 1 to 5 or adjuvant composition of any of items 12 to 23, further comprising one or more antigens or antigen-encoding mRNA.Item 26. The medicament of item 25, wherein the antigens in the medicament comprise, or the antigen-encoding mRNA in the medicament encodes antigens that comprise, a peptide, protein, protein fragment, protein subunit, heptamer, trimer, dimer, VLP, fusion protein, whole- inactivated virus, live attenuated microbe, split-virion, polyvalent antigen or polysaccharide antigen.Item 27. The medicament of item 25 or 26, wherein the medicament is a vaccine for the treatment or prevention of an infectious disease, an auto-immune condition, an allergy or a cancer.Item 28. The medicament of any of items 25 to 27 wherein the vaccine is for the prevention or reduction in severity of one or more indications selected from, influenza, tuberculosis, shingles, herpes, measles, a rhinovirus, SARS-CoV-2, SARS-CoV-1 , rhinovirus, malaria, HIV / AIDS, leishmaniasis, Group A streptococcus, respiratory syncytial virus (RSV), Middle East Respiratory Syndrome virus (MERS), hepatitis, varicella zoster, coronavirus orhuman papilloma virus infection.Item 29. A kit of parts comprising: a) the adjuvant composition of any of items 12 to 23 in a first receptacle, and b) an antigen or antigen-producing mRNA in a second receptacle. Item 30. Use of the immunologically active saponin fraction of any of items 1 to 5, the adjuvant composition of any of items 12 to 23, the medicament of any of items 24 to 28, or the kit of parts of item 29, for the prevention or treatment of an infectious disease, an auto-immune condition, an allergy or a cancer.

Claims

Claims1. An immunologically active saponin composition extracted from Quillaja saponaria Molina comprising: a) at between 50% wt / wt and 80% wt / wt of the total composition, a major peak measured using UHPLC with UV absorbance at 214 nm, comprising a compound detected at 1988 m / z using mass spectroscopy in negative ion mode, and b) one or more compounds corresponding to a peak at 1856 m / z detected using mass spectroscopy in negative ion mode.

2. The immunologically active saponin composition of claim 1 , wherein the active saponin composition has one or more compounds corresponding to a peak at 1726 m / z detected using mass spectroscopy in negative ion mode.

3. The immunologically active saponin composition of claim 1 or 2, wherein the active saponin composition has no compound at 2018 m / z detectable using mass spectroscopy in negative ion mode.

4. The immunologically active saponin composition of any of claims 1 to 3, wherein the active saponin composition has no compound at 2480 m / z detectable using mass spectroscopy in negative ion mode.

5. The immunologically active saponin composition of any of claims 1 to 4, wherein the active saponin composition has one or more of: a) a compound at 1726 m / z detected using mass spectroscopy in negative ion mode, b) a compound at 1856 m / z detected using mass spectroscopy in negative ion mode, c) a compound at 2031 m / z detected using mass spectroscopy in negative ion mode, or d) a compound at 2003 m / z detected using mass spectroscopy in negative ion mode.

6. A method of extracting and at least partially purifying the immunologically active saponin compositions of any of claims 1 to 5, comprising: a) analysis of samples of raw material biomass from Quillaja saponaria Molina using UHPLC with UV absorbance at 214 nm, wherein the biomass is sampled from bark, trunk, leaves, stems, roots, seeds, flowers, fruits or any combination of thereof, and selection of biomass comprising at least 1 .5% by mass saponins comprising at least 50% a compound detectable at 1988 m / z and one or more compounds corresponding to a peak at 1856 m / z as determined using mass spectroscopy in negative ion mode, b) Harvesting biomass from Quillaja saponaria Molina, c) Extraction of saponins from the selected and harvested biomass by a multi-step process comprising:i) Contacting the harvested biomass with water at a temperature between 40°C to 90°C to create a liquid extract, ii) removal of non-saponin components by one or more of adsorption on organic or inorganic adsorbents, precipitation with an inorganic salt, and precipitation with an organic solvent, followed by filtration, iii) further purification of the extract by nanofiltration, ultrafiltration and / or diafiltration to provide a saponin-rich liquid precursor, iv) drying of the saponin-rich liquid precursor to provide a solid extract comprising approximately 70% wt / wt to 99% wt / wt saponins, d) solubilization of the solid saponin extract of step (c) in an aliphatic alcohol followed by filtration and recovery of the filtrate to produce a concentrated extract, e) Performing a first fractionation using liquid chromatography with water and / or an organic solvent, followed by selection and pooling of eluate fractions using UHPLC with UV absorbance at 214 nm, comprising a compound detected at 1988 m / z using mass spectroscopy in negative ion mode, and one or more compounds corresponding to a peak at 1856 m / z detected using mass spectroscopy in negative ion mode, to provide an enriched saponin fraction extract, f) Dissolving the enriched saponin fraction of step (e) in acetonitrile or aqueous methanol 10 to 50% v / v and passing through an inorganic filter to provide a filtered enriched saponin fraction, g) Performing a second fractionation using liquid chromatography with water and / or an organic solvent, followed by selection and pooling of eluate fractions comprising approximately using UHPLC with UV absorbance at 214 nm, comprising a compound detected at 1988 m / z using mass spectroscopy in negative ion mode, and one or more compounds corresponding to a peak at 1856 m / z detected using mass spectroscopy in negative ion mode, to provide a pooled further enriched saponin sub-fraction, h) Concentration and drying of the pooled further enriched saponin sub-fraction of step(g) to provide a solid saponin sub-fraction comprising 85% to 99.7% wt / wt saponin, of which between approximately 50% and approximately 80% w / w of saponin measured using UHPLC with UV absorbance at 214 nm is a compound detected at 1988 m / z using mass spectroscopy in negative ion mode, and further comprises one or more compounds detected at 1856 m / z using mass spectroscopy in negative ion mode, wherein step (a) may be performed before or after step (b) to provide selected harvested biomass for use in step (c).

7. The method of claim 6, wherein the aliphatic alcohol is methanol and the organicsolvent is selected from dichloromethane, acetonitrile, acetic acid or a mixture thereof.

8. The method of claim 6 or 7, wherein the harvested biomass is at least 70% bark.

9. The method of any of claims 6, 7 or 8, wherein the inorganic filter is silica or silica gel.

10. The method of any of claims 6 to 9, wherein the drying is performed by vacuum drying, freeze drying or spray drying.

11. A saponin composition produced by the method of any of claims 6 to 8.

12. An adjuvant composition comprising the immunologically active saponin composition of any of claims 1 to 5, or 10.

13. The adjuvant composition of claim 12, wherein the adjuvant composition comprising the immunologically active saponin composition is formulated as a liposome-based adjuvant formulation or an ISCOM-based adjuvant formulation.

14. The adjuvant composition of claim 12 or 13, wherein the adjuvant composition is a liposome-based adjuvant comprising: a) 0.04 to 10 mg / mL total of one or more lipids, b) 0.1 to 5 mg / mL total of one or more sterols, and c) 0.04 to 0.4 mg / mL of the saponin fraction of any of claims 1 to 5, formulated in an aqueous buffer.

15. The adjuvant composition of claim 12 or 13, wherein the adjuvant composition is an ISCOM-based adjuvant comprising a) 0.002 to 0.8 mg / mL total of one or more lipids, b) 0.008 to 0.08 mg / mL total of one or more sterols, and c) 0.04 to 0.4 mg / mL of the saponin fraction of any claims 1 to 5, formulated in a aqueous buffer.

16. The adjuvant composition of claim 14 or 15, wherein the lipid is one or more phospholipid, DOPC, DSPC, DLPC, DMPC, DMPG, DPPC, DSPC, DLPG, DOPS, DOPE, DEPE, DMPE, DPPE, DMPS or salts thereof, the sterol is cholesterol or sitosterol, and the aqueous buffer is selected from phosphate buffer, succinate buffer, acetate buffer, citrate buffer or TRIS buffer.

17. The adjuvant composition of claim 12, wherein the adjuvant composition is formulated as an emulsion-based adjuvant formulation.

18. The adjuvant composition of claim 17, wherein the adjuvant composition comprises: a) 5 to 50 mg / mL total of one or more oils, b) 5 to 50 mg / mL total of one or more surfactants, c) 0.04 to 0.4 mg / mL of the saponin fraction of any of claims 1 to 5 d) 0.1 to 5 mg / mL total of one or more sterols, dispersed in an aqueous buffer.

19. The adjuvant composition of claim 18, wherein the oil is selected from squalane, squalene, soybean oil or a combination thereof; the one or more surfactants are selected from polyoxyethylene sorbitan esters, polysorbate, polyoxyethylene monostearate, sorbitan esters, or linear or branched EO / PO block copolymers; the sterol is selected from cholesterol, sitosterol or a combination thereof; and the aqueous buffer is selected from phosphate buffer, succinate buffer, acetate buffer, citrate buffer or TRIS buffer.

20. The adjuvant composition of any of claims 12 to 19, further comprising one or more additional immunomodulators, adjuvants, agonists, antagonists, excipients, preservatives, or antioxidants.

21. The adjuvant composition of claim 20, wherein the one or more additional immunomodulators is selected from one or more TLR agonist, MINCLE agonist, STING ligand, NKT cell activator, NOD ligand, NLR ligand, RLR ligand, CLR ligand or CDS ligand.

22. The adjuvant composition of claim 21 , wherein the one or more additional immunomodulators is 3D(6-acyl)-PHAD, PHAD, 3D-PHAD, MPL, or MPLA.

23. The adjuvant composition of claim 20, wherein the one or more excipients is selected from a sugar, gelatin or a salt, and the one or more preservatives or antioxidants is selected from B carotene, vitamin A, vitamin C, vitamin D, vitamin E, co-enzyme Q10, selenium or combinations thereof.

24. Use of the immunologically active saponin composition of any of claims 1 to 5 or adjuvant composition of any of claims 12 to 23 in the preparation of a medicament.

25. A medicament comprising the immunologically active saponin composition of any of claims 1 to 5 or adjuvant composition of any of claims 12 to 23, further comprising one or more antigens or antigen-encoding mRNA.

26. The medicament of claim 25, wherein the antigens in the medicament comprise, or the antigen-encoding mRNA in the medicament encodes antigens that comprise, a peptide, protein, protein fragment, protein subunit, heptamer, trimer, dimer, VLP, fusion protein, whole- inactivated virus, live attenuated microbe, split-virion, polyvalent antigen or polysaccharide antigen.

27. The medicament of claim 25 or 26, wherein the medicament is a vaccine for the treatment or prevention of an infectious disease, an auto-immune condition, an allergy or a cancer.

28. The medicament of any of claims 25 to 27 wherein the vaccine is for the prevention or reduction in severity of one or more indications selected from, influenza, tuberculosis, shingles, herpes, measles, a rhinovirus, SARS-CoV-2, SARS-CoV-1 , rhinovirus, malaria, HIV / AIDS, leishmaniasis, Group A streptococcus, respiratory syncytial virus (RSV), MiddleEast Respiratory Syndrome virus (MERS), hepatitis, varicella zoster, coronavirus or human papilloma virus infection.

29. A kit of parts comprising: a) the adjuvant composition of any of claims 12 to 23 in a first receptacle, and b) an antigen or antigen-producing mRNA in a second receptacle.

30. Use of the immunologically active saponin fraction of any of claims 1 to 5, the adjuvant composition of any of claims 12 to 23, the medicament of any of claims 24 to 28, or the kit of parts of claim 29, for the prevention or treatment of an infectious disease, an auto-immune condition, an allergy or a cancer.