Saponin-based adjuvants and methods of characterization thereof

Abstract

Immunogenic compositions comprising adjuvants formed from nanoparticles of saponins in combination with cholesterol and phospholipids have specific interactions with body tissues and immune cells. These interactions include drainage of increased immune responses in lymph nodes, strong activation of T and B cells in lymph nodes, and rapid clearance of saponin fractions, while cholesterol fractions remain in the system.

Description

Related applications

[0001] This invention claims priority to U.S. Provisional Patent Application No. 63 / 592,974, filed October 25, 2023, and U.S. Provisional Patent Application No. 63 / 560,475, filed March 1, 2024, each of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates in general to adjuvants used with vaccines, particularly saponin-based vaccines, and methods for characterizing them. Background Technology

[0003] Vaccines are among the most successful and cost-effective methods for combating infectious diseases. Importantly, with the emergence of novel pathogens such as the SARS-CoV-2 virus; the cause of the COVID-19 pandemic, vaccines are also a critical part of pandemic preparedness. Such vaccines must generate sufficiently strong stimulation to drive the desired immune response and be clinically effective while maintaining acceptable tolerability and safety for widespread use. One promising approach is recombinant subunit vaccines, which utilize highly purified pathogen-specific recombinant proteins that enable the immune response to be specifically targeted to the relevant epitopes through robust and thermostable formulations. However, such recombinant proteins are often poorly immunogenic and require adjuvants to induce a strong antibody response. Vaccine adjuvants are compounds that can be used in vaccines to increase the strength of the immune response to a specific antigen and / or modulate the quality of that immune response. Matrix-M™ adjuvant is a novel adjuvant and a key component of the NVX-CoV2373 vaccine, which recently received human use authorization from multiple regulatory agencies, including the U.S. Food and Drug Administration and the European Medicines Agency, and is listed on the World Health Organization's Emergency Use Listing for COVID-19 Vaccines. Matrix-M™ adjuvant is also included in several other vaccine candidates that are in clinical development, including vaccines targeting seasonal influenza (alone or in combination with COVID-19), malaria, and Ebola virus disease.

[0004] Matrix-M™ adjuvant is a saponin-based adjuvant made from saponins from the Chilean soapberry tree (Quillaja Saponaria Molina), formulated with cholesterol and phospholipids into cage-like nanoparticles. Two types of these nanoparticles, Matrix-A™ and Matrix-C™ (each made from specific saponin components (fraction-A; Fr-A and fraction-C; Fr-C)), are mixed in a predetermined ratio to form Matrix-M™ adjuvant. The main component is Matrix-A™, which comprises 85% of Matrix-M™, with Matrix-C™ comprising the remaining 15%.

[0005] Matrix-M™ adjuvants offer antigen dose-saving properties and promote balanced Th1 / Th2 CD4+ T cell immune responses, driving the generation of IgG subclasses with distinct effector functions. Importantly, vaccines adjuvanted with Matrix-M™ have demonstrated amplified antibody responses to include cross-protective antibodies. Furthermore, adjuvanting purified protein antigens with Matrix-M™ can also induce antigen-specific CD8+ T cell responses. Crucially, several large clinical trials have demonstrated that vaccines adjuvanted with Matrix-M™ possess acceptable safety profiles.

[0006] To date, our understanding of the pharmacokinetics and biodistribution of saponin-based adjuvants is generally limited, particularly of Matrix-M™ adjuvants. In addition to addressing these issues, results from biodistribution studies can help further define the mechanism of action (MoA) and safety profile of Matrix-M™-adjuvanted vaccines. Therefore, understanding the biodistribution of Matrix-M™ is crucial. Summary of the Invention

[0007] This invention relates in general to saponin-based adjuvants and their effects on tissues and organs.

[0008] One embodiment of the present invention relates to a method of administering a vaccine composition to a mammalian subject, the method comprising injecting the vaccine composition into the subject at an intramuscular injection site, the vaccine composition comprising a saponin-based adjuvant, and the intramuscular injection site being associated with a draining lymph node (dLN). Following injection of the vaccine composition at the intramuscular injection site, the proportion of T cells in the dLN that are positive for i) CD45 and ii) the saponin-based adjuvant has a minimum value that is greater than one hour and less than 24 hours after injection of the vaccine composition at the intramuscular injection site, and that, at 24 hours after injection of the vaccine composition, the proportion of T cells in the dLN that are positive for i) CD45 and ii) the saponin-based adjuvant is greater than the minimum value.

[0009] Another embodiment of the invention relates to a method of administering a vaccine composition to a mammalian subject, the method comprising injecting the vaccine composition into the subject at an intramuscular injection site associated with a draining lymph node (dLN). The vaccine composition comprises a saponin-based adjuvant. The proportion of B cells in the dLN that are positive for i) CD45 and ii) the adjuvant reaches its maximum less than three hours after injection of the mixture at the intramuscular injection site during a period of approximately one hour to approximately twenty-four hours following injection of the vaccine composition.

[0010] Another embodiment of the invention relates to a method for administering a vaccine composition to a mammalian subject, the method comprising injecting the vaccine composition at an intramuscular injection site associated with a draining lymph node (dLN). The vaccine composition comprises an antigen and a saponin-based adjuvant, the saponin-based adjuvant comprising a saponin component and a cholesterol component. Following injection of the vaccine composition at the intramuscular injection site in the subject, the concentration of the saponin component in the subject's plasma peaks less than 3 hours after injection, while the concentration of the cholesterol component in the subject's plasma peaks more than 6 hours after injection.

[0011] Another embodiment of the invention relates to a method for administering a vaccine composition to a mammalian subject, the method comprising injecting the vaccine composition at an intramuscular injection site associated with a draining lymph node (dLN) in the subject. The vaccine composition comprises an antigen and a saponin-based adjuvant. The saponin-based adjuvant comprises a saponin component and a cholesterol component. Following injection of the vaccine composition at the intramuscular injection site in the subject, the concentration of the saponin component in one of the subject's intestines, lungs, and heart remains approximately zero for up to 168 hours post-injection, while the concentration of the cholesterol component in one of the subject's intestines, lungs, and heart peaks more than 24 hours post-injection.

[0012] Another embodiment of the invention relates to a method for administering a vaccine composition to a mammalian subject, the method comprising injecting the vaccine composition into the subject at an intramuscular injection site associated with a draining lymph node (dLN). A cytokine response induced by the vaccine composition at the dLN, measured 24 hours after injection of the mixture, is stronger than a cytokine response at the dLN measured 6 hours after injection of the mixture. Attached Figure Description

[0013] The invention can be more fully understood by taking into consideration the following detailed description of various embodiments of the invention in conjunction with the accompanying drawings, wherein: Figure 1 The representative structures of fraction-A and modified fraction-A are schematically illustrated, wherein the aldehyde at position C-23 on the triterpenoid core is reduced to a tritium-containing hydroxyl group.

[0014] Figure 2A and Figure 2B Mice were subcutaneously immunized on days 0 and 21 with 1 μg rS and 5 μg Matrix-M™, 1 μg rS and 5 μg Matrix-M™ 50 / 50 (formulated from 50% modified and 50% unmodified Fr-A material (Matrix-M™ 50 / 50)), or 1 μg SARS-CoV-2 rS alone (“Ag only”). Evaluation was performed by ELISA 20 days after the initial immunization. Figure 3 A) and 28 days ( Figure 3 B) Antibody titers against rS protein IgG1 and IgG2a, and hACE2 receptor inhibitory antibody titers, were obtained from serum samples. Individual titers for each symbol are shown, with horizontal bars representing the geometric mean titer and error bars representing 95% confidence intervals (CI). Data were analyzed using one-way ANOVA and Tukey's multiple comparisons test. Statistically significant differences between groups were indicated by * (p<0.05), ** (p<0.01), and **** (p<0.0001). Ten mice were tested (n=10).

[0015] Figure 3 The study design for the biodistribution study is presented, describing the group composition and the time points for measuring radioactivity by liquid scintillation counting.

[0016] Figures 4A-4D The distribution of saponins and cholesterol at the injection site and to local lymph nodes is shown. Matrix-M™ adjuvant is radiolabeled. 3 H-saponins or 3 H-cholesterol formulation, administered with or without SARS-CoV-2 rS antigen. Activity was measured using liquid scintillation in the following ways and expressed as % of the injection dose (ID) per gram of tissue: quadriceps femoris, Figure 4A Iliac lymph nodes Figure 4B Inguinal lymph nodes Figure 4C ; and popliteal lymph nodes, Figure 4D These lymph nodes are fused together on both sides. In each chart, each time point has a cluster with three columns of data points. The middle column with dark dots represents those marked with radio waves. 3 H-saponins and SARS-CoV-2 rS antigen (in) Figure 4CThe Chinese text is marked as "Matrix-M ( 3 Data obtained using H-Sap+ SARS-CoV-2 rS). The columns with lighter dots to the left of the middle column indicate saponins (in...) using individual radiolabeled saponins. Figure 4C The Chinese text is marked as "Matrix-M ( 3 Data obtained using H-Sap. The column with the light-colored dots to the right of the middle column represents cholesterol (in radiolabeled form). Figure 4C The Chinese text is marked as "Matrix-M ( 3 Data were obtained using H-cholesterol. Activity (DPM) below the LOQ (703 DPM) was defined as 703 DPM, and %ID / g tissue was determined accordingly. Each data point represents a single animal, and the horizontal bars represent the mean plus / minus standard deviation. Data points below the LOQ are shown as X. Data for each tissue and time point were analyzed individually using two-way ANOVA and Tukey's multiple comparison test. Statistically significant differences between groups were expressed as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. n = 5–6.

[0017] Figures 5A-5J The distribution of saponins and cholesterol to different sites around the body is shown. Matrix-M™ adjuvants are formulated with radiolabeled 3H-saponins or 3H-cholesterol, the former administered with or without the SARS-CoV-2 rS antigen. Activity is measured by liquid scintillation in the following and expressed as % of the injection dose (ID) per milliliter: plasma, Figure 5A Urine Figure 5B ;kidney, Figure 5C ;liver, Figure 5D ;intestinal, Figure 5E ;spleen, Figure 5F ;lung, Figure 5G ;marrow, Figure 5H ;heart, Figure 5I ; and brain, Figure 5J The columns for each data point at each time point are represented in accordance with the above description. Figures 4A-4D Data aggregated in the same manner as described above, i.e., the middle column of dark data points represents data obtained from radiolabeled saponins applied together with the SARS-CoV-2 rS antigen, the column of light data points to the left of the middle column represents data obtained from radiolabeled saponins alone, and the column of light data points to the right of the middle column represents data obtained using radiolabeled cholesterol, such as... Figure 5IAs shown. Activity (DPM) below LOQ (703 DPM) was defined as 703 DPM, and %ID / g tissue was determined accordingly. Each data point represents a single animal, and the horizontal bars represent the mean plus / minus standard deviation. Data points below LOQ are shown as X. Data for each tissue and time point were analyzed separately using two-way ANOVA and Tukey's multiple comparison test. Statistically significant differences between groups are expressed as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. n = 4–6.

[0018] Figures 6A-6C The distribution of saponins and cholesterol to reproductive organs is shown. Matrix-M™ adjuvants are formulated with radiolabeled 3H-saponins or 3H-cholesterols, the former administered with or without the SARS-CoV-2 rS antigen. Activity was measured by liquid scintillation in the following and expressed as % of the injected dose (ID) per gram of tissue: testes (male), Figure 6A ; ovary Figure 6B ; and uterus, Figure 6C The columns for each data point at each time point are represented in accordance with the above description. Figures 4A-4D Data aggregated in the same manner as described above, i.e., the middle column of dark data points represents data obtained from radiolabeled saponins applied together with the SARS-CoV-2 rS antigen, the column of light data points to the left of the middle column represents data obtained from radiolabeled saponins alone, and the column of light data points to the right of the middle column represents data obtained using radiolabeled cholesterol, such as... Figure 6A As shown. Activity (DPM) below LOQ (703 DPM) was set to 703 DPM, and %ID / g tissue was determined accordingly. Each data point represents a single animal, and the horizontal bars represent the mean plus / minus standard deviation. Data points below LOQ are shown as X. Data for each tissue and time point were analyzed separately using two-way ANOVA and Tukey's multiple comparison test. Statistically significant differences between groups are expressed as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. n = 2–3.

[0019] Figure 7 The reaction for generating fluorescently modified fraction-A from fraction-A is illustrated schematically.

[0020] Figure 8A and Figure 8BResults from flow cytometry analysis of different cell types using fluorescently labeled saponins are presented. The x-axis shows the percentage of ten different cell types that were CD45+ and linked to the fluorescently labeled Matrix-A™ at different time points (0.5 h, 1 h, 3 h, 6 h, 18 h, and 24 h). Cell types, from left to right, are neutrophils, monocytes, F4 / 80-monocytes, macrophages, dendritic cells (DCs), natural killer (NK) cells, natural killer T (NKT) cells, B cells, and other cell types. The y-axis shows the percentage of cell types that were positive for both CD45 and the fluorescently labeled Matrix-A™. Figure 8A Measurements taken at the intramuscular injection site are shown. Figure 8B The measurement taken at dLN is shown.

[0021] Figure 9A and Figure 9B A heatmap was presented showing the intramuscular injection site at 6 h, 24 h, 48 h, 72 h, and 168 h after injection of various adjuvants and NIV antigens. Figure 9A ) and dLN ( Figure 9B The presence of various cytokines at the site was assessed. In each case, mice were injected with an antigen containing Matrix-M™, an antigen containing AS01a, an antigen containing alhydrogel, or a single antigen without adjuvant. The cytokines tested were CXCL10, IL-1β, TNF-α, IL-30, IL-6, CXCL1, CCL2, IL-5, CXCL2, CCL3, IL-33, and IFN-γ.

[0022] While the invention may have various modifications and alternatives, its details have been shown by way of example in the accompanying drawings and will be described in detail. However, it should be understood that the purpose is not to limit the invention to the specific embodiments described. Rather, the purpose is to cover all modifications, equivalents, and alternatives that fall within the spirit and scope of the invention as defined by the appended claims. Detailed Implementation

[0023] definition As used herein and in the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a protein” may refer to a single protein or a mixture of such proteins, and reference to “the method” includes reference to equivalent steps and / or methods known to those skilled in the art, and so on.

[0024] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, amplifies or otherwise alters or modifies the immune response induced by that immunogen. Modification of the immune response may include enhancing or broadening the specificity of one or both of the antibody and cellular immune responses.

[0025] As used herein, the terms “about” or “approximately” preceding a numerical value indicate a range of that value plus or minus 10%. For example, “about 100” covers 90 and 110.

[0026] As used herein, “substantially” means the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance constitutes a substantial percentage of the sample containing it. For example, in a sample, the substantially purified component constitutes 85%, preferably 85%-90%, more preferably at least 95%-99.5%, and most preferably at least 99%. If the component is substantially displaced, the amount remaining in the sample is less than or equal to about 0.5% to about 10%, preferably less than about 0.5% to about 1.0%.

[0027] As used herein, the term "treatment" refers to a method for achieving a beneficial or desired outcome, such as a clinical result. For the purposes of this disclosure, a beneficial or desired outcome may include suppressing or inhibiting the onset or progression of an infection or disease; improving or reducing the development of symptoms of an infection or disease; or a combination thereof.

[0028] As used in this article, “prevention” can be used interchangeably with “prophylaxis” and can mean complete prevention of an infection or disease, or prevention of the development of symptoms of such an infection or disease; delay in the onset of an infection or disease or its symptoms; or reduction in the severity of an infection or disease or its symptoms that subsequently develop.

[0029] As used herein, “effective dose” or “effective amount” refers to the amount of antibody sufficient to induce an immune response that reduces at least one symptom of pathogen infection. An effective dose or effective amount can be determined, for example, by measuring the amount of neutralized secreted and / or serum antibodies, such as by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA), or microneutralization assay.

[0030] Matrix-A™ adjuvant One embodiment of the present invention typically relates to the biodistribution of radiolabeled saponins (Fr-A) or cholesterol incorporated into Matrix-A™ particles, comprising 85% of the Matrix-M™ adjuvant. The biodistribution patterns of these two key components are individually identified by head-to-head comparison of Matrix-A™ particles made from radiolabeled Fr-A or radiolabeled cholesterol. Furthermore, it was found that the presence of rS nanoparticle antigens included in the NVX-CoV2373 vaccine does not affect the biodistribution and / or excretion of saponins.

[0031] Vaccine adjuvants are substances that enhance and modulate the immunogenicity of antigens. Due to their ability to activate components of the innate immune system, adjuvants increase potency, broaden specificity, alter the humoral and cellular characteristics of the vaccine-induced immune response, and improve memory responses. These properties enable the development of vaccines against target populations (e.g., infants, the elderly, and immunocompromised individuals) and infectious diseases (e.g., malaria, shingles, and avian influenza), for which conventional vaccines have been unsuccessful or have limited efficacy. Additionally, adjuvants can help reduce the number of doses required for an immunized individual or decrease the amount of antigen required in each dose (dose-saving effect).

[0032] The strong demand for novel adjuvants is driven by the development of vaccines containing highly purified antigens derived from recombinant technologies. These adjuvants offer more precise characterization and are safer than live attenuated or inactivated whole-cell vaccines, but may have lower immunogenicity. In fact, most older live attenuated, inactivated, or toxoid vaccines "have their own adjuvanting function" because they contain intrinsic molecules known as pathogen-associated molecular patterns (MAMBs), which are recognized by cells of the innate immune system, such as monocytes, macrophages, and dendritic cells (DCs), via a series of so-called pattern recognition receptors. In contrast, purified recombinant protein vaccines typically lack these microbial patterns necessary to trigger an innate immune response. Adding an adjuvant to the vaccine antigen triggers a rapid innate immune response at the injection site and / or draining lymph nodes (dLNs) by attracting and activating antigen-presenting cells (APCs). APCs can then process the antigen and present it to CD4+ and CD8+ T cells. Both T cell types are capable of providing effector cellular functions, and CD4+ T cells contribute to the generation of a mature adaptive immune response. This acute response creates the conditions for the development of immune memory, which can persist for years and elicit a more rapid response in subsequent infections. Adjuvants also influence the type of adaptive immune response induced by vaccine antigens by modifying the initial signals provided to the innate immune system. Although adjuvants have been used for over a century, the mechanisms of action of the most widely used human adjuvants (aluminum salts, oil-in-water emulsions, saponin-based adjuvants (SBAs), and Toll-like receptor (TLR) agonists) are not fully understood, and animal models may not fully reflect the more nuanced circumstances found in human data.

[0033] Saponins are a large family of glycoconjugates that share a triterpenoid structure with various glycoside side chains. Most saponins used in vaccine adjuvants and foods are extracted from the bark of the South American soapbark tree using sustainable processes. These bark extracts contain a heterogeneous mixture of dozens of closely related saponins, exhibiting structurally different glycosylation or acylation patterns that also affect their biological activity. Among the many components that can be purified, South American soap treeQS-21 is structurally and functionally most characteristic and associated with potent adjuvant activity. However, QS-21 is chemically unstable under alkaline conditions, exhibits hemolytic activity in vitro, and is associated with immediate pain at the injection site. These limitations of free QS-21 can be mitigated by incorporating it into particles with cholesterol (as in liposome-based adjuvant systems 01 (AS01) and Army Liposome Formulation Q (ALFQ)) or with combinations of cholesterol and phospholipids (as in immunostimulatory complexes (ISCOM) or Matrix-M™ nanoparticles). In addition to “quenching” the hemolytic activity of saponins, formulation into nanoparticles allows for targeted delivery of adjuvants to phagocytes, thereby concentrating stimulation on the appropriate cells. Saponin adjuvants can also synergize with other classes of adjuvants, such as TLR agonists. This property is used in the liposome-based adjuvant AS01, which binds QS-21 and the TLR4 agonist MPL (3-O-deacyl-4'-monophosphoryl lipid A) (a type of Salmonella Minnesota). Salmonella minnesota (A non-toxic derivative of lipopolysaccharide). The QS-21 and MPL components of this adjuvant enhance antigen-specific antibody responses and promote T-cell responses. AS01 is currently used in vaccines against malaria and shingles (both developed by GSK (Rixensart, Belgium)). A synergistic adjuvant effect of saponins and MPL can also be observed when saponins and MPL are incorporated into ISCOM nanoparticles.

[0034] ISCOM initially identified the adjuvant activity present in these glycoproteins and bark extract fractions. soap tree ISCOM was developed through co-formulation with saponins to improve the immunogenicity of membrane-derived viral glycoproteins. The characteristic structure of ISCOM relies on the strong affinity between saponins and cholesterol. ISCOM is composed of... soap treeStable particles formed from saponins, cholesterol, and phospholipids, in which multiple copies of the antigen are physically incorporated into the saponin and lipid matrix. ISCOMs are 40 nm diameter particles with a typical rigid cage-like structure. ISCOMs induce strong and durable antigen-specific humoral and cellular immune responses, including CD4+ helper T cells and CD8+ cytotoxic T cells. However, a limitation of the ISCOM system is that only hydrophobic membrane proteins can be readily incorporated into ISCOMs without modification. Manufacturing challenges that arise when incorporating a wider variety of antigens into ISCOM particles led to the discovery of similar characteristic structures even without antigen incorporation. These complexes, later known as matrices, constitute an effective adjuvant that can be easily mixed with a variety of antigens. Although the antigen is not physically linked to the particle, these formulations retain the ability of ISCOMs to induce strong humoral and cellular immune responses, and because they are not limited to hydrophobic membrane proteins, they have the potential for a wider range of applications.

[0035] This technology was further developed to produce Matrix-M™ formulations. Matrix-M adjuvants consist of two distinct groups of physically stable nanoparticles mixed in a specific ratio (85% Matrix-A + 15% Matrix-C). Matrix-A™ and Matrix-C™ contain different complementary properties. soap tree Saponin fractions. Matrix-C particles contain fraction-C saponins (primarily composed of QS-21), which have strong adjuvant activity but are reactive in mice, as measured by stupor and lethality. Matrix-A particles contain fraction-A saponins, which, at the same dose, have weaker adjuvant activity than fraction-C but are better tolerated in mice. Nanoparticles contain cholesterol and phospholipids. Combinations of these two particle types were tested, and this combination reduced the reactiveness observed in animal models while maintaining adjuvant activity. Additionally, Matrix-M is stable in aqueous solutions at 2–8°C for several years. Matrix-M is an adjuvant used in the NVX2373 SARS-CoV-2 recombinant spike protein vaccine (Novavax) and several vaccines currently or already being evaluated in clinical trials. These trials included COVID-19, seasonal influenza, COVID-19 + seasonal influenza, epidemic (H5N1) influenza, avian (H7N9) influenza, respiratory syncytial virus (RSV), malaria, Ebola, genital herpes simplex virus (HSV), rabies, and Epstein-Barr virus.

[0036] Preparation, administration and delivery In addition to the antigen, the pharmaceutical formulation of the present invention also contains a Matrix-M™ adjuvant. The formulation may contain a naked antibody, immunoconjugate, or fusion protein as an antigen, effective in producing the desired response in a weight or volume unit suitable for administration to a human patient, and is preferably sterile.

[0037] The composition can be formulated with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the bioactivity of the active ingredient. Such preparations may routinely contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also routinely contain compatible solid or liquid fillers, diluents, or encapsulating substances suitable for human administration. When used in pharmaceuticals, salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be conveniently used to prepare their pharmaceutically acceptable salts, and are not excluded from the scope of this invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, etc. Furthermore, pharmaceutically acceptable salts can be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts. The term "carrier" refers to a natural or synthetic organic or inorganic component to which the active ingredient binds to facilitate application. Components of the pharmaceutical composition can also be mixed with the antibodies of the present invention in a manner that prevents interactions from significantly impairing the desired pharmaceutical efficacy.

[0038] According to certain aspects of the invention, the composition can be achieved by combining an antigen having the desired purity with an optional physiologically acceptable carrier, excipient, or stabilizer. Remington's Pharmaceutical Sciences(16th edition, Osol, A. ed. (1999)) is prepared in the form of a lyophilized formulation or an aqueous solution for storage. Acceptable carriers, excipients, or stabilizers are non-toxic to the receptor at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins, etc. Examples of active ingredients include serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN, PLURONICS™, or polyethylene glycol (PEG).

[0039] The composition may also optionally contain suitable preservatives, such as benzalkonium chloride; chlorobutanol; parabens and thimerosal.

[0040] Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation, preferably isotonic with the recipient's blood. This preparation can be formulated using suitable dispersants or wetting agents and suspending agents according to known methods. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents (e.g., solutions of 1,3-butanediol). Acceptable media and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, non-volatile oils are routinely used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid can be used in the preparation of injectable formulations. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc., can be... Remington's Pharmaceutical Sciences It can be found in Mack Publishing Co., Easton, PA.

[0041] Active ingredients can also be encapsulated in microcapsules, for example, prepared by coagulation technology or by interfacial polymerization, such as in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions in hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules. Remington's Pharmaceutical Sciences This technique was disclosed in the 16th edition, Osol, A. (1980).

[0042] Formulations intended for internal administration are typically sterile. This is easily achieved through filtration via sterile filter membranes.

[0043] In some embodiments, the pharmaceutical composition of the present invention is stable at 4°C. In some embodiments, the pharmaceutical composition of the present invention is stable at room temperature.

[0044] The compositions of the present invention may be administered to human patients via any route, including but not limited to intravenous, intradermal, transdermal, subcutaneous, intramuscular, and inhalation (e.g., via The compositions of the invention may be administered via aerosol, oral (e.g., sublingual), topical (i.e., skin and mucous membrane surfaces, including airway surfaces), intrathecal, intra-articular, intrapleural, intracerebral, intra-arterial, intraperitoneal, oral, intralymphatic, intranasal, rectal, or vaginal administration, by perfusion through a local catheter, or by direct intralesional injection. In one embodiment, the compositions of the invention are administered by intravenous bolus or intravenous infusion over a prescribed period (e.g., 0.5 to 2 hours). The compositions of the invention may be delivered by peristalsis or in the form of a reservoir; however, as is well known in the art, the most suitable route in any given case will depend on factors such as the species, age, sex, and general condition of the subject, the nature and severity of the condition being treated, and / or the nature of the particular composition being administered (i.e., dosage, formulation).

[0045] In one embodiment, the dose of the antigen-containing composition is measured in mg (antigen) / kg patient body weight. In other embodiments, the dose of the antigen-containing composition is measured in mg / kg patient lean body weight (i.e., body weight minus body fat percentage). In still other embodiments, the dose of the antigen-containing composition is measured in mg / m 2 The patient's body surface area is measured. In other embodiments, the dose of the composition containing the antigen is measured in mg per dose administered to the patient. Any dose measurement can be used in conjunction with the compositions and methods of the present invention, and the dose units can be converted using standard methods in the art.

[0046] In some embodiments, the amount of adjuvant present in the composition can range from about 1 μg to 500 μg, and in other embodiments, from about 25 μg to about 100 μg. In some embodiments, the composition may contain 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, or 75 μg of adjuvant.

[0047] Those skilled in the art will understand that the dosage can be selected based on many factors, including the subject's age, sex, species, and condition. For example, the effective amount of the compositions of the present invention can be inferred from dose-response curves obtained from in vitro testing systems or animal model (e.g., rats or monkeys) testing systems. Models and methods for evaluating antibody activity are known in the art (Wooldridge et al., Blood , 89(8): 2994-2998 (1997)), this reference is incorporated into this paper in its entirety through citation.

[0048] Examples of dosing regimens that can be used in the methods of the present invention include, but are not limited to, daily, three times a week (intermittent), weekly, every 14 days, monthly, every 6-8 weeks, every 2 months, every 6 months, or annually. Other dosing regimens may include a single dose, or may include a second dose administered at some time after the initial dose, such as at an interval of two weeks, one month, six months, or one year from the initial dose.

[0049] Experimental methods Female BALB / c mice (8-10 weeks old) and female / male CD-1 IGS mice (8 weeks old) were obtained from Charles River Laboratories (CRL, Germany). At termination, the animals were euthanized by cervical dislocation (SVA) or by deep anesthesia with pentobarbital (180 mg / kg, intraperitoneal).

[0050] Matrix-M™ adjuvant consists of two 40 nm cage-like particles made from two separate saponin fractions, Matrix-A™ and Matrix-C™ (85% and 15%, respectively). Matrix-A™ and Matrix-C™ particles are formed by formulating purified saponin fractions Fr-A and Fr-C (derived from the tree soapberry) with cholesterol and phospholipids.

[0051] To track the biodistribution of saponins, all saponins in Fr-A were radiolabeled with tritium (3H). The aldehyde group on the triterpenoid aglycone of quillaic acid (position C-23) was reduced to a hydroxyl group using tritium (3H)-labeled sodium borohydride [3H]-NaBH4, resulting in modified Fr-A materials. Figure 1 A). To synthesize deuterium (2H)-labeled Fr-A used in the adjuvant evaluation of modified materials, sodium borodeuteride (NaBD4) was used as a reducing agent. Modified Fr-A was mixed with unmodified Fr-A at a 50:50 ratio and then formulated into Matrix-A™ adjuvant particles. These particles were subsequently mixed with unmodified Matrix-C™ at an 85:15 ratio to form Matrix-M™ adjuvant [Matrix-M (3H-Sap)], also known as Matrix-M™ 50 / 50. To track the biodistribution of cholesterol, tritium-labeled cholesterol [1-2-3H(N)] (Perkin Elmer, Bridgeport CT) was used to formulate radiolabeled Matrix-A™ particles, which, together with unmodified Matrix-C™, were used to form Matrix-M™ adjuvant [Matrix-M (3H-cholesterol)].

[0052] SARS-CoV-2 rS (constructor BV2373, Novavax, Inc., Gaithersburg, MD) was constructed from the full-length wild-type SARS-CoV-2 S glycoprotein based on the GenBank gene sequence MN908947 (nucleotides 21563–25384). The native full-length S protein was modified to possess protease resistance by mutating RRAR to QQAQ (3Q) at the putative furin cleavage site within the S1 / S2 cleavage domain. Two additional proline amino acid substitutions were inserted at positions K986P and V987P (2P) within the heptad repeat 1 (HR1) domain to stabilize the SARS-CoV-2 rS in the pre-fusion conformation. The synthetic transgenic engineering has been incorporated into the baculovirus vector (BV2373) for expression in the fall armyworm (Spodoptera frugiperda) (Sf9) insect cells, thereby producing the SARS-CoV-2rS protein.

[0053] BALB / c mice were subcutaneously immunized at the base of the tail at a total volume of 100 μL / mouse on days 0 and 21. Three groups of animals (n = 10 each) received 1 μg of SARS-CoV-2 rS either unadjuvanted or in combination with 5 μg of Matrix-M™ adjuvant or 5 μg of Matrix-M™ adjuvant containing modified (2H-labeled) Matrix-A™ (Matrix-M™ 50 / 50). Blood was collected from the lateral tail vein of fully conscious animals on days 20 and 28. Serum was extracted from the blood and stored at -20°C until analysis.

[0054] CD-1 IGS mice were intramuscularly immunized while anesthetized with isoflurane, with a total volume of 40 μL entering the right quadriceps femoris muscle. An overview of the study group and termination time points is provided below. Figure 3 In summary, mice received a single dose of either Matrix-M adjuvant containing radiolabeled saponins (10 μg) [Matrix-M (3H-Sap)], or Matrix-M adjuvant containing radiolabeled saponins mixed with SARS-CoV-2 rS (1 μg) (10 μg) [Matrix-M (3H-Sap) + SARS-CoV-2 rS)], or Matrix-M adjuvant containing radiolabeled cholesterol administered alone (10 μg) [Matrix-M (3H-cholesterol)] [n = 42 per condition, 6 mice (3 females, 3 males) at each time point]. The weight of the syringe was recorded before and after administration to measure the weight of the injected solution. The radioactivity (DPM / g) of the administered formulation was determined by liquid scintillation counting (MicroBeta2, Perkin Elmer), and the injected (radioactive) dose (ID) was calculated and used to determine normalized radioactivity, expressed as a percentage of the injected dose per gram of tissue (as described below).

[0055] On the day of sacrifice, the animal was weighed and then euthanized. Samples were then collected in tare-weighted tubes and weighed. First, blood samples were collected using cardiac puncture to prepare plasma for bioactivity analysis. Immediately after blood sampling, the animal was perfused with heparinized saline (2.5 IU / mL), and tissue samples were collected up to a maximum weight of 200 mg. The following samples were collected: injection site (quadriceps femoris, QF), iliac lymph nodes (LN), inguinal LN, popliteal LN, plasma, urine, kidneys, liver, intestines, spleen, lungs, bone marrow, heart, brain, axillary LN, mandibular LN, mesenteric LN, testes, ovaries, and uterus. Right and left LNs were combined.

[0056] For scintillation analysis of total radioactivity, tissue samples were mixed with 0.1 mL of 0.9% saline and then homogenized at +4 °C at a frequency of 30 s-1 (TissueLyser II, Qiagen) for 2 min. One milliliter of Solvable (PerkinElmer) was added to the homogenized tissue and the samples were incubated at +60 °C for 2 h. Finally, the dissolved tissue homogenate was diluted 1:10 in UltimaGold (Perkin Elmer). Samples of lymph nodes were not homogenized but were directly dissolved in 0.5 mL of Solvable. The processed tissue samples were analyzed in a microplate counter (MicroBeta2; Perkin Elmer).

[0057] The radioactivity of the samples was measured as disintegrations per minute (DPM) and converted to % of injected radioactivity per gram of tissue (normalized %ID / g; relative radioactivity / activity) using the following formula: ((MD / (IS x DR)) x 100) / TW, where MD is the measured dose in DPM, IS is the amount of injected solution in grams (g), DR is the dose radioactivity in DPM / g, and TW is the tissue weight (g). The estimated limit of quantification (LOQ) was 703 DPM, which corresponded to a range of 0.0358–7.15 %ID / g tissue after normalization, depending on the quality of the tissue samples analyzed. Samples with DPM values <LOQ were set to 703 DPM before calculating % ID / g.

[0058] Anti-S IgG1 and IgG2a antibodies in sera from days 20 and 28 were quantified by enzyme-linked immunosorbent assay (ELISA). Ninety-six-well MaxiSorp microplates (Nunc) were coated overnight at 4°C with PBS containing 1.5 μg / mL SARS-CoV-2 rS protein (BV2373). Individual sera were serially diluted in PBS containing 0.05% Tween-20 (PBS-T) and 1% bovine serum albumin (BSA) in deep-well plates. Serum samples were serially diluted 5-fold in eight steps, starting with 1:300 or 1:1000 (day 20) and 1:300 or 1:10,000 (day 28) for IgG1; and starting with 1:15, 1:100 or 1:500 (day 20), and 1:15, 1:100 or 1:1000 (day 28) for IgG2a. Samples were then added, either singly (day 20) or in duplicate (day 28), to antigen-coated microtiter plates and incubated at room temperature for 2 hours. Combined serum from untreated BALB / c mice and serum from mice immunized with SARS-CoV-2 rS (containing Matrix-M adjuvant) served as negative and positive controls, respectively. After washing with PBS-T, diluted HRP-linked secondary antibody, anti-IgG1, or anti-IgG2a (BIORAD Laboratories, Hercules, CA, United States) was added and incubated at room temperature for 2 hours. After washing, TMB substrate was added and incubated for 10 min, then the reaction was terminated with 1.8 M sulfuric acid. Absorbance was measured at 450 nm (SpectraMax M3, Molecular Devices). Anti-S titers were calculated using a four-parameter logic equation (SoftMax software v.6.5.1). The inflection point (EC50 value) of the titration curve was based on an estimated curve fit defined using a four-parameter logic equation. This was used as the titer value. For titers below the limit of detection (LOD), report titers <15 (starting dilution) and assign the value of “15” to the sample to calculate the group average titer.

[0059] The titer of hACE2 receptor blocking antibodies was determined by ELISA. Ninety-six-well plates were coated overnight at 4°C with 1.0 μg / mL SARSCoV-2 rS protein. The coated wells were then blocked for 1 h at room temperature with StartingBlock™ (TBS) blocking buffer (ThermoFisher Scientific). Mouse serum was serially diluted 2-fold at a 1:20 dilution and added to the coated wells, incubated at room temperature for 1 h. After washing, 30 ng / mL histidine-tagged hACE2 (Sino Biologics, Beijing, CN) was added to the wells and incubated at room temperature for 1 h. HRP-conjugated antihistidine IgG was added and incubated for 1 h, followed by the addition of TMB substrate. Absorbance was measured at 450 nm using a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, United States), and data were analyzed using SoftMax Pro 6.5.1 GxP software. The serum antibody titer at which hACE2 achieves 50% inhibition (IC50) of the SARS-CoV-2 rS protein was then determined. For titers below the limit of detection (LOD), half the value of the initial dilution (a value of "10") was allocated to the samples to calculate the group mean titer.

[0060] Statistical analysis was performed on the data using GraphPad Prism software (version 9). For IgG1, IgG2a, and hACE2 data, the geometric mean titer (GMT) with a 95% confidence interval (CI) was calculated for each group, and the log10 transformed mean titer measurements were compared between groups using one-way ANOVA and the Tuki multiple comparison test. Comparisons of %ID / g or %ID / mL between groups at different time points were performed using two-way ANOVA, followed by the Tuki test. A p-value <0.05 was considered statistically significant.

[0061] Flow cytometry experiments were performed in BALB / c mice that underwent intramuscular immunization while anesthetized with isoflurane, with a total volume of 50 μL entering the right quadriceps femoris muscle. Mice received a single dose of Matrix-M adjuvant (5 μg), with 10% fraction-A of Matrix-A fluorescently labeled. Termination was performed at 0.5 h, 1 h, 3 h, 6 h, 18 h, and 24 h. At each time point, five mice were euthanized, and the right quadriceps femoris muscle (injection site) and right iliac lymph nodes (primary draining lymph nodes) were collected. Samples were prepared into single-cell suspensions, and cells were labeled with fluorescently labeled antibodies as described in Tables 1 and 2. Several different immune cell populations (neutrophils, monocytes / macrophages, dendritic cells, and lymphocytes) were analyzed by flow cytometry. Flow cytometry analysis was performed using FlowJo v10 10.9.0 (BDLife Sciences).

[0062] Table 1

[0063] *Note that the specified B-cell markers included in the assay panel are not present. B cells are instead defined as MHC II+ cells, which are neither monocytes, macrophages, nor dendritic cells (DCs), and are negative for neutrophils, T-, NKT-, and NK cell markers. These cells also exhibit lymphocyte characteristics in the FSC vs. SSC plots. Table 2

[0064] To evaluate the cytokine and chemokine responses induced by adjuvants in conjunction with quadrivalent nanoparticle influenza vaccines (NIV) (A / Michigan, A / Singapore, B / Phuket, B / Iowa), 6 µg of hemagglutinin (HA) / NIV strain was administered in the hind leg with three adjuvants: 5 µg Matrix-M1 adjuvant and 3 µg AS01. B Intramuscular immunization was administered to a total of 125 eight-week-old female BALB / c mice (n = 25 mice / group) with either 200 µg of aluminum salt (Alum) or PBS alone. Ag (NIV) and PBS were used as controls. Five mice from each group were sacrificed at 6, 24, 48, 72, and 168 hours after the initial immunization (ppi) to collect samples from the injection site (muscle) and draining lymph nodes (dLN).

[0065] Tissues were collected in tubes and flash-frozen in a dry transport container filled with liquid nitrogen until homogenization was performed to extract soluble proteins. In short, tissues were placed in cold PBS containing a protease inhibitor (Halt™ protease inhibitor cocktail, Thermo Scientific, Gothenburg, Sweden) and homogenized as quickly as possible on ice using a tissue homogenizer to avoid heating the sample. To collect the supernatant, the sample was briefly centrifuged and then transferred to microtubes for homogenization at 10,000 × 10⁻⁶ at 4 °C. g Centrifuge for another 10 minutes. Centrifuge the dLN sample once and the muscle sample twice, transferring the supernatant to a new tube after each centrifugation. Then store the supernatant at -70°C until further analysis.

[0066] Cytokine and chemokine concentrations in samples were analyzed using electrochemiluminescence at the Clinical Biomarker Facility of SciLifeLab, Uppsala University, Sweden, using the V-PLEX PlusMouse Cytokine 19-Plex kit from Meso Scale Discovery (MSD; Rockville, MD, USA). Samples were diluted according to the manufacturer's instructions prior to analysis. The following analytes were measured on a MESO QuickPlex SQ 120: IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-15, IL-17A / F, IL-27p28 / IL-30, IL-33, IP-10, KC / GRO, MCP-1, MIP-1α, MIP-2, and TNF-α.

[0067] result Experiment 1: Adjuvant activity of Matrix-M™ containing partially modified saponins of Matrix-A™ Radiolabeling Fr-A by reducing the aldehyde substituent on the saponin triterpenoid aglycone (position C-23) enables the tracking of the biodistribution of saponins after immunization with Matrix-M™ adjuvant. Figure 1In a two-dose immunization regimen using SARS-CoV-2 rS antigen, the adjuvant properties of unmodified Matrix-M™ adjuvant were evaluated in BALB / c mice compared to Matrix-M™ formulated with 50% modified and 50% unmodified Fr-A material (Matrix-M™ 50 / 50), to confirm retention of adjuvant properties using the regimen illustrated in Figure 2. On day 20 post-primary immunization, similar titers of SARS-CoV-2 rS-specific IgG1 and IgG2a, as well as similar titers of functional human angiotensin-converting enzyme 2 (hACE2) receptor blocking antibodies, were found in the sera of both adjuvanted groups. Figure 3 A), both of which were significantly superior to the unadjuvanted antigen. On day 28, the IgG1 anti-rS and hACE2 receptor inhibitory antibody responses were slightly reduced in the group receiving SARS-CoV-2 rS adjuvanted with Matrix-M™ 50 / 50 ( Figure 3 (B) The IgG2a anti-rS titer remained substantially the same in both adjuvanted groups. Comparison with unadjuvanted SARS-CoV-2 rS immunization showed adjuvant effects of unmodified Matrix-M™ and Matrix-M™ 50 / 50 on rS-specific IgG1 and IgG2a, as well as hACE receptor blocking antibodies, at days 20 and 28. Therefore, while the definitive adjuvant function of Matrix-M™ 50 / 50 as assessed by humoral immunity is slightly reduced, it suggests that modified Matrix-M™ 50 / 50 is suitable as a representative Matrix-M™ adjuvant for tracking the biodistribution of saponins. Of interest, Matrix-M™ containing labeled saponins showed no reduction in its ability to induce antigen-specific IgG2a responses, a hallmark of the known trend of Matrix-M™ supporting strongly Th1-biased T cell responses.

[0068] Experiment 2: Local biological distribution To investigate the local biodistribution and particle integrity of Matrix-M™ adjuvant after intramuscular injection in CD-1 mice, radioactivity in the quadriceps femoris muscle (QF; injection site) and draining hind limb lymph nodes (LN) was analyzed at seven time points from 1 h to 168 h (7 days) after injection of Matrix-M™ adjuvant containing radiolabeled saponins (+ / - without SARSCoV-2 rS antigen) or cholesterol.

[0069] One hour post-injection (pi), high percentages of saponins and cholesterol per gram of tissue-injected radioactive dose (%ID / g) were detected at the injection site and in the iliac lymph nodes. Distribution at the QF injection site, iliac lymph nodes, inguinal lymph nodes, and popliteal lymph nodes was as follows: Figure 4A , Figure 4B , Figure 4C and Figure 4D The distribution in the iliac LN was substantial compared to that in the inguinal and popliteal LNs, thus indicating that the iliac LN was the primary draining LN (dLN) after QF injection. Saponins in both the QF and iliac LN showed a rapid decline starting 1 h post-injection. At 48 h post-injection, saponins in both the QF and iliac LN reached low levels and remained low for the remainder of the experiment. At the injection site, cholesterol counts showed a slower decline compared to saponin counts, resulting in significantly higher cholesterol levels than saponin levels starting 3–6 h post-injection, suggesting at least partial breakdown of Matrix particles at these time points. The presence or absence of the SARS-CoV-2 rS antigen was found to have no effect on the local biodistribution of saponins.

[0070] Experiment 3: Systemic biological distribution Study body fluids and non-local tissues to understand the systemic distribution of Matrix-M™ adjuvants and their constituent compounds, and to indicate possible excretion pathways and their time course. Figures 5A-5J The distribution over time in different parts of the body is shown. In plasma, urine, and kidneys (respectively...) Figures 5A-5C In this study, the retrieved relative radioactivity of labeled saponins differed from that of cholesterol at most time points. The relative activity of labeled saponins in plasma, urine, and kidney peaked 1–3 h post-injection, followed by a rapid decline at 6 h. In contrast, lower relative radioactivity of cholesterol was detected early in plasma and kidney, subsequently increasing to a plateau of approximately 5%–20% ID / g at 24 h post-injection, which persisted until the last measurement at 168 h post-injection, comparable to results in non-drained lymph nodes (LN). Detectable activity of cholesterol was barely observed in urine, as expected for lipophilic compounds such as cholesterol. Notably, the presence of SARS-CoV-2 antigen during immunization led to increased relative activity of saponins in urine at 1 and 3 h post-injection, and in the kidney at 3 h post-injection. In contrast, the absence or presence of the antigen had no effect on saponin activity in plasma, liver, intestine, spleen, lung, bone marrow, heart, and brain at any study time point.

[0071] A unique pattern of relative activity of saponins relative to cholesterol was found in the liver. Figure 5D The two labeled compounds showed low levels 1 hour after injection, then increased to similar activity 3 hours after injection, and then cholesterol showed slightly higher relative activity 6 to 72 hours after injection.

[0072] Bone marrow showed peak saponin levels at 3 and 6 hours post-injection, which decreased at 24 hours post-injection. Figure 5H The relative activity of saponins was found to be low in the intestine, spleen, lung, heart, and brain, some below the LOQ. In the intestine, lung, and bone marrow (… Figure 5E , Figure 5G and Figure 5H The study showed a relative increase in cholesterol activity, reaching a plateau of approximately 5–20% ID / g tissue 24 h post-injection, which remained stable until 168 h post-injection, with comparable levels found in non-drained lymph nodes, liver, and kidneys. A similar pattern of plateauing cholesterol activity from 24 h post-injection was observed in the spleen, heart, and brain, but at lower levels. Figure 5F , Figure 5I and Figure 5J ).

[0073] Therefore, these analyses demonstrate the rapid and substantial breakdown of Matrix particles in vivo, as evidenced by the distinct biodistribution of saponins and cholesterol. Saponins in the Matrix-M™ adjuvant do not accumulate systemically but are rapidly excreted into the urine via the kidneys. In contrast, systemic cholesterol levels plateaued approximately 24 hours post-injection and remained stable until the end of the measurement (168 hours post-injection). This suggests that, as the particles break down, labeled cholesterol enters the body's cholesterol circulation pool before being gradually excreted / metabolized.

[0074] Experiment 4: Biodistribution of the reproductive tract To evaluate the biodistribution of labeled saponins and labeled cholesterol in reproductive organs after injection of Matrix-M™ adjuvant, samples from the testes of male mice and the ovaries and uterus of female mice were analyzed. Figures 6A-6C The distribution of labeled saponins and labeled cholesterol in the testes, ovaries, and uterus is shown separately. The relative activity of saponins was low in all three organs, with the highest relative activity detected 1 h post-injection, followed by a decline at 3 h post-injection, remaining at low levels until the last measurement at 168 h post-injection. Addition of SARS-CoV-2 rS antigen to the adjuvant had no effect on the relative saponin activity in male or female reproductive organs. From 24 h post-injection, the pattern of relative cholesterol activity in all three organs reached a plateau, with the highest relative cholesterol activity detected in the ovaries. Overall, the distribution patterns of saponins and cholesterol in the reproductive tract were comparable to those found in other analyzed solid organs.

[0075] Experiment 5: Cellular distribution of Matrix-M™ adjuvant In experiments used to determine which cells take up Matrix-M™ adjuvant, mice were injected with the antigen and fluorescently modified Matrix-M™ adjuvant. Muscle tissue and dLNs at the injection site were harvested at 0.5 h, 1 h, 3 h, 6 h, 18 h, and 24 h post-injection, and the various cells from the tissue samples were counted by flow cytometry. Figure 8A A graph shows the percentage of CD45-positive and fluorescently tagged Matrix-M™ cell types obtained from the injection site. Results are shown for ten different cell types: neutrophils, monocytes, F4 / 80-monocytes, macrophages, dendritic cells (DCs), natural killer (NK) cells, natural killer T (NKT) cells, B cells, and other cells. For example, the graph shows that approximately 80% of F4 / 80-monocytes were positive for both CD45 and fluorescently tagged Matrix-M™ at 18 h post-injection. While neutrophils, monocytes (particularly F4 / 80-monocytes), and macrophages at the injection site showed Matrix-M™ association, uptake by DCs, NK cells, NKT cells, and T cells was negligible, and association with B cells was minimal.

[0076] Figure 8B The same data for cells derived from dLN are shown. A higher percentage of Matrix-M™-tagged neutrophils and a much lower percentage of Matrix-M™-tagged monocytes were present compared to cells at the injection site. Significant association with T cells and B cells was also observed, which was not seen at the injection site.

[0077] Experiment 6: Cytokine induction at the injection site and dLN Figure 9A A heatmap showing the changes in cytokines induced at the intramuscular injection site over time by various antigen / adjuvant combinations is presented. The antigen (qNIV) was injected with Matrix-M™, AS01, or alhydrogel, or without any adjuvant. Data are shown at five different time points: 6 h, 24 h, 48 h, 72 h, and 168 h. Each time point on the heatmap includes four columns, each related to the adjuvant used. The cytokines examined were CXCL10, IL-1β, TNF-α, IL-30, IL-6, CXCL1, CCL2, IL-5, CXCL2, CCL3, IL-33, and IFN-γ. In muscle, the AS01 combination was found to elicit a stronger cytokine response at 6 h and 24 h. However, at 168 h, the alhydrogel combination induced a greater response for at least some cytokines.

[0078] Figure 9BA similar heatmap is shown, but it focuses on cytokines induced in the dLN. Similar to muscle, the AS01 composition induced the strongest cytokine response at 6 h. The Matrix-M™ composition showed a diminished response at 6 h, while the alhydrogel composition and compositions containing a single antigen showed negligible cytokine responses. However, at 24 h, the Matrix-M™ composition induced a significant response across the cytokine spectrum, a broader response than that produced by the AS01 composition at any given time.

[0079] discuss Monitoring the biodistribution of Matrix-M™ adjuvant (a key component of NVX-CoV2373 and several novel vaccine candidates) is an important step in elucidating the method of action (MoA) and safety profile of Matrix-M™ adjuvant. Using Matrix-M™ adjuvant composed of unlabeled Matrix-C™ particles and Matrix-A™ particles containing either radiolabeled saponins or radiolabeled cholesterol, it was possible to demonstrate a very rapid transfer of Matrix-M™ adjuvant from the injection site to the dLN following intramuscular injection. Typically, the dLN showed a peak in the labeled component within one hour post-injection. Within 48 hours, a sharp decrease in both labeled components was observed at the injection site and in the dLN, while at early time points, there was a transient increase in saponin counts in plasma, urine, and kidneys, but no increase in cholesterol. Low levels of relative cholesterol radioactivity were detected systemically from 24 hours to the most recent measurement of the study (168 hours). Generally, the biodistribution of saponins was independent of the presence of rS nanoparticle antigens in the injection solution. This demonstrates the biodistribution of saponins and cholesterol in Matrix-A™ (the major component of Matrix-M™) after immunization with Matrix-M™. Due to the low content of Matrix-C™ in Matrix-M™, the radiolabeling of the Matrix-C component was insufficient for detection. Therefore, a slightly different biodistribution profile of Matrix-C™, for example, in its kinetics, cannot be ruled out.

[0080] The rapid removal of saponins and cholesterol from the injection site within 48 h post-injection, and their early distribution to the dLN, indicate that the MoA of Matrix-M™ adjuvant is not related to a reservoir effect. Conversely, the rapid distribution of saponins and cholesterol to the iliac LN further reinforces the concept of Matrix-M™ adjuvant as a "setting the stage" for antigens in the dLN. These results are consistent with comparisons of adjuvant activity and attenuated minimal QS-21 saponin adjuvants, demonstrating that active QS-21 preferentially localizes to draining lymph nodes 24 h post-injection. It is noteworthy that pooling iliac LN (and all other LNs) from both the injection and relative sites in the presented radiometric data likely weakens the observed effect, as changes at the draining site are only expected.

[0081] The current findings regarding the rapid translocation of Matrix-M™ adjuvant to the dLN are consistent with previously published data describing Matrix-M™ as an effective inducer of the migration of immune cells (primarily monocytes, dendritic cells, and neutrophils) to the dLN shortly after injection of Matrix-M™ adjuvant, with or without the antigen. These cells acquired the activated phenotype required to initiate antigen-specific adaptive immunity; at 48 h post-subcutaneous immunization, the effect of Matrix-M™ adjuvant was more significant compared to head-to-head comparisons with Alhydrogel, Freund's Complete Adjuvant, and AS03.

[0082] The exact mechanisms by which Matrix-M™ adjuvants and antigens reach the dLN (e.g., the relative contributions of cellular transport and free flow via the lymph) remain to be explored in future research.

[0083] Although the data presented here show a rapid reduction of either of the two labeled Matrix components (saponins and cholesterol) in the QF within 48 h, the faster removal of saponins from the QF than cholesterol indicates significant breakdown of Matrix particles already present at the injection site. The strong affinity between saponins and cholesterol is well-established, and cholesterol is essential for the formation of Matrix particles, which are stable when stored in neutral buffer. However, the fate of Matrix particles in vivo may differ. It is believed that, similar to other saponin-based adjuvants such as ISCMATRIX and AS01, Matrix particles target phagocytes and are processed via an endosome / lysosome pathway, and this processing depends on lysosomal acidification and the enzymatic activity present within the lysosome. During this intracellular processing, Matrix particles may break down at a lower pH within the lysosome, releasing saponins. As demonstrated with other saponin-based adjuvants, a key consequence of saponin release is lysosomal membrane instability. Lysosomal membrane permeation then allows co-administered vaccine antigens to enter the cytosol, thereby enabling cross-presentation of MHC class I molecules and inducing antigen-specific CD8+ T cell responses. Such responses have been demonstrated against Matrix-M™ adjuvanted vaccines in mouse models and in humans after vaccination with NVX-CoV2373.

[0084] Furthermore, the transient peak detection of saponins, rather than cholesterol, in plasma, urine, and kidneys before 24 hours post-injection confirmed distinct excretion pathways for unbound cholesterol and saponins, along with their corresponding degradation products. In the liver, similar relative saponin and cholesterol activity was detected at 1 and 3 hours post-injection. However, this does not necessarily imply that Matrix particles are intact in that particular organ, as fluid and tissue studies in other studies indicate at least partial breakdown of Matrix particles at these time points. Saponins and potential degradation products appeared relatively rapidly in urine and were almost completely cleared within 7 days, thus indicating this as the primary excretion pathway. In contrast, systemic cholesterol was detected from 24 hours to the most recent measurement in this study (168 hours), but systemic cholesterol radioactivity was lower compared to the initial activity in QF and iliac LN. It can be inferred that at least a portion of the labeled cholesterol enters the cholesterol circulation pool as an essential component of the cell membrane, which could explain the systemic detection and slower clearance kinetics. Due to technical limitations, analysis of feces, which typically represent the primary pathway for cholesterol removal, was not possible in this study.

[0085] Generally, no differences in saponin biodistribution dependent on the presence or absence of rS nanoparticle antigens have been observed, as have been described for other antigens and minimal QS-21. This would be expected, as the Matrix formulation consists of formulated particles mixed with the antigen, unlike the saponin-based adjuvant ISCOM (where the antigen is physically incorporated into the particles). However, at several time points, the presence of rS antigens during immunization led to increased relative activity of saponins in urine (1 h and 3 h post-injection) and kidneys (3 h post-injection). Since these differences are small and rare, the overall explanation is that the presence or absence of vaccine antigens does not significantly affect the biodistribution of saponins.

[0086] The limitations and time-restricted nature of Matrix-M™ adjuvant at the injection site, along with its rapid distribution to the dLN, are not only advantageous for effectively inducing a specific memory response to the co-administered antigen, but also beneficial from a safety profile perspective. The observed rapid clearance of saponins without systemic distribution can be incorporated into the benefit-risk assessment of Matrix-M™ adjuvanted vaccines. Notably, no long-term accumulation of saponins in any organs, including the reproductive tract, has been observed. It is reasonable to believe that the incomplete elimination of cholesterol after 168 hours can be explained by cholesterol from Matrix particles entering the endogenous pool as an essential component of cell membranes or metabolites. From a biodistribution perspective, the described overall favorable safety profile confirms the results of mild to moderate and transient reactivity from multiple Phase 1–3 clinical trials evaluating Matrix-M™ adjuvanted vaccines against COVID-19, malaria, influenza, or Ebola virus diseases.

[0087] In summary, the data presented here demonstrate the rapid translocation of Matrix-M™ to the dLN, followed by rapid removal of detected saponins and cholesterol from both the injection site and the dLN. Importantly, this transient presence of the adjuvant in the dLN is sufficient to generate high titers of neutralizing antibodies and immune memory, as well as IgG2a labeling consistent with a strong Th1 T cell response. Head-to-head comparisons of saponins and cholesterol, key components of Matrix particles, indicate rapid particle breakdown at the injection site, which may be crucial for intracellular events such as the lysosomal membrane permeation required for the reported activation of CD8+ T cells in response to Matrix-M™-adjuvanted antigens. Furthermore, the results show a significant difference in the immune response at the dLN compared to the injection site.

[0088] Upon reading this specification, various modifications, equivalent processes, and numerous formulations applicable to the present invention will be apparent to those skilled in the art to which this invention pertains. The claims are intended to cover such modifications and processes.

[0089] As noted above, this invention is applicable to saponin-based adjuvants. Therefore, this invention should not be considered limited to the specific embodiments described above, but should be understood to cover all aspects of the invention as set forth in the appended claims.

[0090] The implementation scheme disclosed herein The following are specific examples of implementation schemes. <1> to <30> For illustrative purposes only and do not further limit the scope of the subject matter disclosed by the claims. These enumerated embodiments cover all combinations, sub-combinations, and multiple references (e.g., multiple dependencies) described herein.

[0091] 1. A method of administering a vaccine composition to a mammalian subject, comprising: The vaccine composition, comprising a saponin-based adjuvant, is injected into the subject at an intramuscular injection site associated with a draining lymph node (dLN). The proportion of T cells in the dLN that are positive for i) CD45 and ii) the saponin-based adjuvant after injection of the vaccine composition at the intramuscular injection site has a minimum value when the injection is more than one hour after injection of the vaccine composition at the intramuscular injection site and less than 24 hours after injection of the vaccine composition at the intramuscular injection site, and the proportion of T cells in the dLN that are positive for i) CD45 and ii) the saponin-based adjuvant is greater than the minimum value when the injection of the vaccine composition is 24 hours after injection.

[0092] 2. The method as described in embodiment 1, wherein, from one hour to twenty-four hours after injection of the vaccine composition at the intramuscular injection site, the minimum value of the proportion of the dLN positive for i) CD45 and ii) the saponin-based adjuvant is greater than about one-tenth of the maximum value.

[0093] 3. The method as described in embodiment 1, wherein one to twenty-four hours after injection of the vaccine composition at the intramuscular injection site, the minimum value of the proportion of the dLN positive for i) CD45 and ii) the saponin-based adjuvant is greater than about one-eighth of the maximum value.

[0094] 4. The method as described in embodiment 1, wherein one to twenty-four hours after injection of the vaccine composition at the intramuscular injection site, the minimum value of the proportion of the dLN positive for i) CD45 and ii) the saponin-based adjuvant is greater than about one-fifth of the maximum value.

[0095] 5. The method according to any one of embodiments 1 to 4, wherein the saponin-based adjuvant comprises soap tree Nanoparticles of saponin fraction A, cholesterol, and phospholipids, and soap tree Nanoparticles containing saponin fractions C, cholesterol, and phospholipids.

[0096] 6. The method as described in any one of embodiments 1 to 5, wherein the mammalian subject is a human.

[0097] 7. The method of any one of embodiments 1 to 6, wherein the saponin-based adjuvant is mixed with the antigen.

[0098] 8. The method of any one of embodiments 1 to 7, wherein the saponin-based adjuvant is mixed with a carrier.

[0099] 9. A method of administering a vaccine composition to a mammalian subject, comprising: The vaccine composition is injected into the subject at an intramuscular injection site associated with a draining lymph node (dLN), and the vaccine composition contains a saponin-based adjuvant. The maximum proportion of B cells in the dLN that are positive for i) CD45 and ii) the adjuvant occurs less than three hours after the intramuscular injection of the mixture, within a period of approximately one hour to approximately twenty-four hours following the injection of the vaccine composition.

[0100] 10. The method of embodiment 9, wherein approximately twenty-four hours after injection of the mixture at the intramuscular injection site, the proportion of B cells in the draining lymph nodes that are positive for i) CD45 and ii) the adjuvant is less than approximately one-tenth of the maximum value.

[0101] 11. The method of embodiment 9, wherein approximately twenty-four hours after injection of the mixture at the intramuscular injection site, the proportion of B cells in the draining lymph nodes that are positive for i) CD45 and ii) the adjuvant is less than approximately one-twentieth of the maximum value.

[0102] 12. The method of any one of embodiments 9 to 11, wherein the saponin-based adjuvant comprises soap tree Nanoparticles of saponin fraction A, cholesterol, and phospholipids, and soap tree Nanoparticles containing saponin fractions C, cholesterol, and phospholipids.

[0103] 13. The method as described in any one of embodiments 9 to 12, wherein the mammalian subject is a human.

[0104] 14. The method of any one of embodiments 9 to 13, wherein the saponin-based adjuvant is mixed with the antigen.

[0105] 15. The method of any one of embodiments 9 to 14, wherein the saponin-based adjuvant is mixed with a carrier.

[0106] 16. A method for administering a vaccine composition to a mammalian subject, comprising: The vaccine composition is injected into the subject at an intramuscular injection site associated with a draining lymph node (dLN); The vaccine composition comprises an antigen and a saponin-based adjuvant, wherein the saponin-based adjuvant comprises a saponin component and a cholesterol component; and After the vaccine composition is injected into the subject at the intramuscular injection site, the concentration of the saponin component in the subject's plasma reaches its peak less than 3 hours after the injection, while the concentration of the cholesterol component in the subject's plasma reaches its peak more than 6 hours after the injection.

[0107] 17. The method of embodiment 16, wherein the saponin-based adjuvant comprises soap tree Nanoparticles of saponin fraction A, cholesterol, and phospholipids, and soap tree Nanoparticles containing saponin fractions C, cholesterol, and phospholipids.

[0108] 18. The method of any one of embodiments 16 to 17, wherein the saponin-based adjuvant is mixed in a carrier.

[0109] 19. The method of any one of embodiments 16 to 18, wherein after the vaccine composition is injected into the subject at the intramuscular injection site, the concentration of the saponin component in the kidney of the subject reaches a peak less than 3 hours after the injection, and the concentration of the cholesterol component in the kidney of the subject reaches a peak more than 24 hours after the injection.

[0110] 20. A method for administering a vaccine composition to a mammalian subject, comprising: The vaccine composition is injected into the subject at an intramuscular injection site associated with a draining lymph node (dLN); The vaccine composition comprises an antigen and a saponin-based adjuvant, wherein the saponin-based adjuvant comprises a saponin component and a cholesterol component; and After the vaccine composition is injected into the subject at the intramuscular injection site, the concentration of the saponin component in one of the subject's intestines, lungs, and heart remains approximately zero for up to 168 hours after the injection, while the concentration of the cholesterol component in one of the subject's intestines, lungs, and heart reaches its peak more than 24 hours after the injection.

[0111] 21. The method of embodiment 20, wherein the saponin-based adjuvant comprises soap tree Nanoparticles of saponin fraction A, cholesterol, and phospholipids, and soap tree Nanoparticles containing saponin fractions C, cholesterol, and phospholipids.

[0112] 22. The method as described in any one of embodiments 20 to 21, wherein the saponin-based adjuvant is mixed in a carrier.

[0113] 23. A method for administering a vaccine composition to a mammalian subject, comprising: The vaccine composition is injected into the subject at an intramuscular injection site associated with a draining lymph node (dLN), and the vaccine composition contains a saponin-based adjuvant. The cytokine response induced by the vaccine composition at the dLN, measured 24 hours after injection of the mixture, was stronger than the cytokine response at the dLN, measured 6 hours after injection of the mixture.

[0114] 24. The method of embodiment 23, wherein the cytokine response is correlated with the abundance of at least one of the following cytokines: CXCL10, IL-1b, TNF-α, IL-30, IL-6, CXCL1, CCL2, IL-5, CCL3, IL-33, and IFN-g.

[0115] 25. The method of any one of embodiments 23 to 24, wherein, compared to 6 hours after the injection, the abundance of at least two of the cytokines selected from the group consisting of: CXCL10, IL-1b, TNF-α, IL-30, IL-6, CXCL1, CCL2, IL-5, CCL3, IL-33, and IFN-g at the dLN is higher at approximately 24 hours after the injection.

[0116] 26. The method of any one of embodiments 23 to 25, wherein, compared to 6 hours after the injection, the abundance of at least three of the cytokines selected from the group consisting of: CXCL10, IL-1b, TNF-α, IL-30, IL-6, CXCL1, CCL2, IL-5, CCL3, IL-33, and IFN-g at the dLN is higher at approximately 24 hours after the injection.

[0117] 27. The method of any one of embodiments 23 to 26, wherein the saponin-based adjuvant comprises soap tree Nanoparticles of saponin fraction A, cholesterol, and phospholipids, and soap tree Nanoparticles containing saponin fractions C, cholesterol, and phospholipids.

[0118] 28. The method of any one of embodiments 23 to 27, wherein the mammalian subject is a human.

[0119] 29. The method of any one of embodiments 23 to 28, wherein the saponin-based adjuvant is mixed with the antigen.

[0120] 30. The method of any one of embodiments 23 to 29, wherein the saponin-based adjuvant is mixed with a carrier.

Claims

1. A method of administering a vaccine composition to a mammalian subject, comprising: The vaccine composition, comprising a saponin-based adjuvant, is injected into the subject at an intramuscular injection site associated with a draining lymph node (dLN). The proportion of T cells in the dLN that are positive for i) CD45 and ii) the saponin-based adjuvant after injection of the vaccine composition at the intramuscular injection site has a minimum value when the injection is more than one hour after injection of the vaccine composition at the intramuscular injection site and less than 24 hours after injection of the vaccine composition at the intramuscular injection site, and the proportion of T cells in the dLN that are positive for i) CD45 and ii) the saponin-based adjuvant is greater than the minimum value when the injection of the vaccine composition is 24 hours after injection.

2. The method of claim 1, wherein, one to twenty-four hours after injection of the vaccine composition at the intramuscular injection site, the minimum value of the proportion of the dLN positive for i) CD45 and ii) the saponin-based adjuvant is greater than about one-tenth of the maximum value.

3. The method of claim 1, wherein, one to twenty-four hours after injection of the vaccine composition at the intramuscular injection site, the minimum value of the proportion of the dLN positive for i) CD45 and ii) the saponin-based adjuvant is greater than about one-eighth of the maximum value.

4. The method of claim 1, wherein, one to twenty-four hours after injection of the vaccine composition at the intramuscular injection site, the minimum value of the proportion of the dLN positive for i) CD45 and ii) the saponin-based adjuvant is greater than about one-fifth of the maximum value.

5. The method of any one of claims 1 to 4, wherein the saponin-based adjuvant comprises soap tree Nanoparticles of saponin fraction A, cholesterol, and phospholipids, and soap tree Nanoparticles containing saponin fractions C, cholesterol, and phospholipids.

6. The method of any one of claims 1 to 5, wherein the mammalian subject is a human.

7. The method of any one of claims 1 to 6, wherein the saponin-based adjuvant is mixed with the antigen.

8. The method of any one of claims 1 to 7, wherein the saponin-based adjuvant is mixed with a carrier.

9. A method of administering a vaccine composition to a mammalian subject, comprising: The vaccine composition is injected into the subject at an intramuscular injection site associated with a draining lymph node (dLN), and the vaccine composition contains a saponin-based adjuvant. The maximum proportion of B cells in the dLN that are positive for i) CD45 and ii) the adjuvant occurs less than three hours after the intramuscular injection of the mixture, within a period of approximately one hour to approximately twenty-four hours following the injection of the vaccine composition.

10. The method of claim 9, wherein approximately twenty-four hours after injection of the mixture at the intramuscular injection site, the proportion of B cells in the draining lymph nodes that are positive for i) CD45 and ii) the adjuvant is less than approximately one-tenth of the maximum value.

11. The method of claim 9, wherein approximately twenty-four hours after injection of the mixture at the intramuscular injection site, the proportion of B cells in the draining lymph nodes that are positive for i) CD45 and ii) the adjuvant is less than approximately one-twentieth of the maximum value.

12. The method of any one of claims 9 to 11, wherein the saponin-based adjuvant comprises soap tree Nanoparticles of saponin fraction A, cholesterol, and phospholipids, and soap tree Nanoparticles containing saponin fractions C, cholesterol, and phospholipids.

13. The method of any one of claims 9 to 12, wherein the mammalian subject is a human.

14. The method of any one of claims 9 to 13, wherein the saponin-based adjuvant is mixed with the antigen.

15. The method of any one of claims 9 to 14, wherein the saponin-based adjuvant is mixed with a carrier.

16. A method for administering a vaccine composition to a mammalian subject, comprising: The vaccine composition is injected into the subject at an intramuscular injection site associated with a draining lymph node (dLN); The vaccine composition comprises an antigen and a saponin-based adjuvant, wherein the saponin-based adjuvant comprises a saponin component and a cholesterol component; and After the vaccine composition is injected into the subject at the intramuscular injection site, the concentration of the saponin component in the subject's plasma reaches its peak less than 3 hours after the injection, while the concentration of the cholesterol component in the subject's plasma reaches its peak more than 6 hours after the injection.

17. The method of claim 16, wherein the saponin-based adjuvant comprises soap tree Nanoparticles of saponin fraction A, cholesterol, and phospholipids, and soap tree Nanoparticles containing saponin fractions C, cholesterol, and phospholipids.

18. The method of any one of claims 16 to 17, wherein the saponin-based adjuvant is mixed in a carrier.

19. The method of any one of claims 16 to 18, wherein after the vaccine composition is injected into the subject at the intramuscular injection site, the concentration of the saponin component in the kidney of the subject reaches a peak less than 3 hours after the injection, and the concentration of the cholesterol component in the kidney of the subject reaches a peak more than 24 hours after the injection.

20. A method for administering a vaccine composition to a mammalian subject, comprising: The vaccine composition is injected into the subject at an intramuscular injection site associated with a draining lymph node (dLN); The vaccine composition comprises an antigen and a saponin-based adjuvant, wherein the saponin-based adjuvant comprises a saponin component and a cholesterol component; and After the vaccine composition is injected into the subject at the intramuscular injection site, the concentration of the saponin component in one of the subject's intestines, lungs, and heart remains approximately zero for up to 168 hours after the injection, while the concentration of the cholesterol component in one of the subject's intestines, lungs, and heart reaches its peak more than 24 hours after the injection.

21. The method of claim 20, wherein the saponin-based adjuvant comprises soap tree Nanoparticles of saponin fraction A, cholesterol, and phospholipids, and soap tree Nanoparticles containing saponin fractions C, cholesterol, and phospholipids.

22. The method of any one of claims 20 to 21, wherein the saponin-based adjuvant is mixed in a carrier.

23. A method for administering a vaccine composition to a mammalian subject, comprising: The vaccine composition is injected into the subject at an intramuscular injection site associated with a draining lymph node (dLN), and the vaccine composition contains a saponin-based adjuvant. The cytokine response induced by the vaccine composition at the dLN, measured 24 hours after injection of the mixture, was stronger than the cytokine response at the dLN, measured 6 hours after injection of the mixture.

24. The method of claim 23, wherein the cytokine response is correlated with the abundance of at least one of the following cytokines: CXCL10, IL-1b, TNF-α, IL-30, IL-6, CXCL1, CCL2, IL-5, CCL3, IL-33, and IFN-γ.

25. The method of any one of claims 23 to 24, wherein, compared to 6 hours after the injection, the abundance of at least two of the cytokines selected from the group consisting of: CXCL10, IL-1b, TNF-α, IL-30, IL-6, CXCL1, CCL2, IL-5, CCL3, IL-33, and IFN-g at the dLN is higher at approximately 24 hours after the injection.

26. The method of any one of claims 23 to 25, wherein, compared to 6 hours after the injection, the abundance of at least three of the cytokines selected from the group consisting of: CXCL10, IL-1b, TNF-α, IL-30, IL-6, CXCL1, CCL2, IL-5, CCL3, IL-33, and IFN-g at the dLN is higher at approximately 24 hours after the injection.

27. The method of any one of claims 23 to 26, wherein the saponin-based adjuvant comprises soap tree Nanoparticles of saponin fraction A, cholesterol, and phospholipids, and soap tree Nanoparticles containing saponin fractions C, cholesterol, and phospholipids.

28. The method of any one of claims 23 to 27, wherein the mammalian subject is a human.

29. The method of any one of claims 23 to 28, wherein the saponin-based adjuvant is mixed with the antigen.

30. The method of any one of claims 23 to 29, wherein the saponin-based adjuvant is mixed with a carrier.

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