WO2023235592A2 - Tlr agonists comprising saponin nanoparticle vaccine adjuvants to improve immunomodulation - Google Patents

Abstract

Provided herein are nanoparticles including a TLR agonist, a saponin, a phospholipid, and a sterol. The composition of the nanoparticles is modularly tunable and can be configured such that the nanoparticles are particularly useful as adjuvants for enhancing a variety of potent and durable immunogenic responses. Also provided are adjuvant compositions and immunogenic compositions including the nanoparticles, and methods for using these compositions to induce an immunogenic response and treat or prevent a disease.

Description

TLR AGONISTS COMPRISING SAPONIN NANOPARTICLE

VACCINE ADJUVANTS TO IMPROVE IMMUNOMODULATION

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority’ to U.S. Provisional Application No. 63/348,326, filed June 2, 2022, the full disclosure of which is incorporated by reference in its entirety for all purposes.

BACKGROUND

[0002] The development of protective vaccines has been at the heart of intensive scientific research for the last hundred years, catalyzed by the emergence of global pandemics such as SARS-CoV-2, HIV, and influenza (J.R. Mascola & A.S. Fauci, Nat. Rev. Immunol. 20, (202.0): 87). However, many commercial vaccines are still formulated with traditional live-attenuated or inactivated vaccines, both of which suffer from lack of affordability, complex manufacturing process, and more importantly, potential safety hazards (P.M. Moyle & I. Toth, ChemMedChem 8, (2013): 360). Over the last, few decades, protein-based subunit vaccine approaches have been shown to offer desirable characteristics in terms of safety, cost effectiveness, scalability, and manufacturability. Subunit vaccines leverage the use of viral protein antigens paired with immune stimulating molecules, also known as adjuvants, that are essential in promoting the magnitude and durability of the immune response.

[0003] To date, only a handful of adjuvants are clinically licensed in vaccine formulations and approved by the Food and Drag Administration (FDA), including the insoluble salt Alum, the synthetic single-stranded DNA CpG-ODN 1018, the liposome AS01, and oil-in-water emulsions such as MF59 or AS03 (B. Pulendran, P.S. Arunachalam & D.T. O’Hagan, Nat. Rev. Drug Discov. 20, (2.021): 454), There are other adjuvant candidates in development, many offering promising improvements in the overall potency of subunit vaccines. A specific class of adjuvants that has been gaining atention is Toll-like receptor agonists (TLRas) (C. Maisonneuve, S. Bertholet, D.J. Philpott & E. De Gregorio, Proc. Natl. Acad, of Sei. I l l, (2014): 12294; M.S, Duthie, H.P. Windish, C.B. Fox & S.G. Reed, Immunol Rev. 239, (2011): 178). They include TLRl/2a Pam3CSK4 (Pam3CysSerLys4), TLR3a Poly(I:C) (Polyinosinic:polycytidylic acid), TLR4a MPLA (Monophosphoryl lipid A), TLR7/8a Resiquimod or 3M-052, and TLR9a CpG-ODN. TLRas differ in physical and chemical properties, and can include lipids, small molecules, single-stranded DNA, and double-stranded RNA. All of these diverse TLRas bind to Toll-like receptors (TLRs), commonly expressed by antigen-presenting cells (APCs), to activate immune signaling pathways. Recent FDA approvals of MPL/Alum-based AS04 from GlaxoSmithKline (GSK) and CpG/ Alum-based adjuvant CpG 1018 from Dynavax, in addition to numerous clinical trials using 3M-052 and other TLRas, reinforce their emergence as promising adjuvants for both vaccines and cancer therapeutics.

[0004] Although adjuvants can be delivered in their soluble form, intensive research has demonstrated the advantage of formulating adjuvants as nanoparticles, improving their stability, solubility, cellular uptake, and immunogenicity to ultimately augment vaccine immune responses (F. Ledesma, B. Ozcan, X, Sun, S.M. Medina & M.P. Landry, Adv. Funct. Mat. 32, (2022): 2107174; S. Wu, Y. Xia, Y. Hu & G. Ma, Adv. Drug Deliv. Rev. 176, (2021 ): 113871; B.S. Ou et al. bioRxiv, (2023): 522505; B.S. Ou, O.M. Saouaf, J. Baillet & E.A. Appel, Adv. Drug Deliv. Rev. 187, (2022): 1 14401 ; B. Sun & T. Xia, J. Mat. Chem. B 4, (2016): 5496; D.J. Irvine, A. Aung & M. Silva, Adv. Drug Deliv. Rev. 158, (2020): 91; D.J. Irvine, M.C. Hanson, K. Rakhra & T. Tokatlian, Chem. Rev. 115, (2015): 11109). As such, the nanoparticulate adjuvant Matrix-M licensed by Novavax was approved by the FDA in October 2022 for its use in Novavax COVID- 19 vaccine, for individuals 12 years or older. These nanostructures, derived from natural triterpene glycosides Quillaja (Quil-A) saponin, leverage the spontaneous self-assembling properties of saponins, a non-TLRa class of adjuvants, when mixed with cholesterol and phospholipids.

[0005] Saponins have been used for decades in vaccine formulations as potent natural adjuvants and are considered as non-toxic when self-assembled with cholesterol and phospholipids into the well-known honeycomb like structure, commonly known as ISCOMATRIX (B. Morein, B. Sundquist, S. Hoglund, K. Dalsgaard & A. Osterhaus, Nature 308, (1984): 457; M. Silva et al., Sci. Immunol. 6, (2021): eabfl 152; K. Lbvgren-Bengtsson & B. Morein, Vaccine Adjuvants: Preparation Methods and Research Protocols', Humana Press: Humana Totowa, NJ (2000); S. Pedersen et al., Biophys. J 102, (2012): 2372; M.T. Sanders, L.E. Brown, G. Deliyannis & MJ. Pearse, Immunol. Cell Biol. 83, (2005): 119; MJ. Pearse & D. Drane, Adv. Drug Deliv. Rev. 57, (2005): 465; E. Maraskovsky et al., Immunol. Cell Biol. 87, (2009): 371). These nanoparticles rely on the specific affinity of saponins and cholesterol in aqueous medium to form particulates of around 40 nm in diameter (with 30-70 nm reported). While their mechanism of immune stimulation has not yet been fully elucidated, recent reports suggest saponin-based nanoparticles could increase lymph flow and lymph node permeability to enhance antigen acquisition by B cells in draining lymph nodes (dLNs) (M. Silva et al., Sci. Immunol. 6, (2021): eabfl 152; E. Maraskovsky et al., Immunol. Cell Biol. 87, (2009): 371).

[0006] Few attempts have been made to combine both TLRas and saponin class adjuvants into the same nanostructure (M. Silva et al., Sci. Immunol. 6, (2021): eabfl 152; N. Van Hoeven et al., NPJ Vaccines 3, (2018): 39; A.M. Didierlaurent et al., Expert Rev. Vaccines 16, (2017): 55; A. Silva et al., J. Immunol 194, (2015): 2199; P. Martinez et al., Set. Reports 5, (2015): 8925; T.K. Raaijmakers et al., J Immunother. Cancer 8, (2020): e000649). For example, GSK uses the liposome AS01 containing MPLA and QS-21 saponin in its licensed Shingrix and Mosquirix vaccines (A.M. Didierlaurent et al., Exp. Rev. Vaccines 16, (2017): 55). Similarly, Irvine and coworkers recently designed ISCOMATRIX incorporating TLRa MPLA and saponins (SMNP) (M. Silva et al., Sci. Immunol. 6, (2021): eabfl 152). In view of these limited investigations, a need exists for improved compositions and methods related to saponin nanoparticles or nanostructures incorporating a wider variety of TL.R agonists and acting as potent and diverse adjuvants. The present disclosure addresses this and other associated needs.

BRIEF SUMMARY

[0007] The following summary provides a high-level overview of various aspects of the invention and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. Covered embodiments of the disclosure are not defined by this summary. The subject mater should be understood by reference to appropriate portions of the entire specification, any or all figures, and each claim. Some of the exemplary embodiments of the present disclosure are discussed below.

[0008] The present disclosure generally relates to a modular saponin based nanoparticle platform, referred to herein as TLRa-SNPs, which incorporates Toll-like receptor (TER) adjuvants (e.g., TLRl/2a, TLR4a, TLR7/8a, or a combination thereof) with saponin nanoparticles (SNPs). The TLRa-SNPs disclosed herein can greatly improve the potency, durability, breadth, and neutralization of vaccines including those against COVID-19 and HIV, suggesting a broad applicability of the provided adjuvant technology to a range of different antigens. Additionally, formulations of TLRa-SNPs can induce unique acute cytokine and immune-signaling profiles, leading to specific T helper (Th) cell responses of interest for the treatment or prevention of target diseases. The modular TLRa-SNPs adjuvant platform thus can have a major impact in modem vaccine strategy.

[0009] In one aspect, the disclosure is to a nanoparticle. The nanoparticle includes a Toll- like receptor (TLR) agonist. The TLR agonist is a non-lipid TLR agonist or a derivative thereof. The nanoparticle further includes a saponin, a phospholipid, and a sterol, i.e., a nanoparticle sterol.

[0010] In another aspect, the disclosure is to an adjuvant composition configured to enhance an immune response of a subject against an antigen. The adjuvant composition includes a plurality of nanoparticles. Each nanoparticle of the plurality of nanoparticles includes a TLR agonist. The TLR agonist is a non-lipid TLR agonist or a derivative thereof. Each nanoparticle further includes a saponin, a phospholipid, and a sterol, i.e., a nanoparticle sterol.

[0011] In another aspect, the disclosure is to another adjuvant composition configured to enhance an immune response of a subject against an antigen. The immune response includes a production by the subject of a higher concentration of IgGl antibody than of IgG2c antibody. The adjuvant composition includes a plurality of nanoparticles. Each nanoparticle of the plurality of nanoparticles includes a TLR agonist, a saponin, a. phospholipid, and a sterol, i.e., a nanoparticle sterol.

[0012] In another aspect, the disclosure is to an immunogenic composition. The immunogenic composition includes any of the adjuvant compositions disclosed herein. The immunogenic composition further includes the antigen that the adjuvant composition is configured to enhance an immune response of a subject, against.

[0013] In another aspect, the disclosure is to a method of inducing an immune response in a subject. The method includes administering to the subject an effective amount of any of the immunogenic compositions disclosed herein.

[0014] In another aspect, the disclosure is to a method of preventing or treating a disease in a subject. The method includes administering to the subject an effective amount of any of the immunogenic compositions disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 presents a schematic representation of saponin nanoparticles SNPs and exemplary’ formulations of Toll-like receptor agonist (TLRa)-SNPs in accordance with provided embodiments: TLRl/2a-SNP incorporating Pam3CSK4, TLR4a-SNP incorporating MPLA, and TLR7/8a-SNP incorporating imidazoquinoline derivative.

[0016] FIG. 2 presents the chemical structure of the cholesteryl imidazoquinoline derivative.

[0017] FIG. 3 present a graph plotting the hydrodynamic diameters of the TLRa-SNPs of FIG. 1 .

[0018] FIG. 4 presents a graph plotting the surface charges of the TLRa-SNPs of FIG. 1.

[0019] FIG. 5 presents cryo-Electron microscopy images of (i) SNP, (ii) TLRl/2a-SNP, (iii) TLR4a-SNP, and (iv) TLR7/8a-SNP, demonstrating the maintenance of SNP structure after introduction of the TLRas. Scale bars: 50 nm.

[0020] FIG. 6 presents a graph ploting the hydrodynamic diameters of tire TLRa-SNPs of FIG. 1 over time, showing the colloidal stability of SNP and TLRa-SNPs over the course of 42. days.

[0021] FIG. 7 presents a graph plotting the polydispersity indices (PDIs) of the TLRa-SNPs of FIG. 1 over time, showing the colloidal stability of SNP and TLRa-SNPs over the course of 42 days.

[0022] FIG. 8 presents a graph plotting the surface charges of the TLRa-SNPs of FIG. 1 over the course of 42 days.

[0023] FIG. 9 presents a series of graphs plotting results from an experiment in which RAW- Blue macrophage cells were incubated with sa.ponin-nanoparticle (SNP) or TLRa incoiporated SNPs (TLRa-SNPs). The plots show' activation curves of (left) soluble TLRl/2aand TLRl/2a- SNP, (center) soluble TLR4a and TLR4a-SNP, and (right) soluble cholesteryl-modified TLR7/8a and TLR7/8a-SNP across a range of TLRa concentrations (0.0091-2.22 μg/mL) with 100,000 RAW-Blue cells (n = 3).

[0024] FIG. 10 presents a graph plotting in vitro RAW-Blue activation curves of relevant solvents and buffers.

[0025] FIG. I I presents a graph plotting in vitro RAW-Blue activation curves of soluble SNP, soluble TLRas, and TLRa-SNPs. [0026] FIG. 12 presents a timeline of immunization and blood collection to determine IgG titers in a study of the in vivo humoral response to RBD NP vaccines adjuvanted with TLRa- SNPs. Mice were immunized on Week 0 and boosted on Week 3 with RBD NP vaccines adjuvanted with CpG/Alum, SNP, or TLRa-SNPs. IgGl, TgG2c, neutralization, and variants titers were determined on Week 5.

[0027] FIG. 13 presents a graph plotting results from the study of FIG. 12 showing anti-RBD

IgG binding endpoint titers of RBD NP vaccines adjuvanted with CpG/Alum, SNP, or TLRa-

SNPs. Data (n = 4-5) are shown as mean +/- SEM.p values were determined using the general linear model followed by Tukey’s HSD comparison procedure on the logged titer values for

IgG titer comparisons. *p < 0.05, **p < 0.01 , ***p < 0.001, and ****p < 0.0001 .

[0028] FIG. 14 presents a graph plotting results from the study of FIG. 12 showing area under the curves (AUCs) of anti-RBD IgG endpoin t antibody titers from Week 0 to Week 1 1 of the different RBD NP vaccines. Data (n ~ 4-5) are shown as mean +/- SEM. p values were determined using the general linear model followed by Tukey’s HSD comparison procedure on the logged titer values for IgG titer comparisons. *p < 0.05, **p < 0.01, ***p < 0.001 , and ****p < 0.0001.

[0029] FIG. 15 presents a graph plotting results from the study of FIG. 12 showing half- maximal binding dilution (ECse) of anti-RBD IgG endpoint titers on Week 5 of the different RBD NP vaccines Data (n = 4-5) are shown as mean +/- SEM. p values were determined using the general linear model followed by Tukey’s HSD comparison procedure on the logged titer values for IgG titer comparisons. *p < 0.05, **p < 0.01, ***p < 0,001, and ****p < 0.0001.

[0030] FIG. 16 presents a graph plotting results from the study of FIG. 12 showing anti-RBD IgG binding endpoint titers a year (D365) after immunization. Data (n = 4-5) are shown as mean +/- SEM.p values were determined using the general linear model followed by Tukey’s HSD comparison procedure on the logged titer values for IgG titer comparisons. *p < 0.05, **p

< 0.01, ***p < 0.001, and ****p < 0.0001.

[0031] FIG. 17 presents a graph plotting results from the study of FIG. 12 showing Anti- RBD IgG endpoint titers drop of different RBD NP vaccines with different adjuvants from D77 to D365.

[0032] FIG. 18 presents a graph plotting results from the study of FIG. 12 showing anti-spike IgG binding endpoint titers from sera collected on Week 5 after the initial immunization. Titers were determined for wildtype WT spike as well as Alpha (B.1.1 .7), Beta. (B.1.351), Delta (B. 1.617.2), and Omicron (B. 1.1.529) variants of the spike protein. Data (n ^:::: 4-5) are shown as mean +/- SEM. p values were determined using the general linear model followed by Tukey’s HSD comparison procedure on the logged titer values for IgG titer comparisons. *p < 0.05, < 0.01, ***p < 0.001, and ****p < 0.0001.

[0033] FIG. 19 presents a graph plotting results from the study of FIG. 12 showing anti-spike IgG endpoint titer drop against SARS-CoV-2 variant of concerns and WT spike titer of RBD NP vaccines with different adjuvants. Boxes shown are interquartile range.

[0034] FIG. 20 presents a graph plotting percent infectivity for SNP vaccine treatment at a range of Week 5 serum dilutions as determined by a SARS-CoV-2 spike-pseudotyped viral neutralization assay. Data (n = 4-5) are shown as mean W- SEM.

[0035] FIG. 21 presents a graph plotting percent infectivity for TLRl/2a-SNP vaccine treatment at a range of Week 5 serum dilutions as determined by a SARS-CoV-2 spike- pseudotyped viral neutralization assay. Data (n = 4-5) are shown as mean +/- SEM. [0036] FIG. 22 presents a graph plotting percent infectivity for TLR4a-SNP vaccine treatment at a range of Week 5 serum dilutions as determined by a SARS-CoV-2 spike- pseudotyped viral neutralization assay. Data (n = 4-5) are shown as mean +/- SEM.

[0037] FIG. 2.3 presents a graph plotting percent infectivity for TLR7/8a-SNP vaccine treatment at a range of Week 5 serum dilutions as determined by a SARS-CoV-2 spike- pseudotyped viral neutralization assay. Data (n = 4-5) are shown as mean +/- SEM.

[0038] FIG . 24 presents a graph plotting percent infectivity for CpG/Alum vaccine treatment at a range of Week 5 serum dilutions as determined by a SARS-CoV-2 spike-pseudotyped viral neutralization assay. Data (n = 4-5) are shown as mean +/- SEM.

[0039] FIG. 25 presents a graph plotting a comparison of ID₅₀ values determined from the neutralization curves of FIGS. 20-24. Dotted line denotes the threshold for which the FDA considers as “high titer.” Data (n = 4-5) are shown as mean +/- SEM. p values were determined using the general linear model followed by Tukey’s HSD multiple comparisons procedure on the logged neutralization ID₅₀ values or relative infectivity comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. [0040] FIG. 26 presents a graph plotting relative percent infectivity of all vaccine formulations of FIGS. 20-24 compared to convalescent human serum at 1: 100 dilution. Data (n = 4-5) are shown as mean +/- SEM.p values were determined using the general linear model followed by Tukey’s HSD multiple comparisons procedure on the logged neutralization ID₅₀ values or relative infectivity comparisons. *p < 0.05, **» < 0.01, ***p < 0.001, and ****p < 0.0001.

[0041] FIG. 27 presents a graph plotting anti-RBD IgGl titers from sera collected on Week 5, 2 weeks after boost, showing response to RBD NP vaccines adjuvanted with TLRa-SNPs. Data (n = 4-5) are shown as mean +/- SEM. p values listed were using the general linear model followed by Tukey’s HSD multiple comparison procedure on the logged titer values. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0042] FIG. 28 presents a graph plotting anti-RBD IgG2c titers from sera collected on Week 5, 2 weeks after boost, showing response to RBD NP vaccines adjuvanted with TLRa-SNPs. Data (n ~ 4-5) are shown as mean +/- SEM. p values listed were using the general linear model followed by Tukey’s HSD multiple comparison procedure on the logged titer values. *p < 0.05, **p < 0.01 , ***p < 0.001 , and ****p < 0.0001 .

[0043] FIG. 29 presents a graph plotting ratios of the anti-RBD IgG2c to IgGl titers of f TGS.

27 and 28. Lower values (below 1) suggest a Th2 response or humoral response, and higher values (above 1) imply a Th 1 response or cellular response.

[0044] FIG. 30 presents a timeline of immunization and blood collection to measure IgG titers in a study of the vivo humoral response to HIV GP120 vaccines adjuvanted with TLRa- SNPs. Mice were immunized on Week 0 and boosted on Week 4 with GP120 vaccines adjuvanted with Alum, SNP, or TLRa-SNPs. IgG l and IgG2c titers were determined on Week 6.

[0045] FIG. 31 presents a graph plotting results from the study of FIG. 30 showing anti- GP120 IgG binding endpoint titers of GPI20 vaccines adjuvanted with Alum, SNP, or TLRa- SNPs. Data (n ~ 5) are shown as mean +/- SEM. p values were determined using the general linear model followed by Tukey’s HSD comparison procedure on tire logged titer values for IgG titer comparisons, *p < 0.05, **p < 0,01, ***p < 0.001 , and ****p < 0.0001 ,

[0046] FIG. 32 presents a graph plotting results from the study ofFIG. 30 showing area under the curves (AUCs) of anti-GP120 IgG endpoint antibody titers from Week 0 to Week 6 of different GP120 vaccines. Data (n = 5) are shown as mean +/- SEM. p values were determined using the general linear model followed by Tukey’s HSD comparison procedure on the logged titer values for IgG titer comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0047] FIG. 33 presents a graph plotting results from the study of FIG. 30 showing anti- GP120 IgGl titers from sera collected on Week 6, 2 weeks after boost. Data (n = 5) are shown as mean +/- SEM. p values -were determined using the general linear model followed by Tukey’s HSD comparison procedure on the logged titer values for IgG titer comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0048] FIG. 34 presents a graph plotting results from the study of FIG. 30 showing anti-

GP120 IgG2c titers from sera collected on W eek 6, 2 weeks after boost. Data (n = 5) are shown as mean +/- SEM, p values were determined using the general linear model followed by

Tukey’s HSD comparison procedure on the logged titer values for IgG titer comparisons. *p <

0.05, **p < 0.01, ***p < 0.001, and *p < 0.0001

[0049] FIG. 35 presents a graph plotting ratios of the anti -GP 120 IgG2c to IgGl titers of

FIGS. 33 and 34. Lower values (below I) suggest a. Th2 response or humoral response, and higher values (above 1) imply a Thl response or cellular response.

[0050] FIG. 36 presents a timeline of immunization and draining lymph node (dLN) analysis in a study of dLNs post-immunization with RBD NP vaccines adjuvanted with SNP or TLRa- SNPs. Luminex analysis was performed 1 day (24 hours) after immunization and germinal center B cells (GCBCs) were measured 12 days after immunization.

[0051] FIG. 37 presents a graph of results from the study of FIG. 36 showing that all three

TLRa-SNPs induced significantly higher beneficial proinflammatoiy cytokine levels of IFN-y, IP10, IL03, and IL-15 in the dLNs compared to SNP, reported in median fluorescence intensity (MFI). Data (n = 4-5) are shown as mean +/- SEM. p values for the Luminex assay were from a generalized maximum entropy estimation regression adjusted to control the false discovery- rate .

[0052] FIG. 38 presents a graph of results from the study of FIG. 36 showing a dimensional reduction analysis of the full murine Luminex 48-plexed cytokine assay (FIG. 38) in the form of a penalized supervised star (PSS) plot of the RBD NP vaccines. The results show's clear separation of vaccines containing different adjuvants, with each number signifying an individual sample from the treatment groups. Vectors are the projection coefficients for each individual cytokine.

[0053] FIG. 39 presents a series of graphs showing foil artifact-corrected Luminex dataset for serum cytokine levels 24 hours after vaccine immunization in logarithm Median Fluorescence Intensity (MFI). Ploted data are detrended for covariates of cage and nonspecific bindings and bars shown are means.

[0054] FIG. 40 presents a graph plotting results from the study of FIG. 36 showing total

GCBC count from RBD NP vaccines. Data (n = 4-5) are shown as mean +/- SEM, p values for

FACS were determined using the general linear model followed by Tukey’s HSD comparison procedure on the logged values.

[0055] FIG. 41 presents a graph plotting results from the study of FIG. 36 showing frequency of GCBC from all B cells. Data (n ~ 4-5) are shown as mean +/- SEM.p values for FACS were determined using the general linear model followed by Tukey’s HSD comparison procedure on the logged values.

[0056] FIG. 42 presents a timeline of immunization and blood collection to determine IgG titers in a study showing that RBD NP vaccines adjuvanted with a 1 : 1 mixture of TLR4a~SNP and TLR7/8a-SNP did not elicit stronger humoral responses. Mice were immunized on Week 0 and boosted on Week 3 with RBD NP vaccines adjuvanted with SNP, TLR4a-SNPs, TLR7/8a-SNP, or equal mixture of TLR4a-SNP and TLR7/8a-SNP. All mice received equal amount of saponin content. IgGl, IgG2c, and variants titers were determined on Week 5.

[0057] FIG. 43 presents a graph plotting results from the study of FIG. 42 showing anti-RBD IgG binding endpoint titers of RBD NP vaccines with different adjuvants. Data (n = 4-6) are shown as mean SEM.

[0058] FIG. 44 presents a graph plotting results from the study of FIG. 42 showing anti-RBD IgGl titers from sera collected on Week 5, 2 weeks after boost. Data (n = 4-6) are shown as mean +/- SEM.

[0059] FIG. 45 presents a graph plotting results from the study of FIG. 42 showing anti-RBD IgG2c titers from sera collected on Week 5, 2 weeks after boost. Data (n ~ 4-6) are shown as mean +/- SEM. [0060] FIG. 46 presents a graph plotting ratios of the anti-RBD IgG2c to IgG 1 titers of FIGS. 44 and 45. Lower values (below 1) suggest a Th2 response or humoral response, and higher values (above 1) imply a Thl response or cellular response.

[0061] FIG. 47 presents a graph ploting results from the study of FIG. 42 showing anti-spike IgG binding endpoint titers from sera collected on Week 5 after the initial immunization. Titers were determined for wildtype WT spike as well as Delta (B. 1.617.2), and Omicron (B. 1.1.529) variants of the spike protein. Data (n = 4-6) are shown as mean +/- SEM.

[0062] FIG. 48 presents a timeline of the experimental setup and spleen collection on Week 5 to determine antigen-specific CD8+ T cell population in an analysis of antigen-specific IFN- y producing splenocytes with ELISpot.

[0063] FIG. 49 presents a graph ploting results from the study of FIG. 48 showing the number of IFN-y producing CD8-f- T cells upon antigen stimulation of 800,000 splenocytes/well of the vaccine groups. Data (n = 6) are shown as mean +/- SEM.

[0064] FIG. 50 presents a schematic representation of a saponin nanoparticle SNP encapsulating both TLR4a and TLR7/8a in the same nanoparticle. Also presented is a graph plotting anti-RBD endpoint titers for various nanoparticle compositions, demonstrating that nanoparticles encapsulating both TLR4a and TLR7/8a can elicit superior titers.

[0065] FIG. 51 presents images of SNP formulations with TLR7/8a derivatives having either a cholesteryl or a stearyl moiety. A visual inspection showed the formation of stable nanoparticles with formulations including the cholesteryl derivative, but not for formulations including the the stearyl derivative, for which precipitation occurred. This observation demonstrates the non-trivial and delicate nature of the SNPs self-assembly. Stearyl- imidazoquinoline derivative was synthetized as follow: Briefly, 4-amino-2-(ethoxy rnethyl)- lH-iniidazo[4, 5-C]quinoline~l-butylamine (80.0 mg, 0.255 mmol) was dissolved in 5 nil of anhydrous CH2CI2/DMF (80:20) under nitrogen atmosphere. EDC (58.7 mg, 0.306 mmol, 1.2 eq.), DMAP (9.3 mg, 0.076 mmol, 0.3 eq.) and stearic acid (79.9 mg, 0.281 mmol, 1.1 eq.) were added and the reaction mixture was stirred at room temperature for 4 hours. Solvents were evaporated under reduced pressure and the resulting solid was purified by column chromatography on silica gel eluting with a step gradient of MeOH/EtsN (99: 1) from 0 % to 30 % v/v in CH2CI2. The derivative was obtained as a white solid foam (quantitative yield). DETAILED DESCRIPTION

I. General

[0066] Over the past few decades, modem vaccinology pushed its efforts towards the use of subunit protein-based vaccines over more traditional live-attenuated or inactivated vaccines due to the former’s affordability, ease of manufacturing, and high safety profile (P.M. Moyle & I. Toth, ChemMedChem 8, (2013): 360). However, some protein antigens’ poor immunogenicity reinforces the need for developing more potent and modular adjuvants. The present disclosure provides compositions and methods related to an adjuvant platform combining three approaches in adjuvant design and selection: (i) molecular TLRa adjuvants, which resemble pathogen associated molecular patterns found on bacteria, viruses, and other foreign invaders our immune system is trained to recognize, (ii) saponin adjuvants, natural chemicals that improve lymphatic flow and drainage, and (iii) particulate design, displaying both adjuvants together with improved kinetics and cellular uptake. Although previous studies have reported the design of combined TLR4a MPLA and saponins into nano-objects, such as AS 01 and SMNP, a tunable platform approach must consider incorporating other clinically relevant TLRas. In this regard, the disclosure advantageously demonstrates that lipid TLRas, such as MPLA and Pam3CSK4 can be readily incorporated with clinically relevant stoichiometric ratios due to the hydrophobic nature of SNPs. Non-lipid TLRas, such as TLR7/8a Resiquimod, can be incoqiorated by conjugating a cholesterol motif that hydrophobical ly interacts with SNPs. The facile assembly of TLRas with saponins allows for incorporation of more than one TLRa in the same particle, such as the synergetic combination of TLR4a and TLR7/8a (M.S. Duthie, H.P. Windish, C.B Fox & S.G. Reed, Immunol. Rev. 2.39, (201 1): 178; S.P. Kasturi et al., Nature 470, (2011): 543).

[0067] Numerous preclinical and clinical studies have utilized the receptor binding domain nanoparticle (RBD NP) antigen RBD-16GS-I53-50 developed by King and coworkers (T.K. Raaijmakers et al., J Jmmunother. Cancer 8, (2020): e000649; P.S. Arunachalam et al., Nature 594, (2021): 253; L. Grigoryan et al., NPJ Vaccines 7, (2022): 55; A.C. Wails et al.. Cell 193, (2020): 1367). In the studies described in the examples of this disclosure, tire same RBD NP antigen was used in a dose- and schedule- matched study design, allowing for a direct comparison of the potency of TLRa-SNPs with previously screened adjuvants (T.K. Raaijmakers et al., J. Immunother. Cancer 8, (2020): e000649; P.S. Arunachalam et al., Nature 594, (2021): 2.53; L. Grigoryan et al., NPJ Vaccines 7, (2022): 55; A.C. Walls et al.. Cell. 193, (2020): 1367). From those studies, AS03 and CpG/Alum have been reported to induce comparable humoral responses such as robust neutralizing responses, and both outperformed other clinically relevant adjuvants such as AS37, AddaVax, and Essai O/W 1849101 (L. Grigoryan et al., NPJ Vaccines 7, (2022): 55). The use of CpG/Alum as a control in the present disclosure remarkably highlights the superiority of provided TLRa-SNPs (e.g, TLRl/2a-SNP, TLR4a-SNP, TLR7/8a~SNP, and TLR4a-TLR7/8a-SNP) to generate potent, durable, broad, and neutralizing antibody responses compared to previously screened adjuvants (L. Grigoryan et al., NPJ Vaccines 7, (2022): 55). This observation is even more striking considering that the dosage of CpG/Alum was doubled compared to that used in literature (L. Grigoryan et al., NPJ Vaccines I, (2022): 55). Additionally, mice adjuvanted with TLRa-SNPs achieved complete neutralization results, in contrast to convalescent human plasma which resulted in a quantifiable level of infectivity. This is despite the fact that all human patients had previously received up-to date COVID- 19 vaccines, including original prime and boost and an additional booster from either Modema or Pfizer COVID-19 mRNA vaccines, before contracting COVID- 19 8-12 weeks before sample collection. Previous reports have demonstrated that these patients have superior neutralizing antibodies than those from infected patients that have not been previously vaccinated (S.J. Oh et al., J. Clin. Virol. 155, (2022): 105253; T.A. Bates et al., Set. Immunol. 7, (2.022): eabn8014; ¥. Yu et al, Sei. Rep. 12, (2022): 2628; H.W, Jeong et al.. Cell Rep. Med. 3, (2022): 100764; T. Lechmere et al, mBio 13, (2022): e0379821). The data provided herein thus shows the significant benefits that can be realized with the TLRa- SNPs adjuvant technology in larger animals.

[0068] Moreover, in an NHP study, groups receiving AS03 adjuvanted RBD NP antigen vaccines had extended antibody titers and more durable Omicron variant protection when compared to those vaccinated with commercial Pfizer-BioNTech and Modema mRNA vaccines (P.S. Arunachalam et al. Nature 594, (2021): 253). Nonetheless, antibody titers waned to pre-booster magnitude by 6 months post-boost, necessitating additional boosters to maintain a robust protection (P.S. Arunachalam et al. Nature 594, (2.021): 2.53), Using the provided compositions and methods, however, mice maintained antibody endpoint titers in similar magnitude to those adjuvanted with SNP and CpG/Alum on Week 11 for a full year following the vaccination with TLRa-SNPs adjuvanted RBD NP vaccines. These unexpected results were further emphasized by the rapid seroconversion of all mice after a single immunization (i.e, the original prime), thereby enabling single-immunization vaccines adjuvanted with TLRa-SNPs. The rapid and complete seroconversions were further verified with HIV GP120 protein antigen, showing the superiority and potential broad application of TLRa-SNPs compared to SNP and clinical controls. Notably, no substantial differences were observed between the two clinical controls, CpG/Alum, mimicking Dynanvax’s CpG1018, and SNP, similar in composition and structure to Novavax’s Matrix M. Although their adjuvant potencies remained similar, their differences in immune stimulating mechanism can create synergistical effects when combined, as illustrated by the potent, durable, broad, and neutralizing humoral responses seen to be induced by TLRa-SNPs. Overall, TLRa-SNPs as adjuvants can rapidly lead to protective levels of neutralizing antibodies and therefore strongly decrease the need for costly booster shots, which is crucial to protect worldwide populations rapidly and broadly during a pandemic.

[0069] Also beneficially, the provided TLRa-SNP formulations produce unique acute cytokine induction profiles and Th-skewed responses, where the type of induction profile and response for each TLRa-SNP formulation is consistent with that previously described (B. Pulendran, P.S. Arunachalam & D.T. O’Hagan, Nat. Rev. Drug Discov. 20, (2021 ): 454; M.S. Duthie, H.P. Windish, C.B. Fox & S.G. Reed, Immunol. Rev. 239, (2011): 178; M. Kwissa, M. H I. Nakaya, H. Oluoch & B. Pulendran, Blood 1 19, (2012): 2044). For example, TLR l/2a- SNP, TLR4a-SNP, and TLR7/8a-SNP generate robust IgG2c titers indicative of a strong Th1 response. Indeed, all three TLRas can induce Th1 response by activating the NF-KB pathway. Elevated levels of IL-12 and IL Ip, hallmark cytokines of NF-KB activation, in the Lunnnex assay results detailed in the provided examples further verify the incorporation of these three TLRas and their impact on immune signaling (B. Pulendran, P.S. Arunachalam & D.T. O’Hagan, Nat. Rev. Drug Discov. 20, (2021): 454). Additionally, from among these particular TLRa-SNP formulations, only TLRI/2a-SNP elicited higher IgGl titer, leading to an overall Th2 skewed response. The activation of TLR1 and TLR2 triggers the ERK 1 and ERK2 pathways, which typically results in higher Th2 responses (B. Pulendran, P.S. Arunachalam & D.T. O’Hagan, Nat. Rev. Drug Discov. 20, (2021): 454). Similarly, the initiation of endosomal TLRs such as TLR7 and TLR8 activates the IRF-pathway, which in turn induces strong type I interferon response such as the secretion of IFN-a and IFN-p. Assay results provided herein verify the success of TLRas incorporation to the SNP as well as the unique Th responses induced from the library of TLRa-containing saponin-based nanoparticles. In this regard, literature has shown that while Th2~skewed responses resulted in more favorable clinical outcomes for some infectious diseases such as Rabies virus (S.M. Moore, M.J. Wilkerson, R.D. Davis, C.R. Wyatt & D.J. Briggs, J. Clin. Immunol. 26, (2006): 533; M. Garenne & M. Lafon, Perspect. Biol. Med. 41, (1998): 176), Till -skewed responses were favorable for diseases such as COVID-19 (F.J. Gil-Etayo et al., Biomedicines 10, (2022): 296; F.J. Gil-Etayo et al., Front. Cell. Infect. Microbiol. 11, (2021): 624483; F.H.N. Howard et al,, Viruses 14, (202.2): 1493; C. Infante-Duarte & T. Kamradt, Semin. Immunopathol. 21, (1999): 317). Thus, having a library of TLRa-SNPs with tailored Th-skewed responses can have profound clinical implications for which a specific adjuvant formulation can be selected during the early stage of vaccine development depending on the disease the vaccine is designed to prevent.

[0070] The TLRa-SNPs of the compositions and methods disclosed herein thus provide significant advantages for improving the potency, durability, breadth, and neutralization of vaccines, offering the potential of preventing infection by complex immune evasive viruses such as SARS-CoV-2 and HIV. Notably, different formulations induce unique acute cytokine and immune -signaling profiles, leading to different Th-re spouses. A robust and tunable adjuvant library’ is therefore available to reinforce global efforts to rapidly develop and manufacture vaccines in response to an infectious agents currently unknown to cause human disease.

II. Definitions

[0071] As used herein, the term “adjuvant” refers to a substance or composition that increases an effect or potency of a drug or enhances an immune response to an antigen or a vaccine. An adjuvant may modify the strength, longevity, or breadth of a drag effect or immune response.

[0072] As used herein, the term “administering” refers to oral administration, administration as a suppository, , topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject.

[0073] As used herein, the term “agonist” refers to a substance that has an affinity for the active site of a receptor and thereby preferentially stabilizes the active state of the receptor, or a substance that produces activation of receptors and enhances signaling by those receptors.

[0074] As used herein, the term “composition” refers to a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. A “pharmaceutically acceptable composition” is one in which each ingredients, e.g., a earner, diluent, or excipient, is compatible with the other ingredients of a formulation composition and not deleterious to the recipient thereof.

[0075] As used herein, the term “nanoparticle" refers to any solid particulate -with a size that is in the range of nanometers. For example, a nanoparticle can have a diameter of less than 1 micron (1000 nm), or less than about 100 nm.

[0076] As used herein, the terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” refer to a substance that aids the administration of an active agent to and absorption by a subject and may be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the subject. Non-limiting examples of pharmaceutically acceptable excipients and carriers include water, NaCl, normal saline solutions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, and the like. One of skill in the art will recognize that other pharmaceutically acceptable excipients and carriers are useful in the present disclosure.

[0077] As used herein, the term “saponin” refers to a glycosidic molecule comprising a triterpene or steroid aglycone with one or more sugar chains. The term encompasses naturally derived saponins (e.g., plant-derived saponins), synthetic saponins, and saponin derivatives; including semi-synthetic saponin derivatives.

[0078] As used herein, the term “subject” refers to a vertebrate, and preferably to a mammal. Mammalian subjects for which the provided composition is suitable include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets. In some embodiments, the subject is human. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult. In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 years of age, or above 80 years of age. In some embodiments, the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age.

[0079] As used herein, the term “therapeutically effective amount” refers to an amount or dose of a compound, composition, or formulation that produces therapeutic effects for which it is administered. The exact amount or dose will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques. [0080] As used herein, the terms “treat,” “treating,” and “treatment” refer to a procedure resulting in any indicia of success in the elimination or amelioration of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of one or more symptoms. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination or laboratory test.

[0081] As used herein, the term “vaccine” refers to a composition comprising at least one antigen or immunogen, or comprising a nucleic acid molecule encoding at least one antigen or immunogen, in a pharmaceutically acceptable carrier, that is useful for inducing an immune response against the antigen or immunogen in a subject, for the purpose of improving immunity against a disease and/or infection in the subject.

[0082] As used herein, the terms “including,” “comprising,” “having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited. As used herein, the phrase “consisting of’ is closed and excludes any element, step, or ingredient not explicitly specified. As used herein, the phrase “consisting essentially of” limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.

[0083] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a Toll-like receptor agonist” optionally includes a combination of two or more Toll-like receptor agonists, and the like.

III. Nanopartides

[0084] In one aspect, the present disclosure provides various nanoparticles that generally include a Toll-like recetor (TLR) agonist, a saponin, a phospholipid, and a sterol, i.e., a “nanoparticle sterol” that is a sterol associated with the underlying structure of the nanoparticle. Tire particular combination and relative amounts of these components provide the nanoparticles with several surprising improvements in various important characteristics, including a modularly tunable ability to enhance a variety of potent and durable immune responses when a plaurality of the nanoparticles are used as an adjuvant composition, e.g., as any of the adjuvant compositions described in more detail in Section IV.

[0085] The provided nanoparticles can have hydrodynamic diameters that are, for example, between 35 nm and 65 nm, e.g., between 35 nm and 53 nm, between 38 nm and 56 nm, between 41 nm and 59 nm, between 44 nm and 62. nm, between 47 nm and 65 nm. In terms of upper limits, the nanoparticle diameter can be, for example, less than 65 nm, e.g., less than 62 nm, less than 59 nm, less than 56 nm, less than 53 nm, less than 50 nm, less than 47 nm, less than 44 nm, less than 41 nm, or less than 38nni. In terms of lower limits, the nanoparticle diameter can be, for example, greater than 35 nm, e.g., greater than 38 nm, greater than 41 nm, greater than 44 nm, greater than 47 nm, greater than 50 nm, greater than 53 nm, greater than 56 nm, greater than 59 nm, or greater than 62 nm. Laregr diameters, e.g., greater than 65 nm, and smaller diameters, e.g., less than 35 nm, are also contemplated.

[0086] The provided nanoparticles can have negative surface charges that are, for example, between -24 mV and -44 mV, e.g., between -24 mV and -36 mV, between -26 mV and -38 mV, between -28 mV and -40 mV, between -30 mV and -42 mV, or between -32 mV and -44 mV. In terms of upper limits, the nanoparticle nehative surface charge can be, for example, less than -24 mV, e.g., less than -26 mV, less than -28 mV, less than -30 mV, less than -32 mV, less than -34 mV, less than -36 mV, less than -38 mV, less than -40 mV, or less than -42 mV. In terms of lower limits, the nanoparticle negative surface chanrge can be, for example, greater than - 44 mV, e.g., greater than -42 mV, greater than -40 mV, greater than -38 mV, greater than -36 mV, greater than -34 mV, greater than -32 mV, greater than -30 mV, greater than -28 mV. or greater than -26 mV. Higher negative surface charges, e.g., greater tahn -24 mV, and lower negative surface charges, e.g., less than -44 mV, are also contemplated.

[0087] The provided nanoparticles generally include a TLR agonist. In some embodiments, each of the provided nanoparticles includes only one species of TLR agonist. Alternatively, each of the provided nanoparticles can include two or more structurally different species of TLR agonists. In this way, the nanoparticles can incorporate multiple types of TLR agonists that can act synergisticly, e.g., in inducing an immune response. The incorporation of multiple types of TLR agonists in a single nanoparticle can also or instead allow the nanoparticle, when used in an adjuvant composition, to elicit different types of immune responses, e.g., immune responses characterized by different cytokine and/or IgG production profiles. A provided nanoparticle can include, for example, two or more different species of TLR agonists, e.g., three or more, four or more, five or more, six or more, seven or more, seven or more, eight or more, nine or more, or ten or more.

[0088] Tire molar fraction of the one or more TLR agonists in the nanoparticle can be, for example, between 1% and 30%, e.g., between 1 % and 7.7%, between 1 .4% and 11%, between 2% and 15%, between 2.8% and 2.1%, or between 3.9% and 30%. In terms of upper limits, the TLR agonist mole fraction in the nanoparticle can be, for example, less than 30%, e.g., less than 21%, less than 15%, less than 1 1%, less than 7.7%, less than 5.5%, less than 3.9%, less than 2.8%, less than 2%, or less than 1.4%. In terms of lower limits, the TLR agonist mole fraction in the nanoparticle can be, for example, greater than 1%, e.g., greater than 1.4%, greater than 2%, greater than 2.8%, greater than 3.9%, greater than 5.5%, greater than 7.7%, greater than 11%, greater than 15%, or greater than 21%. Higher mole fractions, e.g., greater than 30%, and lower mole fractions, e.g., less than 1%, are also contemplated.

[0089] In some embodiments, at least one TLR agonist of a provided nanoparticle is a non- lipid TLR agonist, or a derivative thereof. In some embodiments, each TLR agonist of a nanoparticle is a non-lipid TLR agonist, or a derivative thereof. The non-lipid TLR agonist or derivative can include or consist of, for example, a TLR1 agonist, a TRL2 agonist, a TRL3 agonist, a TLR4 agonist, a TRL5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, or a TLR10 agents. The non-lipid TLR agonist or derivative can include or consist of an agonist of multiple TLR types. For example, the non-lipid TLR agonist or derivative can include a TLR7/TLR8 agonist and/or a TLR1/TLR2 agonist. The non-lipid TLR agonist or derivative can include or consist of a polynucleotide, e.g., a naturally occurring or synthetic single- or double-stranded RNA or DNA. The non-lipid agonist or derivative can include or consist of a polypeptide, e.g., a bacterial or viral protein or a fragment thereof. The non-lipid agonist can include or consist of a small synthetic molecule. In some embodiments, at least one TLR agonist or derivate includes or consists of an imidazoquinoline compound. The TLR agonist or derivative can include or consist of, for example, Imiquimod, Resiquimod, and/or Gardiquimod.

[0090] In some embodiments, the provided nanoparticle includes a derivative of a non-lipid TLR agonist, where the derivative is a product of conjugating a non-lipid TLR agonist to a sterol, i.e., an agonist sterol, referred to as such to distinguish the sterol from the sterol that is a primary component of the nanoparticle. By conjugating the non-lipid TLR agonist to a sterol, the resulting derivative molecule can hydrophobically interact with other components of the provided nanoparticle advantageously enabling the formation of the nanoparticle having the improved properties described herein. Advantageously and unexpectedly, the conjugation of a non-lipid TLR agonist to a sterol promotes the self-assembly of the provided nanoparticle, whereas conjugation of the non-lipid TLR agonist to a non-sterol lipid does not (FIG. 51). The sterol can include, for example, a cholesterol, an ergosterol, a phytosterol (e.g., a campesterol, a sitosterol, a stigmasterol, and/or a brassicasterol), a lanosterol, a cycloartenol, a hopanoid (e.g., a diploptene and/or a bacteriohopanetetrol), a calusterol, a stenbolone, an ecdysteroid (e.g., an ecdysone, a 20-hydroxyecdysone, and/or a ponasterone A), a cardenolide (e.g., a digitoxin, a digoxin, and/or an oubain), or any combination thereof.

[0091] In some embodiments, the provided nanoparticle includes a derivative of a non-lipd

TLR agonist, where the agonist is conjugated to a cholesterol. For example, the derivative can have the chemical structure:

[0092] In some embodiments, at least one TLR agonist of a provided nanoparticle is a lipid TLR agonist, or a derivative thereof. In some embodiments, each TLR agonist of a nanoparticle is a lipid TLR agonist, or a derivative thereof. The lipid TLR agonist or derivative can include or consist of, for example, a TLR1 agonist, a TRL2 agonist, a TRL3 agonist, a TLR4 agonist, a TRL5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, or a TLR10 agents. Tire lipid TLR agonist or derivative can include or consist of an agonist of multiple TLR types. For example, the lipid TLR agonist or derivative can include a TLR7/TLR8 agonist and/or a TLR1/TLR2 agonist. The lipid TLR agonist or derivative can include or consist of a lipopolysaccharide, e.g., a lipid A analog such as monophosph oryl lipid A (MPLA). The lipid TLR agonist or derivative can include or consist of a macroamphiphile, e.g., a lipoteichoic acid. The lipid TLR agnost or derivative can include or consist of a lipopeptide, e.g., Pam3-Cys-Ser- Lys4 (Pam3CSK4), Pam2-Cys-Ser-Lys4 (Pam2CSK4), and/or macrophage-activating lipopeptide-2 (MALP-2). The lipid TLR agonist can include or consist of a phospholipid, e.g., phosphatidylserine and/or phosphatidylglycerol. [0093] The provided nanoparticles further generally include a saponin. In some embodiments, each of the provided nanoparticles includes one species of saponin. Alternatively, each of the provided nanoparticles can include two or more structurally different species of saponins. A provided nanoparticle can include, for example, two or more different saponins, e.g., three or more, four or more, five or more, six or more, seven or more, seven or more, eight or more, nine or more, or ten or more, The saponin can include or consist of a Quiliaja saponin, e.g., Quil A and. or Quil B. The saponin can include or consist of a ginsenoside, e.g., ginsenoside Rgl, ginsenoside Rbl , ginsenoside Rg3, ginsenoside Rhl, and/or ginsenoside Rh2. The saponin can include or consist of a soyasaponin, e.g., soyasaponin Al, soyasaponin A2, soyasponin I, soyasaponin Bb, and/or soyasaponin Be. The saponin can include or consist of a dioscin, e.g., dioscin and/or protodioscin . Tire saponin can include or consist of an aescin, e.g., alpha-aescin, beta-aescin, aescin la, aescin lb, and/or aescin Ila. The saponin can include or consist of a hederagenin, e.g., hederacolchiside Al, hederacolchiside E, hederacolchiside F, hederosaponin B, and/or hedysarosaponin A.

[0094] The molar fraction of the saponin in the nanoparticle can be, for example, between 7% and 87%, e.g., between 7% and 55%, between 15% and 63%, between 23% and 71 %, betw een 31% and 79%, or between 39% and 87%. In terms of upper limits, the saponin mole fraction in the nanoparticle can be, for example, less than 87%, e.g., less than 79%, less than 71%, less than 63%, less than 55%, less than 47%, less than 39%, less than 31%, less than 23%, or less than 15%. In terms of lower limits, the saponin fraction in the nanoparticle can be, for example, greater than 7%, e.g., greater than 15%, greater than 23%, greater than 31 %, greater than 39%, greater than 47%, greater than 55%, greater than 63%, greater than 71%, or greater than 79%. Higher mole fractions, e.g., greater than 87%, and lower mole fractions, e.g., less than 7%, are also contemplated

[0095] The provided nanoparticles further generally include a phospholipid. In some embodiments, each of the provided nanoparticles includes one species of phospholipid. Alternatively, each of the provided nanoparticles can include two or more structurally different species of phospholipids. A provided nanoparticle can include, for example, two or more different phospholipids, e.g., three or more, four or more, five or more, six or more, seven or more, seven or more, eight, or more, nine or more, or ten or more. The phospholipid can include or consist of a phosphatidylcholine, e.g., dipalmitoylphosphatidylcholine (DPPC), polyene phosphatidylcholine (PPC), soybean phosphatidylcholine (SPC), egg phosphatidylcholine (EPC), and/or L-alpha-phosphatidylcholine (L-alpha-PC) , The phospholipid can include or consist of a phosphatidylglycerol, e.g., dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), monopalmitoylphosphatidylglycerol (MPPG), dilinoleoylphosphatidylglycerol (DLPG), and/or dioleoylphosphatidylglycerol (DOPG). The phospholipid can include or consist of a phosphatidylethanolamine, e.g., dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLPE), and/or soybean phosphatidylethanolamine (SPE). The phospholipid can include or consist of a phosphatidylserine, e.g., dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), monopalmitoylphosphatidylserine (MPPS), dioleoylphosphatidylserine (DOPS), and/or dilinoleoylphosphatidylserine (DTPS). The phospholipid can include or consist of a lecithin, e.g., soybean lecithin, egg lecithin, and/or sunflower lenithin.

[0096] The molar fraction of the phospholipid in the nanoparticle can be, for example, between 1% and 55%, e.g., between 1% and 11 %, between 1.5% and 17%, between 2.2% and 25%, between 3.3% and 37%, or between 5% and 55%. In terms of upper limits, the phospholipid mole fraction in the nanoparticle can be, for example, less than 55%, e.g., less than 37%, less than 25%, less than 17%, less than 11%, less than 7.4%, less than 5%, less than 3.3%, less than 2.2%, or less than 1.5%. In terms of lower limits, the phospholipid fraction in the nanoparticle can be, for example, greater than 1%, e.g., greater than 1.5%, greater than 2,2%, greater than 3.3%, greater than 5%, greater than 7.4%, greater than 11 %, greater than 17%, greater than 25%, or greater than 37%. Higher mole fractions, e.g., greater than 55%, and lower mole fractions, e.g., less than 1 %, are also contemplated

[0097] Tire provided nanoparticles further generally include a sterol, i.e., a nanoparticle sterol, referred to as such to distinguish the sterol from any optionally included in the TLR agonist. In some embodiments, each of the provided nanoparticles includes one species of nanoparticle sterol. Alternatively, each of the provided nanoparticles can include two or more structurally different species of nanoparticle sterols. The nanoparticle sterol can include or consist of, for example, cholestreol, ergosterol, sitosterol, stigmasterol, or any combination thereof.

[0098] The molar fraction of the sterol in the nanoparticle can be, for example, between 7% and 87%, e.g., between 7% and 55%, between 15% and 63%, between 23% and 71%, between

31% and 79%, or between 39% and 87%. In terms of upper limits, the sterol mole fraction in the nanoparticle can be, for example, less than 87%, e.g., less than 79%, less than 71 %, less than 63%, less than 55%, less than 47%, less than 39%, less than 31%, less than 23%, or less than 15%. In terms of lower limits, the sterol fraction in the nanoparticle can be, for example, greater than 7%, e.g., greater than 15%, greater than 23%, greater than 31%, greater than 39%, greater than 47%, greater than 55%, greater than 63%, greater than 71%, or greater than 79%. Higher mole fractions, e.g., greater than 87%, and lower mole fractions, e.g., less than 7%, are also contemplated.

IV. Adjuvant Compositions

[0099] In another aspect, the present disclosure provides various adjuvant compositions specifically configured to enhance an immune response of a subject against an antigen. The provided adjuvant compositions each generally include a plurality of any of the nanoparticles disclosed herein, e.g., any of the nanoparticles described in more detail in Section III. For example, the adjuvant composition can include a plurality of nanoparticles that each have a non-lipid TLR agonist and/or a lipid TLR agonist, a saponin, a phospholipid, and a nanoparticle sterol. Becuase the adju vant compositions include the provided nanoparticles, the compositions exhibit several surprising improvements in various important characteristics, including a modular and tunable ability to enhance a variety of potent and durable immune responses.

[0100] In some embodiments, the plurality of nanoparticles in the provided aduvant composition includes apluarlity of only one species of nanoparticle. Alternatively, the plurality of nanoparticles can include two or more different species of nanoparticles, e.g,, nanoparticles that are assembled with different TLR agonists, saponins, phospholipids, and/or nanoparticle sterols. In this way, the adjuvant composition can incorporate multiple types of nanoparticles that can act synergisticly, e.g., in inducing an immune response. The incorporation of multiple types of nanoparticles in a single adjuvant composition can also or instead allow the adjuvant composition to elicit different types of immune responses, e.g., immune responses characterized by different cytokine and/or IgG production profiles. A provided adjuvant composition can include, for example, two or more different species of nanoparticles, e.g., three or more, four or more, five or more, six or more, seven or more, seven or more, eight or more, nine or more, or ten or more.

[0101] In some embodiments, a provided adjuvant composition is specifically configured to enhance an immune response against an antigen associated with an infectious disease or a cancer. In some embodiments, the adjuvant composition is configured to enhance an immune response against an infectious disease antigen. Tire infectious disease antigen can include, for example, a bacterial antigen, a viral antigen, a fungal antigen, a protozoal antigen, a helminthic antigen, or a combination thereof. In some embodiments, the adjuvant composition is configured to enhance an immune response against a viral antigen. The viral antigen can include, for example, a severe acute respiratory syndrome coronavirus (SARS-CoV) antigen, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen, a Middle East respiratory syndrome coronavirus (MERS-CoV) antigen, a simian immunodeficiency virus (SIV) antigen, a human immunodeficiency virus (HIV) antigen, a hepatitis C virus antigen, a herpes simplex virus antigen, an Epstein-Barr virus antigen, a cytomegalovirus antigen, an influenza virus antigen, or a combination thereof.

[0102] In some embodiments, the provided adjuvant composition is particularly configured to enhance a Th2-skewed immune response that can be more effective in treating certain infectious diseases such as rabies. These adjuvant compositions can be configured to enhance immue responses in which a subject produces higher IgGl concentrations than IgG2c concentrations, The immune response can be characterized as having an IgGl-to-IgG2c ratio that is, for example, between 1 and 20, e.g., between 1 and 6, between 1 .3 and 8.1 , between 1 .8 and 11, between 2.5 and 15, or between 3.3 and 20. In terms of upper limits, the immune response IgGl-to-IgG2c ratio can be, for example, less than 20, e.g., less than 15, less than 1 1, less than 8.1, less than 6, less than 4.5, less than 3.3, less than 2.5, less than 1.8, or less than 1 ,3. In terms of lower limits, the immune response IgGl-to-IgG2c ratio can be, for example, greater than 1 , e.g., greater than 1.3, greater than 1.8, greater than 2.5, greater than 3.3, greater than 4.5, greater than 6, greater than 8.1, greater than 11, or greater than 15. Higher ratios, e.g., greater than 20, are also contemplated.

[0103] In some embodiments, the provided adjuvant composition is particularly configured to enhance a Th 1 -skewed immune response that can be more effective in treating certain infectious diseases such as COVID-19. These adjuvant compositions can be configured to enhance immue responses in which a subject produces higher IgG2c concentrations than IgGl concentrations. The immune response can be characterized as having an IgG2c-to-IgGl ratio that is, for example, between 1 and 20, e.g., between 1 and 6, between 1 .3 and 8.1 , between 1 .8 and 11, between 2.5 and 15, or between 3.3 and 20. In terms of upper limits, the immune response IgG2c-to-IgGl ratio can be, for example, less than 20, e.g., less than 15, less than 1 1, less than 8.1, less than 6, less than 4.5, less than 3.3, less than 2.5, less than 1.8, or less than 1 ,3. In terms of lower limits, the immune response IgG2c-to-IgGl ratio can be, for example, greater than 1 , e.g., greater than 1.3, greater than 1.8, greater than 2.5, greater than 3.3, greater than 4.5, greater than 6, greater than 8.1, greater than 11 , or greater than 15. Higher ratios, e.g., greater than 20, are also contemplated.

V. Immunogenic Compositions and Vaccines

[0104] In another aspect, the present disclosure provides various immunogenic compositions specifically configured to induce an immune response of a subject against an antigen. The provided immunogenic compositions each generally’ include an antigen and any of the adjuvant compositions disclosed herein, e.g., any of the adjuvant compositions described in more detail in Section IV. In some embodiments, the immunogenic composition has the form and function of a vaccine. The particular combination and relative amounts of these components provide the immunogenic compositions with several surprising improvements in various important characteristics, including a modularity tunable ability to induce a variety of potent and durable immune responses.

[0105] In some embodiments, the provided immunogenic composition further includes a pharmaceutically acceptable carrier. In some embodiments, the provided immunogenic composition further includes a pharmaceutically acceptable excipient. In some embodiments, the provided immunogenic composition includes a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient. The pharmaceutically acceptable carrier can include or consist of one or more substances for providing the formulation with stability, sterility and isotonicity. For example, the carrier can include or consist of one or more antimicrobial preservatives, antioxidants, chelating agents and/or buffers. The pharmaceutically acceptable carrier can include or consist of one or more substances for preventing the growth or action of microorganisms. For example, the carrier can include or consist of one or more antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like. The pharmaceutically acceptable carrier can include or consist of one or more substances for providing the formulation with a more palatable or edible flavor. In some instances, the pharmaceutically acceptable excipient is an agent that facilitates the delivery of an antigen to a target cell or tissue. One of skill in the art will recognize that other pharmaceutical earners or excipients are useful in the provided embodiments.

[0106] The provided immunogenic composition can include one or more buffers, e ,g., neutral buffered saline or phosphate buffered saline; one or more carbohydrates, e.g., glucose, mannose, sucrose or dextrans; mannitol; one or more proteins, polypeptides or amino acids such as glycine; one or more antioxidants, e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, or butylated hydroxyanisole; one or more bacteriostats; one or more chelating agents, e.g., EDTA or glutathione; one or more solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a recipient; one or more suspending agents; one or more thickening agents; one or more preservatives; one or more flavoring agents; one or more sweetening agents; one or more coloring compounds; or any combination thereof.

[0107] The immunogenic compositions disclosed herein can be provided in a desired dosage formulation, suitable, for example, to be administered in a therapeutically or prophylactically effective manner and amount. The quantity to be administered can depend at least in part on a variety of factors including, e.g., the age, body weight, physical activity, hereditary characteristics, general health, sex, and diet of the individual subject; the condition or disease to be treated or prevented; and the stage or severity of the condition or disease. In certain embodiments, the size of the dose may also be determined by the existence, nature, and extent, of any adverse side effects that accompany the administration in a particular individual. Other factors that can influence the specific dose level and frequency of dosage for any particular patient, include the activity of the specific compound employed, the metabolic stability⁷ and length of action of that compound, the mode and time of administration, and the rate of excretion.

[0108] In some embodiments, the immunogenic composition dose can take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages. As used herein, the term "‘unit dosage form^'' refers to physically discrete units suitable as unitary dosages, e.g., an ampoule, for humans and other mammals with each unit containing a predetermined quantity calculated to produce the desired onset, tolerability , and/or therapeutic or prophylactic effects, in association with a suitable pharmaceutical excipient. In addition, more concentrated dosage forms may' be prepared, from which the more dilute unit dosage forms may then be produced, The more concentrated dosage forms thus will contain substantially⁷ more than, e.g., at least, one, two, three, four, five, six, seven, eight, nine, ten, or more than ten times the amount of the immunogenic composition. The dosage forms typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like. VI. Methods for Inducing an Immune Response

[0109] In another aspect, the present disclosure provides various method for inducing an immune response in a subject. The methods generally include administering to the subject any of the immunogenic compositions disclosed herein, e.g., any of the immunogenic compositions described in more detail in Section V. For example, the immunogenic composition can be a vaccine as described herein, and the administration of the immunogenic composition can include vaccinating the subject with the vaccine.

[0110] The disclosed immunogenic compositions can be administered using the provided method as a single dose or as multiple doses, for example, two doses administered at an interval of about one week, two w eeks, three weeks, one month, about two months, about three months, about six months, or about 12 months. Other suitable dosage schedules can be determined by a medical practitioner. In some embodiments, additional compounds or medications can be co- administered to the subject. Such compounds or medications can be co-administered to, for example, alleviate signs or symptoms of the disease being treated, or to reduce side effects caused by induction of the immune response.

[0111] In some embodiments, the immune response induced by the provided method is assessed by immunophenotyping or by characterizing specific T-cell responses from the subject, for example by using flow cytometry. In some embodiments, the immune response is assessed by detecting antibodies obtained from the subject, for example using an antigen binding assay such as an enzyme-linked immunosorbent assay (ELISA) , In some embodiments, the immune response includes generation of antibodies that recognize the antigen, where the antigen can be any of those described herein, e.g., any of the antigens described in more detail in Section IV .

[0112] In some embodiments, the immune response induced by the provided method is greater than an immune response induced using a corresponding immunogenic composition that does include an adjuvant composition as described in Section IV, i.e., that does not include nanoparticles as described in Section III.

VII. Methods for Treating or Preventing a Disease

[0113] In another aspect, the present disclosure provides various method preventing or treating a disease in a subject. The methods generally include administering to the subject any of the immunogenic compositions disclosed herein, e.g., any of the immunogenic compositions described in more detail in Section V. Tire disease treatment or prevention methods therefore generally include inducing an immune response in the subject as described in more detail in Section VI. In some embodiments, the treating of the disease in the subject includes decreasing or eliminating one or more signs or symptoms of the disease.

[0114] In some embodiments, the disease prevented or treated with the provided method is an infectious disease or cancer. The infectious disease can be one associate with any of the antigens disclosed herein, e.g., any of the antigens described in more detail in Section IV. The infectious disease can be caused by, for example, a bacterial infection, a viral infection, a fungal infection, a protozoal infection, a helminthic infection, or a combination thereof. In some embodiments, the infection is a SARS-CoV-2 infection.

[0115] In some embodiments, the provided method further includes obtaining a test sample from the subject in which an immune response is induced. The test sample can include, for example, a blood sample, a tissue sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, or a combination thereof. In some embodiments, the provided method further includes determining the level of one or more biomarkers in the obtained test sample. Determining the presence or level of biomarkers(s) can be used to, as non-limiting examples, determine response to treatment or to select an appropriate composition for the prevention or treatment of the disease.

[0116] In some embodiments, the provided method further includes comparing the determined level of the one of more biomarkers in the obtained test sample to the level of the one or more biomarkers in a reference sample. The reference sample can be obtained, for example, from the subject in which the immune response is induced, with the reference sample being obtained prior to the obtaining of the test sample, e.g., prior to the administering to the subject of the therapeutically effective amount of the provided immunogenic composition. In this way, the reference sample can provide information about baseline levels of the biomarkers in the sample before the immune response against the antigen is induced in the subject, and the test sample can provide information about levels of the biomarkers after the immune response is induced.

[0117] Alternatively, the reference sample can be obtained, for example, from a different subject, e.g., a subject in which the immune response against the antigen is not induced according to the provided methods. In this way, the reference sample can provide information about baseline levels of the biomarkers without immune response inducement, and the test sample can provide information about levels of the biomarkers with immune response inducement. The reference sample can also be obtained, for example, from a population of subjects, e.g., subjects in which the immune response against the antigen is not induced according to the provided method. In this way, the reference sample can provide population- averaged information about baseline levels of the biomarkers without immune response inducement, and the test sample can provide information about levels of the biomarkers with immune response inducement.

[0118] The reference sample can also be obtained from an individual or a population of individuals after an immune response against the antigen is induced, and can serve as, for example, a positive control sample. In some embodiments, the reference sample is obtained from normal tissue. In some embodiments, the reference sample is obtained from abnormal tissue.

[0119] Depending on the biomarker, an increase or a decrease relative to a normal control or reference sample can be indicative of the presence of a disease, or response to treatment for a disease. In some embodiments, an increased level of a biomarker in a test sample, and hence the presence of a disease, e.g., an infectious disease or cancer, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least, 1. 1-fold, e.g., at least 1 ,2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1,7- fold, at least 1 .8-fold, at least 1 .9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fokl, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 1 1- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold higher in comparison to a negative control. In other embodiments, a decreased level of a biomarker in the test sample, and hence the presence of the disease, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1 ,4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold lower in comparison to a negative control.

[0120] The biomarker levels can be detected using any method known in the art, including the use of antibodies specific for the biomarkers. Exemplary methods include, without limitation, polymerase chain reaction (PCR), Western Blot, dot blot, ELISA, radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, FACS analysis, electrochemiluminescence, and multiplex bead assays, e.g., using Luminex or fluorescent microbeads. In some instances, nucleic acid sequencing is employed.

[0121] In certain embodiments, the presence of decreased or increased levels of one or more biomarkers is indicated by a detectable signal, e.g., a blot, fluorescence, chemiluminescence, color, or radioactivity, in an immunoassay or PCR reaction, e.g., quantitative PCR. Tins detectable signal can be compared to the signal from a reference sample or to a threshold value .

[0122] In some embodiments, the results of the biomarker level determinations are recorded in a tangible medium. For example, the results of diagnostic assays, e.g., the observation of the presence or decreased or increased presence of one or more biomarkers, and the diagnosis of whether or not there is an increased risk or the presence of a disease, e.g., an infectious disease or cancer, or whether or not a subject is responding to treatment can be recorded, for example, on paper or on electronic media, e.g., audio tape, a computer disk, a CD-ROM, or a flash drive.

[0123] In some embodiments, the provided method further includes the step of providing to the subject a diagnosis and/or the results of treatment.

VIII. Kits

[0124] In another aspect, the disclosure provides various kits, e.g., kits for inducing an immune response as described in more detail in Section VI and/or fort treating or preventing a disease as described in more detail in Section VII. The kit includes any of the immunogenic compositions disclosed herein and described in further detail in Section V,

[0125] The provided kit can be packaged in a way that allows for safe or convenient storage or use. The kit can be packaged, for example, in a box or other container having a lid. Typically, the provided kit includes one or more containers, with each container storing a particular kit component such as, for example, a reagent or a control sample. The choice of container will depend on the particular form, e.g., liquid form, solid form, suspension form, or powder form, of its contents. Furthermore, containers can be made of materials that are designed to maximize the shelf-life of the kit components. As a non-limiting example, kit components that are light- sensitive can be stored in containers that are opaque.

[0126] In some embodiments, the provided kit contains one or more elements, e.g., a syringe, useful for administering the disclosed pharmaceutical composition to a subject, e.g., using a provided method. In some embodiments, the kit further includes one or more elements, e.g., test tubes or slides, useful for obtaining and/or processing one or more samples obtained from the subject. In some embodiments, the kit further includes instructions for use, e.g., directions for the practice of a provided method. While the instructional materials typically include written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to electronic storage media, e.g., magnetic discs, tapes, cartridges, chips; optical media, e.g., CD-ROM; and the like. Such media can include internet addresses.

IX. Exemplary Embodiments

[0127] The following embodiments are contemplated. All combinations of features and embodiments are contemplated.

[0128] Embodiment 1: A nanoparticle comprising a Toll-like receptor (TLR) agonist, a saponin, a phospholipid, and a nanoparticle sterol, wherein the TLR agonist is a non-lipid TLR agonist or a derivative thereof.

[0129] Embodiment 2: An embodiment of embodiment 1 , wherein the derivative comprises the non-lipid TLR agonist conjugated to an agonist sterol.

[0130] Embodiment 3: An embodiment of embodiment 2, wherein the derivative comprises the non-lipid TLR agonist conjugated to an agonist cholesterol.

[0131] Embodiment 4: An embodiment of any one of embodiments 1-3, wherein the non- lipid TLR agonist comprises an agonist of one or both of TLR7 and TLR8.

[0132] Embodiment 5: An embodiment of any one of embodiments 1-4, wherein the non- lipid TLR agonist comprises an imidazoquinoline compound.

[0133] Embodiment 6: An embodiment of any one of embodiments 1-5, wherein the derivative has the structure:

[0134] Embodiment 7: An embodiment of any one of embodiments 1 -6, wherein the saponin comprises Quit A.

[0135] Embodiment 8: An embodiment of any one of embodiments 1-7, wherein the phospholipid comprises dipalmitoylphospatidylcholine (DPCC).

[0136] Embodiment 9: An embodiment of any one of embodiments 1-8, wherein the nanoparticle sterol comprises a nanoparticle cholesterol.

[0137] Embodiment 10: An embodiment of any one of embodiments 1-9, wherein the TLR agonist is a first TLR agonist, and wherein the nanoparticle further includes a second TLR agonist having a different chemical structure than the first TLR agonist.

[0138] Embodiment 11 : An embodiment of embodiment 10, wherein the second TLR agonist comprises a lipid TLR agonist.

[0139] Embodiment 12: An embodiment of embodiment 11, wherein the lipid TLR agonist comprises a TLR4 agonist

[0140] Embodiment 13: An adjuvant composition configured to enhance an immune response of a subject against an antigen, wherein the adjuvant composition comprises a plurality of the nanoparticle of any one of embodiments 1-12.

[0141 ] Embodiment 14: An adjuvant composition configured to enhance an immune response of a subject against an antigen, wherein the adjuvant composition comprises a plurality of nanoparticles, each nanoparticle of plurality of nanoparticles comprising a TLR agonist, a saponin, a phospholipid, and a nanoparticle sterol; and wherein the immune response comprises a production by the subject of a higher concentration of IgGl than of IgG2c.

[0142] Embodiment 15: An embodiment of embodiment 14, wherein the TLR agonist comprises a lipid TLR agonist. [0143] Embodiment 16: An embodiment of embodiment 15, wherein the lipid TLR agonist comprises an agonist of one or both of TLR1 and TLR2.

[0144] Embodiment 17: An embodiment of embodiment 15 or 16, wherein the TLR agonist comprises Pam3-Cys-Ser-Lys4 (Pam3CSK4).

[0145] Embodiment 18: An embodiment of any one of embodiments 15-17, wherein the lipid TLR agonist comprises a TLR4 agonist.

[0146] Embodiment 19: An embodiment ofany one of embodiments 15-18, wherein the TLR agonist comprises a non-lipid TLR agonist or a derivative thereof.

[0147] Embodiment 20: An embodiment of embodiment 19, wherein the derivative comprises the non-lipid TLR agonist conjugated to an agonist sterol.

[0148] Embodiment 21: An embodiment of embodiment 20, wherein the derivative comprises the non-lipid TLR agonist conjugated to an agonist cholesterol.

[0149] Embodiment 22: An embodiment of any one of embodiments 19-21 , wherein the non- lipid TLR agonist comprises an agonist of one or both of TLR7 and TLR8.

[0150] Embodiment 23: An embodiment of any one of embodiments 19-22, wherein the non- lipid TLR agonist comprises an imidazoqmnoline compound.

[0151] Embodiment 24: An embodiment of any one of embodiments 14-23, wherein the saponin comprises Quil A.

[0152] Embodiment 25: An embodiment of any one of embodiments 14-24, wherein the phospholipid comprises DPCC.

[0153] Embodiment 26: An embodiment of any one of embodiments 14-25, wherein the nanoparticle sterol comprises a first cholesterol.

[0154] Embodiment 27: An embodiment of any one of embodiments 13-26, wherein the plurality of nanoparticles is a first plurality’ of nanoparticles, and wherein the adjuvant composition comprises a second plurality of nanoparticles having a different TLR agonist than the TLR agonist of the first plurality of nanoparticles

[0155] Embodiment 28: An embodiment of any’ one of embodiments 13-27, wherein the antigen comprises a protein or a portion thereof. [0156] Embodiment 29: An embodiment of any one of embodiments 13-28. wherein the antigen comprises a bacterial antigen, a viral antigen, a fungal antigen, a protozoal antigen, a helminthic antigen, or a combination thereof.

[0157] Embodiment 30: An embodiment of any one of embodiments 13-29, wherein the infectious disease antigen comprises a severe acute respiratory syndrome coronavirus (SARS- CoV) antigen, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen, a Middle East respiratory syndrome coronavirus (MERS-CoV) antigen, a simian immunodeficiency vims (SIV) antigen, a human immunodeficiency virus (HIV) antigen, a hepatitis C virus antigen, a herpes simplex virus antigen, an Epstein-Barr virus antigen, a cytomegalovirus antigen, an influenza virus antigen, or a combination thereof.

[0158] Embodiment 31 : An immunogenic composition comprising the adjuvant composition of any one of embodiments 13-30, and the antigen.

[0159] Embodiment 32: An embodiment of embodiment 31, wherein the immunogenic composition is a vaccine.

[0160] Embodiment 33: A method of inducing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition of embodiment 31 or 32.

[0161] Embodiment 34: An embodiment of embodiment 33, wherein the immune response is greater than a baseline immune response induced by administering to the subject an equivalent amount a corresponding immunogenic composition lacking the adjuvant composition.

[0162] Embodiment 35: An embodiment of embodiment 33, wherein the immune response comprises a production of a higher concentration of IgGl antibody than of IgG2c antibody.

[0163] Embodiment 36: A method of preventing or treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition of embodiment 31 or 32,

[0164] Embodiment 37: An embodiment of embodiment 36, wherein the disease is an infectious disease or a cancer. [0165] Embodiment 38: An embodiment of embodiment 37. wherein the infectious disease is caused by a bacterial infection, a viral infection, a fungal infection, a protozoal infection, a helminthic infection, or a combination thereof.

[0166] Embodiment 39: An embodiment of embodiment 37, wherein the infectious disease is caused by a SARS-CoV virus, a SARS-CoV-2 virus, a MERS-CoV virus, an SIV virus, an HIV virus, a hepatitis C virus, a herpes simplex virus, an Epstein-Barr virus, a cytomegalovirus, an influenza virus, or a combination thereof

EXAMPLES

[0167] The present disclosure will be better understood in view of the following non-limiting examples. The following examples are intended for illustrative purposes only and do not limit in any way the scope of the present invention.

[0168] The following examples provide analytical results related to the provided platform of saponin nanoparticles incorporating Toll-like receptor agonists (TLRa-SNPs). Formulations of TLRa-SNPs were created, where the formulations incorporated Pam3CSK4 (TLRl/2a-SNP), MPLA (TLR4a-SNP), imidazoquinoline derivative (TLR7/8a-SNP), as well as a mixture of both MPLA and imidazoquinoline derivative (TLR4a-TLR7/8a-SNP). Compared to traditional 1SCOMATRIX (plain saponin nanoparticle, SNP), all four TLRa-SNPs elicited improved humoral immune responses when used as adjuvants for SARS-CoV-2 and HIV vaccines in mice. TLRa-SNPs led to more potent antibody titers, improved antibody durability, enhanced breadth protection against variants of concerns (VOCs) and induced better neutralizing responses when compared to SNPs or clinical control CpG/Alum . Notably, TLRa-SNPs induced unique acute cytokine profiles, leading to distinct Th-skewed responses depending on the nature of the TLRa adjuvant incorporated. Overall, the results demonstrate a potent and modular TLRa-saponin nanoparticle platform that improves humoral immune responses and generates tunable long-term Th-skewed responses, a pertinent feature in preventing diseases for which different Th-skewed responses lead to better clinical outcome.

Example 1. Formulation and characterization of TLRa-SNPs

[0169] Plain saponin nanoparticles (SNPs), traditionally known as ISCOMATRIX, were formulated as reported in literature: mixing Quil-A saponin, dipalmitoylphosphatidylcholine (DPPC), and cholesterol at a molar ratio of 10: 10:5 in aqueous medium to trigger spontaneous self-assembly into honeycomb like nanoparticles of 30-70 nm in diameter (B. Moreinet ak, Nature 308, (1984): 457; K. Lovgren-Bengtsson & B. Morein, Vaccine Adjuvants: Preparation

Methods and Research Protocols,' Humana Press: Humana Totowa, NJ (2000); S. Pedersen et al., Biophys. J. 102, (2.012): 2372). Lipid-based TLRas were readily incorporated with SNPs at a specific stoichiometry via hydrophobic effects, and non-lipidated TLRas were conjugated with a cholesterol motif to specifically interact with saponins. In this regard, TLRl/2a Pam3CSK4, TLR4a synthetic MPLA (MPLAs), and cholesteryl TLR7/8a imidazoquinoline derivative were integrated with SNPs by mixing the desired TLRa adjuvant, Quil-A saponin, DPPC, and total cholesterol content at a molar ratio of 1: 10:2.5: 10 across all formulations (FIG.

[0170] Cholesteryl imidazoquinoline derivative (FIG. 2) was synthesized by first dissolving 100 mg of 4-amino-2-(ethoxy methyl)- !H-imidazo[4, 5-C[quinoline-l-butylamine (100 mg, 0.319 mmol) in 8 mL of anhydrous CH₂Cl₂/DMF (85:25). In parallel, cholesteryl chloroformate (145 mg, 0.322 mmol, 1 ,01 eq.) was dissolved in 3 mL of anhydrous CH2CI2 and triethylam ine (53 pL, 0.383 mmol, 1.2 eq.) was then added. This second solution was added dropwise at 0 °C into the first one and the reaction was stirred at room temperature for 16 hours. Solvents were removed under reduced pressure and the resulting solid was purified by column chromatography on silica gel eluting with a. step gradient of MeOH/EtsN (99: 1) from 0 % to 6 % v/v in CH2CI2 (Rf: 0.5, 9/1 CHzCb./'MeOH). The title compound was obtained as a white solid foam (90 mg, 39 %). ^;H NMR (500 MHz, CD₂CI₂) <5 (ppm): 0.69 (s, 3H), 0.86 (d, J--- 2.0 Hz, 3H), 0.87 (d, J= 2.2. Hz, 3H), 0.90-1.62 (m, 28H), 1.65-1.75 (m, 2H), 1.78-1.89 (m, 3H), 1.91-2.08 (m, 6H), 2.19-2.34 (m, 2H), 3.16-3.27 (m, 2H), 3.60 (q, J= 7.2 Hz, 2H), 4.37-4.47 (m, 1H), 4.52-4.60 (m, 21 1). 4.78 (s, 1H), 4.82-4.90 (m, 2FI), 5.34-5.39 (m, 1H), 5.61 (bs, 1FI), 7.31-7.36 (m, 1H), 7.50 (m, 1H), 7.75 (dd, ./ 1.4, 8.3 Hz, 1H), 7.95 (d, J= 8.3 Hz, 1H). ^!3C NMR (125 MHz, CD2CI2) S (ppm): 12.0, 15.3, 18.9, 19.5, 21.4, 2.2.7, 22.9, 24.2, 24.6, 27.6, 27.8 28.4, 28.5, 28.8, 32.2, 32.3, 36.2, 36.5, 36.9, 37.4, 38.9, 39.9, 39.9, 40.1, 40.5, 42.7, 46.3, 50.5, 56.5, 57.1, 65.5, 66.6, 74.6, 115.8, 120.4, 12.2.7, 127.0, 127.3, 127.6, 134.3, 140.4, 145.2, 149.5, 151.8, 156.4.

[0171] Cholesterol and DPPC (20 mg/mL) were each dissolved in a solution of 20% (w/v) of MEGA-10 detergent in Milli-Q water. Quil-A saponin (20 mg/mL) was dissolved in pure Milli-Q water. All solutions were heated at 50 °C until complete dissolution before use. When required, Cholesteryl imidazoquinoline, MPLA, and Pam3CSK4 (2 mg/mL) were each dissolved in a solution of 20% (w/v) of MEGA- 10 detergent in Milli-Q water and heated at 70 °C under sonication until dissolution. All formulations were obtained after quickly mixing the appropriate volume of each compound at 60 °C without stirring in this order: Cholesterol/DPPC/Adjuvant/Quil-A saponin followed by its dilution with PBS IX to reach a final concentration of 1 mg/mL. in cholesterol. Solutions were left for equilibration overnight at room temperature and dialyzed against PBS I X (MWCO 10 kDa) for 5 days. Solutions were then filtered using 0.2 pm ACRODISC® Syringe Filters and concentrated using Centricon spin filters (MWCO 50 kDa, 3000 RCF, 40 mins) in stock solutions of 5 mg/ml in Quil-A Saponin.

[0172] The nanoparticles resulting from integrating the TLRas with SNPs showed monodisperse populations of hydrodynamic diameters between 40 and 60 nm and negative surface charges equal to or below -30 mV (FIGS. 3 and 4). Cryo-electron microscopy confirmed all nanoparticles conserved the signature honeycomb-like structure after introduction of TLRa adjuvants (FIG. 5). Moreover, TLRa-SNPs maintained their colloidal stability for at least 6 weeks when stored at 4 °C and for several months when stored at -20 °C (FIGS. 6-8).

Example 2. In vitro activation of TLRa-SNPs

[0173] To further confirm that TLRas had been incorporated with SNPs and maintained their bioactivities, a RAW-Blue transgenic mouse macrophage in vitro cell assay was performed as a confirmation of TLR activation. RAW-Blue cells were incubated with different formulations of TLRa-SNPs (SNPs, TLR 1 /2a-SNPs, TLR4a-SNPs, or TLR7/8a-SNPs) or the corresponding soluble TLRa adjuvants. Activation of the TLRs by the TLRas would lead to downstream activation of NF-KB and AP-1 pathways, which can be assessed with QUANTI -Blue for a colorimetric output. For this assay, RAW-blue cells were cultured at 37 °C with 5% CO₂ in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with L-glutamme (2 mM), D- glucose (4.5 g/L), 10% heat-inactivated fetal bovine serum (HI-FBS), and penicillin (100 U/mL)/streptomycin (100 pg). Every other passage, zeocin (100 pg/mL) was added to the culture medium. Serial dilutions of solvent controls, soluble SNP components (cholesterol, DPPC, saponin), soluble TLRas (TLR1/2 Pam3CSK4, TLR4 MPLAs, and TLR7/8 cholesteryl imidazoqmnoline derivative), SNP, and TLRa-SNPs were added to a 96-well tissue culture treated plate to achieve final concentrations between 40 and 0,01 pg/mL of TLRas (or equivalent concentration of individual component based on TLRa-SNPs added for the negative controls). About 100,000 cells were added to each well in 180 pL of medium and were incubated for 24 h at 37 °C in a 5% CO₂ incubator. [0174] Concentration-dependent NF-KB and AP-1 activation curves were generated by stimulating the cells with a range of soluble TLRa or TLRa-SNP concentrations (n-3; equivalent TLRas concentration of 2.22-0.0091 pg/mL; FIGS. 9-1 1). No activation with plain SNPs or relevant buffer controls was observed from the dilution curves. In contrast, all TLRa- SNPs generated concentration dependent NF-kB and AP-1 activation. Interestingly, TLRa- SNPs produced more potent activation compared to their soluble counterparts, most likely due to nanoparticles’ ability to multivalently display TLRas which results in enhanced cellular uptake. All three different TLRas could be incorporated into SNPs and maintain their bioactivity in vitro, demonstrating the success of this platform approach.

Example 3. Potent and broad humoral responses generated by receptor-binding domain nanoparticle vaccines adjuvanted with TLRa-SNPs

[0175] The immune responses generated by different TLRa-SNPs were investigated using SARS-CoV-2 vaccines formulated with receptor-binding domain nanoparticles (RBD NP) antigen and TLRa-SNPs as nanoparticle adjuvants. Tire RBD NP used in this study, also called RBD-16GS-I53-50, has been extensively studied in mice, nonhuman primates (NHPs), and human clinical trials (P.S. Arunachalam et al,, Set. Transl. Med. 14, (2022): eabq4130; P.S. Arunachalam et al.. Nature 594, (2021): 253; L. Grigoryan et al., NPJ Vaccines 7, (2022): 55; A.C. Walls et al., Cell 183, (2020): 1367; B.S. Ou et al, bioRxiv, (2022): 520166). It has also been shown to generate potent neutralizing humoral responses when formulated with AS03 or CpG/Alum adjuvants, leading to the Korean Ministry of Food and Drug Safety approving RBD NP adjuvanted with AS03 as COVID- 19 vaccines for adults 18 years or older. Following the same immunization regime as previous studies (L. Grigoryan et al, NPJ Vaccines 7, (2022): 55; A.C. Walls et al. Cell 183, (2020): 1367), 7-8 week old C57BL/6 mice (n ~ 5) were subcutaneously (s.c.) immunized with vaccines comprising 1 .5 pg RBD NP and 10 pg of TLRa- SNP formulations (SNP, or TLRl/2a-SNP, or TLR4a-SNP, or TLR7/8a-SNP). For comparison, a clinical control containing CpG/Alum (20 pg and 100 pg, respectively) was evaluated. Mice were immunized on Week 0 (prime) followed by a boost at Week 3, and sera were collected from Week 0 to Week 11 (FIG. 12).

[0176] Anti-RBD total IgG titers over time were first evaluated (FIG. 13), On Week 3, prior to boosting, mice immunized with RBD NP vaccines adjuvanted with CpG/Alum or SNP elicited inconsistent antibody titers, with around half the mice’s anti-RBD IgG endpoint below' the limit of detection. In contrast, on Week 3 all mice immunized with vaccines adjuvanted with TLRa-SNPs (TLRl/2a-SNP, TLR4a-SNP, and TLR7/8a-SNP) seroconverted and generated antibody titers significantly higher than the CpG/Alum and SNP controls {p values in Table 1). Two weeks post-boost (Week 5), no significant difference in titers was observed between the two controls, CpG/Alum and SNP, having average endpoint titers of 2.1 x 10³ and 9.6 x IO⁴, respectively. However, TLRa-SNPs induced significantly higher titers for which the average endpoint titers were 9.1 x 10⁵ (TLRl/2a-SNP), 5.2 x 10⁵ (TLR4a-SNP), and 4.3 x 10⁵ (TLR7/8a-SNP), Additionally, TLRa-SNP vaccinated mice maintained significantly higher anti-RBD IgG endpoint titers at all timepoints post boost and produced robust titers with little deviations across animals (p values in Table 1). In contrast, vaccination of mice with CpG/Alum resulted in variable titer responses up to two orders of magnitude apart, leading to a standard error of the mean (SEM) at least double that of the other treatments after boosting.

Table 1. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for specific anti-RBD endpoint IgG titers time points compared between different RBD MP vaccines (referring to FIG. 13).

[0177] The area under the curve (AUG) values of the endpoint titers over the 1 1 -week period were also calculated to estimate the mean IgG production. No significant difference in AUG titers between CpG/Alum and SNP was observed (FIG. 14; p values in Table 2). While all TLRa-SNPs resulted in significantly higher AUCs than CpG/Alum, only TLRl/2a-SNP produced a significantly higher AUG than SNP. ECso titers (half-maximal binding dilution) were also measured two weeks post-boost (Week 5) to ensure the endpoint titers reported correlated with the functional potency of the antibody (FIG. 15). Similar to comparisons of endpoint titers, CpG/Alum and SNP induced low and variable ECso titers compared to TLRa- SNPs, which all generated significantly higher average ECso (p values in Table 3) . The endpoint titers of the vaccinated mice a year after immunization were also assessed (D365, FIG. 16). While the average endpoint titers from Week 11 to D365 drastically dropped by 24-fold for CpG/Alum (2.6 x 10³) and 13-fold for SNP (3.9 x 10³), TLRa-SNPs’ endpoint titers remained high with only a 7-fold (6.7 x 10⁴) and 5-fold (5.4 x IO⁴) decrease for TLRl/2a-SNP and TLR4a-SNP, respectively . TLR7/8a-SNP vaccinated mice were excluded from the comparison due to an insufficient sample size that remained (n=2 at D365 due to murine dermatitis leading to euthanasia criteria; statistical power calculated using Mead’s Resource Equation) (J. Charan & N.D. Kanthana, J. Pharmacol. Pharmacother. 4, (2013): 303; W.N. Arifin & W.M. Zahiruddin, Malays. J. Med. Sci. 24, (2017): 101). Overall, the titers measured at D365 from mice receiving vaccines adjuvanted with TLRa-SNPs were significantly higher than from mice that received vaccines adjuvanted with either SNP or CpG/Alum controls. More importantly, these titers were at the same order of magnitude as the endpoint titers measured for tire SNP and CpG/Alum controls on Week 11, demonstrating the durability of RBD NP vaccines adjuvanted with TLRa-SNPs (FIG. 1 l,p values in Table 4).

Table 2. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for area under the curves (AUCs) of anti-RBD endpoint IgG titers compared between different RBD NP vaccines (referring to FIG. 14).

Table 3. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for Week 5 half-maximal binding dilution of anti-RBD IgG titers compared between different RBD NP vaccines (referring to FIG. 15).

Table 4. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for D365 anti-RBD endpoint IgG titers compared between different RBD NP vaccines (referring to FIG. 16)

[0178] Wild-type Spike titers were next determined to confirm the antibodies produced from RBD NP adjuvanted with TLRa-SNP vaccines could bind to native spike proteins. Moreover, the breadth of the antibody response was assessed by measuring the endpoint titers against different SARS-CoV-2 VOCs including Alpha (B. l.1.7), Beta (B. 1.351), Delta (B.1.617.2), and Omicron (B.1.1.529) spike proteins two weeks post-boost (Week 5) compared to CpG/Alum (FIG. 18). While no differences in endpoint titers were observed between SNP and CpG/Alum regardless of variants (p values in Table 5), TLRl/2a-SNP led to significantly higher titers against all variants. Overall improved titers against variants were also observed for TLR4a-SNP and TLR7/8a-SNP. Notably, the ratio of titers against different variants compared to wild-type spike titer (percent drop) showed more consistent and higher breadth protection tor TLRa-SNPs compared to CpG/Alum and SNP controls for which more variable and larger interquartile ranges were observed (FIG. 19). Overall, these findings show' that SNP alone as an adjuvant does not perform better than the clinical control CpG/Alum in generating humoral responses. In contrast, the provided TLRa-containing SNPs elicit more potent and durable titer responses with better breadth of protection.

Table 5. p values from a general linear model (GLM) followed by Tukey HSD comparisons procedure for Week 5 anti-spike variant endpoint IgG titers compared between different CpG/Alum to other vaccine groups (referring to FIG. 18).

Example 4. Production of strong neutralizing antibodies by receptor-binding domain nanopartide vaccines adjuvanted with TLRa-SNPs

[0179] Since mice immunized with RBD NP vaccines adjuvanted with TLRa-SNPs generated potent, durable, and broad antibody responses, additional studies were earned out to measure the neutralizing activities of the sera. Week 5 sera neutralization was evaluated by utilizing a SARS-CoV-2 spike pseudotyped lentivirus to measure serum-mediated inhibition of viral entry into HeLa cells overexpressing ACE2 and TMPRSS2 (FIG. 12). Neutralization assays were conducted as described previously (A.E. Powell et al., ACS Cent. Sci. ”, (2021): 183). Briefly, SARS-CoV-2 spike-pseudotyped lentivirus was produced in HEK239T cells. Six million cells were seeded one day prior to transfection. A five-plasmid system (K.H.D. Crawford et al.. Viruses 12, (2020): 513) and BioT (Bioland Scientific) were used for viral production per manufacturer’s protocol with the full-length wild-type spike sequence from the Wuhan-Hu- 1 strain of SARS-CoV-2. Virus-containing culture supernatants were harvested approximately 72 hours after transfection by centrifugation and filtered through a 0.45-pm syringe filter. Stocks were stored at -80 °C prior to use. For the neutralization assay, ACE2/HeLa cells were plated 1--2 days prior to infection (T.F. Rogers et al., Science 369, (2020): 956. Mouse and human sera were heat inactivated at 56 °C for 30 min prior to use. Sera and viruses were diluted in cell culture medium and supplemented with a polybrene at a final concentration of 5 μg/mL. Serum/virus dilutions were incubated at 37 °C for 1 h. After incubation, media was removed from cells and replaced with serum/vims dilutions and incubated at 37 °C for 2 days. Cells were then lysed using BriteLite (Perkin Elmer) luciferase readout reagent, and luminescence was measured with a BioTek plate reader. Each plate was normalized by wells with cells or virus only and curves were fit with a three-parameter non- linear logistic regression inhibitor curve to obtain IC50 values. Seram dilution curves display mean infectivity ± SEM for each individual mouse at each serum dilution. Normalized values were fit with a three-parameter non-linear logistic regression inhibitor curve in GraphPad Prism 8.4.1 to obtain IC50 values. Fits were constrained to have a value of 0% at the botom of the fit.

[0180] Seram neutralization ID₅₀ was measured through neutralizing activities of a range of sera concentrations to determine the half-maximal inhibition of infectivity (NT₅₀) (FIGS. 20- 25). Consistent with previous findings (B.S. Ou et ah, bioRxiv, (2022): 520166). mice immunized with RBD NP adjuvanted with CpG/Alum resulted in highly variable neutralizing responses, with two mice’s neutralizing activities below the limit of detection. In contrast to CpG/Alum, RBD NP vaccines adjuvanted with SNP and TLRa-SNPs induced significantly higher neutralizing ID₅₀ (p values in Table 6). Notably, TLRa-SNPs elicited higher ID₅₀ than the “high titer” classification according to FDA’s recommendation (ID₅₀ ~ IO²⁴) (J. Charan & N.D. Kantharia, J. Pharmacol. Pharmacother. 4, (2013): 303). Moreover, mice sera neutralization activities were compared with human patients’ convalescent plasma by testing the pseudo viruses’ infectivity at the lowest sera dilution (1: 100 dilution; FIG. 26). High infectivity indicates antibodies from the sera failed to prevent the spike-pseudotyped lentivirus from entry into ACE2 and TMPRSS2 expressing HeLa cells. In this regard, variable results were observed for SNP and CpG/Alum controls where not all mice receiving vaccines with these two adjuvants reached 0% infectivity. Mice immunized with vaccine adjuvanted with CpG/Alum resulted in significantly higher infectivity than those immunized with vaccines adjuvanted with TLRa-SNPs, with an average comparable to mice that received non- adjuvanted RBD NP vaccine (p values in Table 7). On the contrary, 0% infectivity was measured from all mice receiving vaccines adjuvanted with TLRa-SNPs, indicating neutralization of all lentivirus and prevention of their entry into and infection of HeLa cells. Notably, the sera were even more neutralizing than the human patients’ convalescent plasma, for which the average infectivity was 15%. Overall, TLRa-SNPs as vaccine adjuvants generated superior humoral responses in potency, durability, breadth, and neutralization compared to both SNP and CpG/Alum controls. Table 6. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for Week 5 specific ID₅₀ neutralization titers compared between different RED NP vaccines (referring to FIG. 25).

Table 7. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for relative percent infectivity at 1: 100 dilution compared between different RBD MP vaccines (referring to FIG. 26).

Example 5. Tunable Th-skewed responses elicited by TLRa-SNPs

[0181] Additional studies evaluated whether the nature of TLRa adjuvant affected the immune response of the TLRa-SNPs, since TLRas have been shown to generate unique immune signaling and T-helper responses (J. Fornefett et al., BMC Microbiol . 18, (2018): 45; R.L. Coffin an, A, Sher & R.A. Seder. Immunity 33, (2010): 492). To confirm that different TLRa-SNPs can elicit unique levels of IgGl and IgG2c titers, as these isotypes are respectively strong indicators of Th2- and Th I -skewed responses, IgG isotypes were measured across the different vaccine treatments. Indeed, while all TLRa-SNPs led to improved total IgG antibody responses compared to SNP and CpG/Alum controls, they generated different levels of IgG isotypes. While TLR4a-SNP and TLR7/8a-SNP generated similar IgGl responses compared to SNP and CpG/Alum, TLR l/2a-SNP induced significantly higher IgGl titers compared to all other groups, with average titers an order of magnitude higher compared to those generated by SNPs (FIG. 27, p values in Table 8). On the other hand, all TLRa-SNPs elicited higher IgG2c titers, with at least a 5-fold increase in average titers compared to the controls (FIG. 28, p values in Table 9). Tire differences in IgGl and IgG2c titers suggested that different TLRa-SNPs resulted in different “flavors” of antibody responses, which we measured by calculating the ratio of IgG2c to IgGl titers (FIG. 29). In this regard, TLRl/2a-SNP elicited a Th2-skewed response whereas TLR4a-SNP and TLR7/8a-SNP induced a Th 1 -skewed response. These results are drastically different from the balanced Thl/Th2 responses generated by SNP and CpG/Alum controls. The tailored Th-responses induced from the different TLRas thereby position TLRa-SNPs as a potent and modular adjuvant platform of interest for inducing immunity against other challenging viruses.

Table 8. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for Week 5 anti-RBD endpoint IgGl titers between different RBD NP vaccines (referring to FIG. 27).

Table 9. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure tor Week 5 anti-RBD endpoint IgG2c titers between different RBD NP vaccines (referring to FIG. 28).

Example 6. Induction of robust humoral responses with TLRa-SNPs as GP120 vaccine adjuvants

[0182] To confinn that TLRa-SNPs can elicit robust immune responses in an antigen- independent manner, C57BL/6 mice (n=5) were subcutaneously vaccinated with HIV GP120 antigen adjuvanted with TLRl/2a-SNP and TLR4a-SNP, the two groups that elicited highest antibody titers from RBD NP vaccines. Titer responses were compared to the two controls, GP120 vaccines adjuvanted with SNP or Alum. Mice were immunized and boosted on Week 0 and Week 4 and sera were collected from Week 0 to Week 6 to assess total anti-GP120 IgG and subtype titers (FIG, 30). Before boosting, most mice that received Alum or SNP adjuvanted vaccines did not have detectable triers, but all mice that received vaccines adjuvanted with TLRl/2a-SNP or TLR4a-SNP seroconverted and elicited significantly higher titers (FIG. 31, p values m Table 10). Two weeks post-boost (Week 6), vaccines with TLRa-SNP adjuvants generated significantly higher antibody titers compared to those with Alum, with average endpoint titers around 2000-fold higher. Moreover, TLRa-SNPs also elicited over 3-fold higher titers than SNP. Similarly, we observed higher overall antibody production from mice adj uvanted with TLRa-SN Ps by determining the AUCs of titers over the 6 w eeks period (FIG. 32, p values in Table 11). Table 10. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for specific anti-GP120 endpoint IgG titers time points compared between different GP120 vaccines (referring to FIG, 31).

Table 11. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for area under the curves (AUCs) of anti-GP120 endpoint IgG titers compared between different GP120 vaccines (referring to FIG. 32).

[0183] To confinn that the unique Th-skewed responses observed from different TLRa-SNPs in RBD NP vaccines was due to influences of TLRas in immune signaling, the Week 6 IgGl and IgG2c titers from mice immunized with GP120 vaccines were measured. Consistent with previous observations, mice adjuvanted with both TLRl/2a-SNP and TLR4a-SNP induced robust IgG2c responses while mice adjuvanted with only TLRl/2a-SNP generated significantly higher IgGl titers compared to SNP (FIGS. 33 and 34, p values in Table 12 and Table 13). This again resulted in a more Th2. -skewed response from mice adjuvanted with TLRl/2a-SNP and a more Th 1 -skewed response from mice adjuvanted with TLR4a-SNP (FIG. 35). Overall, these results confirmed TLRa-SNPs’ ability to generate potent and robust antibody responses with distinctive Th-skewed responses regardless of the antigen.

Table 12. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for Week 5 anti-GP120 endpoint IgGl titers between different GP120 vaccines (referring to FIG. 33).

Table 13. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for Week 5 anti -GP 120 endpoint IgG2c titers between different GP120 vaccines (referring to FIG. 34).

Example 7. Induction of unique acute cytokine induction in the draining lymph node and robust germinal center response by different TLRa-SNPs

[0184] To measure the acute immune response post-vaccination, the dLN cytokine profile was assessed 1 day (24 hours) post-immunization through a Luminex assay (FIG. 36). Instead of reporting the results in pg/mL, which can suffer from plate/batch/lot inconsistencies, nonspecific binding, differences between standards and real biological samples, and other experimental artifacts, the raw fluorescence signal was analyzed by correcting for nonspecific binding as a covariate in the regression analysis to better infer the true biological patterns in Luminex data (Y. Rosenberg -Hasson, L. Hansmann, M. Liedtke, I. Herschmann & H.T. Maecker, Immunol. Res. 58, (2014): 224; E.J. Breen, W. Tan & A. Khan, Set. Rep. 6, (2016):

26996; D.J. Phillips, S.C. League, P. Weinstein & W.C. Hooper, Cytokine 36, (2006): 180; H.T. Maecker, Y. Rosenberg-Hasson, K.D. Kolstad, V.D. Steen & L.S. Chung, J. Immunol.

204, (2020): 3425).

[0185] According to previously reported methodology (G.A, Roth et al ., .4CS¹ Cent. Sei. 6, (2020): 1800), mice were euthanized using CO₂ 12 days post immunization. Inguinal lymph nodes were collected and dissociated into single cell suspensions. Cells were stained for viability using Ghost Dye Violet 510 (Tonbo Biosciences, Cat: 13-0870-T100) for 5 min on ice and washed with F ACS buffer (PBS with 2% FBS, 1 mM EDTA). Fc receptor was blocked using anti-CDl 6/CD38 (clone: 2.4G2, BD Biosciences, cat: 553142) for 5 min on ice and then stained with fluorochrome conjugated antibodies: CD19 (PerCP-Cy5.5, clone: 1D3, BioLegend, cat: 152406 ) CD95 (PE-Cy7, clone: Jo2, BD Biosciences, cat: 557653), CD38 (BUV395, clone: 90, BD Bsosciences, Cat: 740245), CXCR4 (BV421, clone: L276F12 , BioLegend, cat: 14651 1), CD86 (BV785, clone: GL1, BioLegend, cat: 105043), GL7 (AF488, clone: GL7, BioLegend, cat: 144613), CD3 (AF700, clone: 17A2, BioLegend, cat: 100216), CD4 (BV650, clone: GK1.5, BioLegend, cat: 100469), CXCR5 (BV711, clone: L138D7, BioLegend, cat: 145529) and PD1 (PE-DAZZLE^IM594, clone: 29F.1A12, BioLegend, cat: 135228) for 30 mins on ice (Table S2). Cells were washed, fixed with 4% PF A on ice, washed again and analyzed on an LSRII flow cytometer (BD Biosciences). Data were analyzed with Flow! 10.

[0186] Sufficient levels of cytokines in the dLN are necessary to mount a successful vaccine response, and different cytokines induced can result in different Th responses. Comparing to mice adjuvanted with SNP, those that received TLRa-SNPs (TLRl/2a-SNP, TLR4a-SNP, or TLR7/8a-SNP) all induced significantly higher median fluorescence intensities (MFIs) of IFN- y, IP10 (CXCL10), IL3, and IL15 (FIG. 37, p values in Table 14). These are critical proin flammatory cytokines for initiating strong immune responses and overall improved Th response (T.R. Mosmann & S. Sad, Immunol Today 17, (1996): 138). Elevated levels of IL6, IL25, and IL31 observed with TLRl/2a-SNP adjuvanted vaccine could explain the observed Th-2 skewed response. On the other hand, elevated levels of cytokines such as IFN-y, IP 10, IL18, IL27, and GMCSF observed with TLR4a-SNP could be the cause of the observed Thl- skewed response. [0187] Notably, of the 48 cytokines measured in the Luminex assay, only 1 1 did not show significant differences between SNP and at least one of the TLRa-SNPs (FIG. 39). Thus, to better understand the overall cytokine induction profile of each adjuvant treatment, a dimensional reduction analysis was performed by creating a penalized supervised star plot (PSS, FIG. 38). Cytokine MFI data were detrended for cage and nonspecific binding effects using ordinary least squares (J.A.R. Blais, Math. Probl. in Engineering 2.010, (2010): 1). Detrended data were used to construct a penalized supervised star plot (T.H. Holmes, P.B, Subrahmanyam, W. Wang & H.T. Maecker, Viral Immunol. 32, (2019): 102). Analyses were conducted in SAS® v.9.4 (SAS® Institute, Cary, North Carolina, USA) and R (www.r- project.org). Star plots were constructed with R packages maptools, matrixcalc, piotrix, JPEN,and VCA. Distinct and separate clusters were generated for each adjuvant. The results demonstrated that, not only did TLRa-SNPs induce higher effector cytokine production in the dLNs, but different formulations of TLRa-SNPs also led to unique acute cytokine profiles post- vaccination.

Table 14. p values from a generalized maximum entropy estimation regression adjusted to control the false discovery rate for murine Luminex 48-plexed cytokine assay between SNP and TLRa-SNPs (FIGS. 37 and 39).

[0188] Germinal center activity in the dLN w as assessed 12 days after immunizing with RBD NP adjuvanted with TLRa-SNPs (FIG. 36). While mice treated with CpG/Alum adjuvanted vaccines had the highest count of germinal center B cells (GCBCs), they also had the lowest percent of GCBCs of total B cells, indicating CpG/Alum ’s inability to convert B cells to GCBCs (FIGS. 40 and 41, p values in Table 15 and Table 16). On the contrary, mice immunized wdth vaccines adjuvanted with TLRa-SNPs resulted in higher percents of GCBCs as well as higher total GCBC counts compared to mice receiving vaccines adjuvanted with SNP, suggesting robust geminal center activities. Since robust germinal center activity is important for generating better neutralizing and higher affinity antibodies, these data are consistent with the vaccination results that demonstrated increased magnitude and higher neutralizing antibodies with the TLRa-SNP groups. These experiments suggest TLRa-SNPs are superior in generating unique early proinflammatory responses and robust germinal center activity compared to SNP and CpG/Alum, resulting in an overall improved potency, durability, and breadth of humoral responses.

Table 15. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for GCBC count compared between different RBD NP vaccines (referring to FIG. 40).

Table 16. p values from a general linear model (GLM) followed by Tukey’s HSD multiple comparisons procedure for % GCBC of B cells compared between different RBD NP vaccines (referring to FIG. 41).

[0189] The data disclosed herein clearly indicate a stronger expression of IFN-a (demonstrated by the vector) in the TLR7/8a-SNP cluster (cluster 4, FIG. 38). While the Luminex panel did not include IFN~P, other strong interferon responses, such as high IFN-y, IP 10, and IL28 production, were nonetheless observed from mice adjuvanted with TLR7/8a- SNP. The overall balanced but slightly Th-1 skewed response is consistent with prior findings (B. Pulendran, P.S. Arunachalam & D.T. O ’Hagan, Nat. Rev. Drug Discov. 20, (2021): 454; D. Smirnov, J. J. Schmidt, J.T. Capecchi & P.D. Wightman, Vaccine 29, (2011): 5434; M. Kwissa, HI. Nakaya, H. Oluoch & B. Pulendran, Blood 119, (2012) 2044). Likew ise, the Till - skewed response observed with TLR4a-SNP is consistent with the pre viously reported SMNP, for which only IgG2a titers, but not IgGl , were significantly higher than the SNP control (M. Silva et al., Set Immunol. 6, (2021 ): eabfl 152. of different TLRa species in the same TLRa-SNP

[0190] Co-encapsulating TLR4a and TLR7/8a into the same SNP elicited superior ELISA binding titers. A molar ratio of 0.5:0.5: 10:2.5:9.5 of TLR4a:TLR7/8a- cholesteryl:DPPC:cholesterol were used to formulate TLR4a-TLR7/8a-SNP for co- encapsulating two synergestic TLRas to the same particle. Post RAID NP vaccine immunization, mixtures of TLR4a-SNP and TLR7/8a-SNP were not sufficient to elicit higher antibody responses. Only the co-encapsulated formulation of TLR4a-TLR7/8a-SNP elicited higher titers compared to mice adjuvated with TLR4a-SNP or TLR7/8a-SNP.

[0191] Although the foregoing disclosure has been described in some detail by way of illustration and example for purpose of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications within the spirit and scope of the disclosure may be practiced, e.g., within the scope of the appended claims. It should also be understood that aspects of the disclosure and portions of various recited embodiments and features can be combined or interchanged either in whole or in part. In the foregoing descriptions of the various embodiments, those embodiments which refer to another embodiment may be appropriately combined with other embodiments as will be appreciated by one of skill in the art. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only and is not intended to limit the disclosure. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A nanoparticle comprising a Toll-like receptor (TLR) agonist, a saponin, a phospholipid, and a nanoparticle sterol, wherein the TLR agonist is a non-lipid TLR agonist or a derivative thereof.

2. The nanoparticle of claim 1, wherein the derivative comprises the non- lipid TLR agonist conjugated to an agonist sterol.

3. The nanoparticle of claim 2, wherein the derivative comprises the non- lipid TLR agonist conjugated to an agonist cholesterol.

4. Tire nanoparticle of any one of claims 1-3, wherein the non-lipid TLR agonist comprises an agonist of one or both of TLR7 and TLR8.

5. The nanoparticle of any one of claims 1-4, wherein the non-lipid TLR agonist comprises an imidazoquinoline compound.

6. The nanoparticle of any one of claims 1-5, wherein the derivative has the structure:

7. Tire nanoparticle of any one of claims 1-6, wherein the saponin comprises Quil A.

8. The nanoparticle of any one of claims 1 -7 , wherein the phospholipid comprises dipalmitoylphospatidylcholine (DPCC).

9. The nanoparticle of any one of claims 1-8 , wherein the nanoparticle sterol comprises a nanoparticle cholesterol.

10. The nanoparticle of any one of claims 1-9, wherein the TLR agonist is a first TLR agonist, and wherein the nanoparticle further includes a second TLR agonist having a different chemical structure than tire first TLR agonist.

11. The nanoparticle of claim 10, wherein the second TLR agonist comprises a lipid TLR agonist.

12. The nanoparticle of claim 11, wherein the lipid TLR agonist comprises a TLR4 agonist.

13. An adj uvant composition configured to enhance an immune response of a subject against an antigen, wherein the adjuvant composition comprises a plurality of the nanoparticle of any one of claims 1-12.

14. An adjuvant composition configured to enhance an immune response of a subject against an antigen, wherein tire adjuvant composition comprises a plurality of nanoparticles, each nanoparticle of plurality of nanoparticles comprising a TLR agonist, a saponin, a phospholipid, and a nanoparticle sterol; and wherein the immune response comprises a production by tire subject of a higher concentration of IgGl than of IgG2c.

15. The adjuvant composition of claim 14, wherein the TLR agonist comprises a lipid TLR agonist.

16. The adjuvant composition of claim 15, wherein the lipid TLR agonist comprises an agonist of one or both of TLR1 and TLR2.

17. The adjuvant composition of claim 15 or 16, wherein the lipid TLR agonist comprises Pam3-Cys-Ser-Lys4 (Pam3CSK4).

18. The adjuvant composition of any one of claims 15-17, wherein the lipid TLR agonist comprises a TLR4 agonist.

19. Tire adjuvant composition of any one of claims 15-18, wherein the TLR agonist comprises a non-lipid TLR agonist or a derivative thereof.

20. The adjuvant composition of claim 19. wherein the derivative comprises the non-lipid TLR agonist conjugated to an agonist sterol.

21 . The adjuvant composition of claim 20, wherein the derivative comprises the non-lipid TLR agonist conjugated to an agonist cholesterol.

22. Tire adjuvant composition of any one of claims 19-21 , wherein the non- lipid TLR agonist comprises an agonist of one or both of TLR7 and TLR8.

23. The adjuvant composition of any one of claims 19-22, wherein the non- lipid TLR agonist comprises an imidazoquinoline compound.

24. The adjuvant composition of any one of claims 14-23, wherein the saponin comprises Quil A.

25. The adjuvant composition of any one of claims 14-24, wherein the phospholipid comprises DPCC.

2.6. The adjuvant composition of any one of claims 14-25, wherein the nanoparticle sterol comprises a first cholesterol.

27. The adjuvant composition of any one of claims 13-26, wherein the plurality of nanoparticles is a first plurality of nanoparticles, and wherein the adjuvant composition comprises a second plurality of nanoparticles having a different TLR agonist than the TLR agonist of the first plurality of nanoparticles.

28. The adjuvant composition of any one of claims 13-27, wherein the antigen comprises a protein or a portion thereof.

2.9. The adjuvant composition of any one of claims 13-28, wherein the antigen comprises a bacterial antigen, a viral antigen, a fungal antigen, a protozoal antigen, a helminthic antigen, or a combination thereof.

30. The adjuvant composition of any one of claims 13-29, wherein the infectious disease antigen comprises a severe acute respiratory syndrome coronavirus (SARS- CoV) antigen, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen, a Middle East respiratory syndrome coronavirus (MERS-CoV) antigen, a simian immunodeficiency vims (SIV) antigen, a human immunodeficiency virus (HIV) antigen, a hepatitis C virus antigen, a herpes simplex virus antigen, an Epstein-Barr virus antigen, a cytomegalovirus antigen, an influenza virus antigen, or a combination thereof.

31. An immunogenic composition comprising the adjuvant composition of any one of claims 13-30, and the antigen .

32. The immunogenic composition of claim 31, wherein the immunogenic compos! ti on i s a vaccine .

33. A method of inducing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition of claim 31 or 32.

34. The method of claim 33, wherein the immune response is greater than a baseline immune response induced by administering to the subject an equivalent amount a corresponding immunogenic composition lacking the adjuvant composition.

35. The method of claim 33, wherein the immune response comprises a production of a higher concentration of IgGl antibody than of IgG2c antibody.

36. A method of preventing or treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition of claim 31 or 32.

37. Tire method of claim 36, wherein the disease is an infectious disease or a cancer.

38. The method of claim 37, wherein the infectious disease is caused by a bacterial infection, a viral infection, a fungal infection, a protozoal infection, a helminthic infection, or a combination thereof.

39. The method of claim 37, wherein the infectious disease is caused by a SARS-CoV virus, a SARS-CoV-2 virus, a MERS-CoV virus, an SIV virus, an HIV virus, a hepatitis C virus, a herpes simplex virus, an Epstein-Barr vims, a cytomegalovirus, an influenza virus, or a combination thereof.