Lipid nanoparticle adjuvant compositions

Abstract

Improved vaccine compositions and methods are provided, which incorporate the use of lipid nanoparticles which maintain or improve vaccine efficacy while reducing vaccine toxicity and / or side effects.

Description

LIPID NANOPARTICLE ADJUVANT COMPOSITIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 665,165 filed June 27, 2024, the specification of which is incorporated herein in their entirety by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. 75N93022C00054 awarded by National Institute of Allergy and Infectious Diseases (NIAID). The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present invention relates to compositions and methods that exhibit increased or maintained efficacy while exhibiting reduced toxicity or side effects, with applications in vaccines, immunotherapies, and cancer treatments.BACKGROUND OF THE INVENTION

[0004] Vaccinology applies principles from immunology, microbiology, infectious diseases, and epidemiology to develop vaccines targeting both established and emerging pathogens, as well as broadly applicable vaccine platforms. Pathogens possess multiple components recognized by the immune system during infection, including: (1) antigens, often proteins that bind to host receptors; (2) pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), muramyl dipeptide (MDP), CpG DNA, and polyl:C RNA; and (3) accessory microbial factors, such as toxins, quorum sensing molecules, and factors involved in microbial adhesion and persistence.

[0005] Based on these features and the corresponding immune responses, various vaccine classes have been developed, including inactivated or attenuated organisms, protein subunits, nucleic acid-based vaccines, and recombinant viral vectors. However, as most vaccines do not contain live microorganisms, they lack certain microbial features critical for robust immune activation and often require adjuvants to enhance immunogenicity. Adjuvants facilitate activation of innate immune cells such as macrophages, dendritic cells, and lymphoid fibroblasts, and promote adaptive immune responses by stimulating key immune signaling pathways.

[0006] While adjuvants are effective at augmenting immune responses, their use can also be associated with increased toxicity and adverse side effects. Accordingly, there remains a need for vaccine compositions and adjuvants that maintain or enhance immunogenicity while reducing toxicity and minimizing undesirable side effects.BRIEF SUMMARY OF THE INVENTION

[0007] It is an objective of the present invention to provide compositions and methods that allow for increased vaccine efficacy and reduced vaccine toxicity, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0008] Vaccine development remains limited by the inability to consistently induce strong protective immune responses without provoking undesirable inflammatory or systemic toxicities. While various adjuvants have been incorporated into vaccine formulations to enhance immunogenicity, optimization of these formulations has largely been empirical, requiring a balance between efficacy and tolerability. Despite these efforts, no approaches have been described that successfully eliminate systemic toxic side effects while preserving or enhancing the desired adaptive immune response. The present invention provides vaccine adjuvant compositions that not only enhance immune responses but also reduce or minimize associated inflammatory and systemic side effects.

[0009] In some embodiments, the present invention features an adjuvant composition, such as a lipid nanoparticle (LNP) adjuvant. In some embodiments, the adjuvant composition comprises a lipid nanoparticle having a lipid bilayer that includes a plurality of cationic lipids and a plurality of structural lipids. The composition further comprises at least one agonist associated with the lipid nanoparticle, which activates a pattern recognition receptor (PRR). In some embodiments, the agonist (e.g., at least one agonist) is either incorporated into or encapsulated into the lipid nanoparticle.

[0010] In certain embodiments, the present invention may feature an adjuvant composition comprising a lipid nanoparticle having a lipid bilayer that includes a plurality of cationic lipids (e.g., DOTMA) and structural lipids (e.g., DSPC or DOPE), a toll-like receptor 4 (TLR4) agonist incorporated into the lipid nanoparticle, and a toll-like receptor 9 (TLR9) agonist encapsulated within the lipid nanoparticle. In some embodiments, the TLR4 agonist is monophosphoryl lipid A (MPLA) or an MPLA analogue, and the TLR9 agonist is a CpG oligodeoxynucleotide (CpG).

[0011] Alternatively, in other embodiments, the present invention may feature an adjuvant composition comprising a lipid nanoparticle having a lipid bilayer comprising a plurality of ionizable cationic lipids (e.g., ALC-0315) and structural lipids (e.g., DSPC or DOPE), a toll-like receptor (TLR)-4 agonist incorporated into the lipid nanoparticle, and a TLR-9 agonist encapsulated within the lipid nanoparticle. In some embodiments, the TLR4 agonist is monophosphoryl lipid A (MPLA) or an MPLA analogue, and the TLR9 agonist is a CpG oligodeoxynucleotide (CpG).

[0012] In further embodiments, the present invention may also feature an adjuvant composition comprising an oil-in-water nanoemulsion comprising a squalene lipid core and at least one agonist which activates a pattern recognition receptor. In some embodiments, the adjuvant composition comprises an oil-in-water nanoemulsion comprising a squalene lipid core and at least two agonists. In other embodiments, the adjuvant composition comprises an oil-in-water nanoemulsion comprising a squalene lipid core and two or more agonists. In some embodiments, the agonist activates a pattern recognition receptor (PRR).

[0013] In some embodiments, the present invention may further feature a vaccine composition comprising an adjuvant as described herein and an antigen. In some embodiments, the adjuvant comprises a lipid nanoparticle (LNP) adjuvant or a nanoemulsion adjuvant, both as described herein. The antigen may be a protein antigen, a polypeptide antigen, or an mRNA encoding the antigen.

[0014] Likewise, in some embodiments, the present invention features a method of inducing an immune response in a subject in need thereof, the method comprising administering a vaccine composition as described herein. In certain embodiments, the vaccine composition is administered via injection. In other embodiments, the vaccine composition is administered via inhalation.

[0015] One of the unique and inventive technical features of the present invention is the use of lipid nanoparticles with vaccine adjuvants and antigens to produce synergistic compositions and methods that increase vaccine efficacy while reducing vaccine toxicity and / or side effects. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for maintenance of vaccine adjuvants within the lymphatic system of a subject into which the vaccine compositions of the present are introduced, thereby reducing systemic toxicity while maintaining efficacy. None of the presently known prior references or work has the unique, inventive technical feature of the present invention. For example, typical vaccine adjuvants are squalene nanoemulsions. Surprisingly, LNP formulations also improve the immunogenicity of vaccine antigens similar to squalene nanoemulsions.

[0016] Moreover, the prior references teach away from the present invention. For example, incorporation of MPLA into nanoemulsion-based adjuvants requires a multi-step process. Specifically, it is necessary to first prepare an MPLA-containing liposome formulated with an inert co-lipid, such as DOPG, at a defined molar ratio (e g., 1 :5 MPLA to DOPG). Upon subsequent mixing with the nanoemulsion, the MPLA and DOPG transfer to the interface of the nanoemulsion particles, enabling incorporation of MPLA into the final formulation. Surprisingly,the present invention eliminates the need for this additional liposome preformation step. The inventors have demonstrated that in cationic or ionizable lipid nanoparticle (LNP) formulations, such as those comprising DOTMA or ALC lipids, MPLA can be incorporated directly into the lipid bilayer during LNP preparation. This streamlined process simplifies manufacturing, reduces processing time, and provides an efficient means of incorporating MPLA into LNP-based adjuvants without the use of additional carrier liposomes.

[0017] Furthermore, the inventive technical features of the present invention contributed to a surprising result. For example, it has been assumed, based on a large body of research evidence, that vaccine potency is associated with vaccine associated systemic cytokines and transient side effects. The formulations described herein show that vaccine induced acute systemic cytokines and the associated side effects are not necessary to induce a potent and efficacious immune response.

[0018] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0019] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0020] FIG. 1 compares the immunogenicity of the IVAX adjuvant (e.g., CpG / MPLA nano-emulsion), the DOTMA CpG / MPLA lipid nanoparticle (LNP) formulation, and the ALC CpG / MPLA LNP. Specifically, the H5 vaccine response was evaluated against 28 H5N1 variant hemagglutinin (HA) antigens, with each bar in the graph representing one H5N1 variant. The DOTMA CpG / MPLA LNP induced a more potent antibody response at 14 days post-immunization compared to IVAX.

[0021] FIG. 2A and 2B shows IgG profiling of day 14 (d14) , day 28 (d28), and day (d56) serum samples on protein microarray. C57BL / 6 female mice were immunized intramuscularly twice with various formulations on day 0 (dO) and day 21 (d21 ) , serum samples were collected at d 14, d28, and d56 for IgG profiling against H5 variant antigens on protein microarray. FIG. 2A shows IgG antibody against the immunizing H5 antigen. Bars on the graph represent the following treatment groups, shown left to right for each time point (Day 14, Day 28, and Day 56): (1) Buffercontrol, (2) H5 antigen alone, (3) H5 + IVAX, (4) H5 + IVAX-CHOL, (5) H5 + DOTMA LNP, (6) H5 + CpG / MPLA in DOTMA LNP, (7) H5 + CpG in DOTMA LNP, (8) H5 + ALC LNP, (9) H5 + CpG in ALC LNP, and (10) H5 + CpG / MPLA in ALC LNP FIG. 2B shows IgG antibodies against 28 H5N1 variant HA Ag, with each bar in the graph representing one H5N1 variant. For clarity, each group labeled on the graph corresponds to the ten treatment groups outlined above in FIG. 2A, shown left to right. All IVAX, DOTMA, and ALC LNP formulations containing CpG or CpG / MPLA elicited strong IgG antibody responses against the immunizing H5 antigen and its variants at 28 days and 56 days post-immunization, as detected by protein microarray, whereas lower responses were observed in groups receiving empty DOTMA or ALC LNPs.

[0022] FIG. 3 shows total IgG midpoint titers at d21 , d28, and d56 against the vaccine antigen H5 were measured using ELISA and calculated using Sigmoidal fit. Bars on the graph represent antibody responses at the following time points, shown left to right for each treatment group: Day 14, Day 28, Day 42, and Day 56. The treatment groups are as follows: (1) Buffer control, (2) H5 antigen alone, (3) H5 + IVAX, (4) H5 + IVAX-CHOL, (5) H5 + DOTMA LNP, (6) H5 + CpG / MPLA in DOTMA LNP, (7) H5 + CpG in DOTMA LNP, (8) H5 + ALC LNP, (9) H5 + CpG in ALC LNP, and (10) H5 + CpG / MPLA in ALC LNP. All IVAX, DOTMA, and ALC LNP formulations containing CpG or CpG / MPLA induced high IgG titers against the H5 immunizing antigen, as measured by ELISA, while empty DOTMA or ALC LNPs generated markedly lower titers.

[0023] FIG. 4 shows the total and subtype IgG midpoint titers at d28 against the vaccine antigen H5, measured using ELISA and calculated using a Sigmoidal fit. For each treatment group, the three bars represent the midpoint titers for total IgG, IgG 1 , and lgG2c, shown left to right. The treatment groups are as follows: (1) Buffer control, (2) H5 antigen alone, (3) H5 + IVAX, (4) H5 + IVAX-CHOL, (5) H5 + DOTMA LNP, (6) H5 + CpG / MPLA in DOTMA LNP, (7) H5 + CpG in DOTMA LNP, (8) H5 + ALC LNP, (9) H5 + CpG in ALC LNP, and (10) H5 + CpG / MPLA in ALC LNP. All IVAX, DOTMA, and ALC LNP formulations containing CpG or CpG / MPLA induced lgG2c-biased responses, while empty ALC LNP induced IgG 1 - biased and empty DOTMA LNP induced balanced lgG1 / lgG2c responses.

[0024] FIG. 5A and 5B show transient weight changes following prime (FIG. 5A) and boost (FIG. 5B) immunizations. Weight was monitored for 7 days following each immunization to assess reactogenicity.

[0025] FIG. 6 shows inflammatory cytokine levels three hours following prime and boost immunizations. Blood samples were collected and inflammatory cytokines were measured using LEGENDplex™ Mouse Inflammation Panel multiplex assay. In some embodiments, the innate cytokine response correlated with transient weight loss. For example, IVAX-1 and ALCCpG / MPLA LNP induced elevated cytokine and chemokine levels, while the DOTMA CpG / MPLA LNP induced minimal cytokine and chemokine responses.

[0026] FIG. 7A-7F shows intracellular staining of cytokines following T cell recall with H5 antigen (10 g / mL). Splenocytes were collected 14 days post-boost (Day 35). Squares indicate samples stimulated with H5 antigen; circles indicate unstimulated controls. FIG. 7A shows the frequencies of IFNy -secreting Th1 cells in CD4 (CD4+ / IFNy+), FIG. 7B shows naive (CD62L+ / CD44-), effector memory (CD62L- / CD44+), and central memory (CD62L+ / CD44+) cell populations in CD4 cells, FIG. 7C shows the frequencies of IFNy-secreting CD4 effector memory cells (CD4+ / CD62L-CD44+ / IFNy+), FIG. 7D shows the frequencies of IL-17A-secreting CD4 effector memory cells (OD4+ / CD62L- / CD44+ / IL17+), FIG. 7E shows the frequencies of TNFa-secreting cells in CD4 (CD4+ / TNFa+), and FIG. 7F shows the frequencies of TNFa-secreting CD4 effector memory cells (CD4+ / CD62L- / CD44+ / TNFa+). Overall, IVAX-1 , IVAX with Cholesterol CpG, DOTMA, and ALC LNPs containing CpG or CpG / MPLA significantly enhanced the production of IFN-y, IL-17, and TNF-a in CD4+ T cells and CD4+ effector memory cells.

[0027] FIG. 8 shows virus lung titers by qPCR 4 days after challenge with H5N1 . Each group is compared to PBS via Mann Mann-Whitney test, followed by Benjamini-Hochberg correction for multiple comparisons. *, p<0.05.**, p<0.01. Mice immunized with H5 antigen formulated with IVAX-1 , IVAX with cholesterol-conjugated CpG, DOTMA LNP, or ALC LNP exhibited significantly reduced lung viral titers compared to buffer-treated controls, indicating protection against H5N1 infection.

[0028] FIG. 9 shows protection against SARS-CoV-2 (Wuhan strain) challenge in hamsters. Female hamsters (n = 9 per group) were immunized intramuscularly on Days 0 (dO) and 14 (d14) and challenged on Day 28 (d28). Weight was monitored for 8 days post-challenge (n = 6 per group), and lung viral titers were measured by qPCR 3 days post-challenge (n = 3 per group). Hamsters immunized with SARS-CoV-2 Spike protein formulated with IVAX-1 , DOTMA LNP, or ALC LNP showed improved protection, as evidenced by reduced weight loss and significantly lower lung viral titers compared to animals that received Spike protein alone or buffer.

[0029] Overall, the DOTMA CpG / MPLA LNP adjuvants induced lower systemic inflammatory cytokine and chemokine responses, comparable adaptive immune responses, and protective immunity relative to the IVAX reference standard.DETAILED DESCRIPTION OF THE INVENTION

[0030] Disclosed are various peptides, solvents, solutions, carriers, and / or components to beused to prepare compositions to be used within the methods disclosed herein. Also disclosed are the various steps, elements, amounts, routes of administration, symptoms, and / or treatments that are used or observed when performing the disclosed methods, as well as the methods themselves. These and other materials, steps, and / or elements are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, that while specific reference of each various individual and collective combination and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0031] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Stated another way, the term "comprising" means "including principally, but not necessary solely". Furthermore, variation of the word "comprising", such as "comprise" and "comprises", have correspondingly the same meanings. In one respect, the technology described herein related to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").

[0032] Suitable methods and materials for the practice and / or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.), the disclosures of which are incorporated in their entirety herein by reference.

[0033] A “subject” is an individual and includes, but is not limited to, a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), a fish, a bird, a reptile or an amphibian. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included. A “patient” is a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0034] The terms “administering” and “administration” refer to methods of providing a pharmaceutical preparation, composition, or formulation to a subject. The compositions described herein can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Such methods are well known to those skilled in the art and include, but are not limited to, administering the compositions orally, intranasally, parenterally (e.g., intravenously and subcutaneously), by intramuscular injection, by intraperitoneal injection, intrathecally, transdermally, extracorporeally, topically or the like.

[0035] The terms “treating” or “treatment” refer to any indicia of success or amelioration of the progression, severity, and / or duration of a disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a patient’s physical or mental well-being.

[0036] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.

[0037] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to belimiting.

[0038] Referring now to FIGs. 1-9, the present invention features a vaccine composition, such as a lipid nanoparticle, comprising an encapsulated adjuvant and either a protein antigen or an mRNA encoding the antigen. The antigens may be derived from different microorganisms, including viruses, bacteria, or parasites, thereby allowing the formulation of a single vaccine effective against multiple pathogens. In other embodiments, the antigens may be derived from tumor tissue that needs to be eliminated.

[0039] Without wishing to limit the present invention to any theory or mechanism, it is believed that free CpG, as used in other vaccine formulations such as IVAX, may diffuse away from the site of delivery (e.g., an injection site) and contribute to systemic reactogenicity. In contrast, when CpG is encapsulated within lipid nanoparticles (LNP, e.g., DOTMA LNPs), it remains localized at the injection site and within the lymphatic compartment, thereby minimizing systemic exposure. This compartmentalization supports effective immune stimulation while reducing inflammatory side effects. MPLA, an amphipathic molecule, stably integrates into the lipid nanoparticles, while soluble CpG typically lacks such association and is prone to systemic diffusion. Accordingly, adjuvant formulations that retain CpG within nanoparticles and restrict it to lymphoid tissues offer a strategic advantage in promoting immune activation with reduced systemic toxicity.

[0040] LNP Vaccine Adjuvants:

[0041] The present invention features an adjuvant composition, such as a lipid nanoparticle (LNP) adjuvant. In some embodiments, the adjuvant composition comprises a lipid nanoparticle having a lipid bilayer that includes a plurality of cationic lipids and a plurality of structural lipids. The composition further comprises at least one agonist associated with the lipid nanoparticle, which activates a pattern recognition receptor (PRR). In some embodiments, the agonist (e.g., at least one agonist) is either incorporated into or encapsulated into the lipid nanoparticle.

[0042] In some embodiments, the adjuvant composition comprises a lipid nanoparticle (LNP) having a lipid bilayer that includes a plurality of cationic lipids and a plurality of structural lipids, as well as at least two agonists associated with the LNP. In other embodiments, the adjuvant composition comprises a lipid nanoparticle (LNP) having a lipid bilayer that includes a plurality of cationic lipids and a plurality of structural lipids and two or more agonists associated with the LNP. In some embodiments, the agonist activates a pattern recognition receptor (PRR). In some embodiments, at least one agonist is either incorporated into or encapsulated into the lipid nanoparticle.

[0043] Non-limiting examples of agonists may include, but are not limited to, toll-like receptor (TLR) agonists, nucleotide oligomerization domain (NOD)-like receptor (NLR) agonists, or cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway agonists. In certain embodiments, the agonist is a toll-like receptor (TLR) agonist.

[0044] In some embodiments, the agonist is a TLR4 agonist, such as monophosphoryl lipid A (MPLA) or an MPLA analogue, which may be incorporated into the lipid nanoparticle. In other embodiments, the agonist is a TLR9 agonist, such as a CpG oligodeoxynucleotide (CpG), which may be encapsulated within the lipid nanoparticle. Various types of TLR9 agonists may be used, as their performance can vary across species, such as between mice and humans.

[0045] In some embodiments, the present invention features an adjuvant composition comprising an LNP having a lipid bilayer that includes a plurality of cationic lipids and a plurality of structural lipids, and two agonists associated with the LNP, e.g., two different agonists associated with the LNP. For example, the adjuvant composition may comprise a TLR4-agonist and a TLR9-agonist. In some embodiments, the TRL4-agonist may be incorporated into the lipid nanoparticle (e.g., within the lipid bilayer), and the TLR9-agonist is encapsulated within the lipid nanoparticle. In certain embodiments, the TLR4 agonist is MPLA or an analogue thereof, and the TLR9 agonist is CpG.

[0046] Likewise, the present invention may feature an adjuvant composition (e.g., a DOTMA CpG / MPLA LNP) comprising a lipid nanoparticle having a lipid bilayer that includes a plurality of cationic lipids (e.g., DOTMA) and structural lipids (e.g., DSPC or DOPE), a toll-like receptor 4 (TLR4) agonist incorporated into the lipid nanoparticle, and a toll-like receptor 9 (TLR9) agonist encapsulated within the lipid nanoparticle. In some embodiments, the TLR4 agonist is monophosphoryl lipid A (MPLA) or an MPLA analogue, and the TLR9 agonist is a CpG oligodeoxynucleotide (CpG).

[0047] Alternatively, the present invention may feature an adjuvant composition (e.g., an ALC CpG / MPLA LNP) comprising a lipid nanoparticle having a lipid bilayer comprising a plurality of ionizable cationic lipids (e.g., ALC-0315) and structural lipids (e.g., DSPC or DOPE), a toll-like receptor (TLR)-4 agonist incorporated into the lipid nanoparticle, and a TLR-9 agonist encapsulated within the lipid nanoparticle. In some embodiments, the TLR4 agonist is monophosphoryl lipid A (MPLA) or an MPLA analogue, and the TLR9 agonist is a CpG oligodeoxynucleotide (CpG).

[0048] In some embodiments, the agonist is covalently associated with the lipid bilayer. In other embodiments, the agonist is non-covalently associated with the lipid bilayer. In certainembodiments, the non-covalent association involves ionic interactions, hydrophobic interactions, or a combination thereof.

[0049] In some embodiments, the cationic lipids within the lipid bilayer comprise1 .2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA). In other embodiments, the lipid nanoparticles (LNPs) include ionizable cationic lipids, such as [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), or other ionizable lipids. The LNPs described herein may contain a plurality of cationic lipids, a plurality of ionizable cationic lipids, or a combination of both.

[0050] In some embodiments, the structural lipids within the lipid bilayer may include1 .2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or1.2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In other embodiments, the lipid bilayer may further comprise phosphatidylcholines (PC), phosphatidylethanolamines (PE), cholesterol, and / or PEG-lipids. The present invention is not limited to the lipids listed above and may include other lipids known in the art to be capable of forming lipid nanoparticles.

[0051] In some embodiments, the lipid bilayer is a disordered polymorphic lipid bilayer.

[0052] In some embodiments, the adjuvants described herein (e.g., DOTMA CpG / MPLA, ALC CpG / MPLA, etc.) exhibit greater cross-reactive potency than a control adjuvant (e.g., IVAX), while demonstrating minimal transient weight loss and no significant increase in systemic cytokines associated with reactogenicity. The DOTMA CpG / MPLA formulation may bind and multimerize the vaccine antigen, enhancing immunogenic potency while reducing reactogenicity compared to a control adjuvant (e.g., IVAX). Similarly, ALC CpG / MPLA formulations may also induce antigen multimerization. In some embodiments, multimerization may depend on the physical properties of an antigen. LNP-induced multimerization is experimentally measurable.

[0053] In some embodiments, the lipid nanoparticles (LNPs) described herein have a diameter of less than 200 nm. Without wishing to limit the present invention to any theory or mechanism it is believed that LNPs of this size meet two desirable criteria: they are sufficiently small to permit sterile filtration (e.g., through filters with a pore size of approximately 220 nm) and fall within the optimal size range (approximately 10-200 nm) for effective drainage into lymph nodes.

[0054] Table 1 (below) shows non-limiting CpG and MPLA encapsulation*D: DSPC; C: CHOL; P: DMG-PEG

[0055] In certain embodiments, the present invention provides a positively charged lipid nanoparticle that encapsulates 100% of the input TLR9 agonist (e.g., CpG) and 100% of the TLR4 agonist (e.g., MPLA or an analogue thereof). In some embodiments, CpG encapsulation is assessed using the Oligreen assay, a fluorescence-based method for detecting single-stranded DNA. In certain embodiments, MPLA is incorporated into the lipid film during LNP preparation and achieves complete (100%) incorporation.

[0056] Without wishing to limit the present invention to any theory or mechanism, it is believed that the cationic lipid facilitates nanoparticle formation and promotes binding of the input protein antigen to the nanoparticle surface. Thus, each nanoparticle includes all components necessary to stimulate an adaptive immune response. The nanoparticles may remain localized within the lymphatic system and therefore cannot enter systemic circulation without first passing through the lymphatic network and draining lymph nodes, where the antigens are presented to immune cells. This configuration enables efficient antigen presentation and activation of immune responses while minimizing systemic inflammatory toxicity.

[0057] In some embodiments, the LNPs described herein have a charge ratio of about 0.5 to 2. In some embodiments, the LNPs described herein have a charge ratio of about 0.5. In some embodiments, the LNPs described herein have a charge ratio of about 1. In some embodiments, the LNPs described herein have a charge ratio of about 1.5. In some embodiments, the LNPs described herein have a charge ratio of about 2.

[0058] In some embodiments, after condensation with nucleic acids or other negatively charged molecules, the present invention features a lipid bilayer system that is not a pure classical bilayer system. Instead, in some embodiments, the present invention features lipid nanoparticle adjuvant compositions that are highly condensed and that form polymorphic structures withbilayer and hexagonal-2 phases.

[0059] Nanoemulsion Vaccine Adjuvants:

[0060] The present invention may also feature an adjuvant composition comprising an oil-in-water nanoemulsion comprising a squalene lipid core and at least one agonist which activates a pattern recognition receptor. In some embodiments, the adjuvant composition comprises an oil-in-water nanoemulsion comprising a squalene lipid core and at least two agonists. In other embodiments, the adjuvant composition comprises an oil-in-water nanoemulsion comprising a squalene lipid core and two or more agonists. In some embodiments, the agonist activates a pattern recognition receptor (PRR).

[0061] Non-limiting examples of agonists may include, but are not limited to, toll-like receptor (TLR) agonists, nucleotide oligomerization domain (NOD)-like receptor (NLR) agonists, or cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway agonists. In certain embodiments, the agonist is a toll-like receptor (TLR) agonist.

[0062] In some embodiments, the agonist is a TLR4 agonist, such as monophosphoryl lipid A (MPLA) or an MPLA analogue, which may be incorporated into the interface of an oil-in-water nanoemulsion. In other embodiments, the agonist is a TLR9 agonist, such as a CpG oligodeoxynucleotide (CpG), which may be present in soluble form or may be incorporated into the nanoemulsion interface through the inclusion of a lipophilic tail attached to the CpG. In certain embodiments, the lipophilic tail comprises a cholesterol molecule or a phosphatidyl moiety.

[0063] In some embodiments, the present invention may also feature an adjuvant composition comprising an oil-in-water nanoemulsion comprising a squalene lipid core and two agonists, e g., two different agonists. In some embodiments, at least one agonist is incorporated into the interface of an oil-in-water nanoemulsion. For example, the adjuvant composition may comprise a TLR4-agonist and a TLR9-agonist. In some embodiments, the TLR-4 agonist is incorporated into the interface of an oil-in-water nanoemulsion. In some embodiments, the TLR-9 agonist may be present in soluble form or may be incorporated into the nanoemulsion interface through the inclusion of a lipophilic tail (e.g., a cholesterol molecule or a phosphatidyl moiety) attached thereto. In certain embodiments, the TLR4 agonist is MPLA or an analogue thereof, and the TLR9 agonist is CpG.

[0064] Likewise, the present invention may feature an adjuvant composition comprising: an oil-in-water nanoemulsion comprising a squalene lipid core, a toll-like receptor (TLR)-4 agonist incorporated into the interface of an oil-in-water nanoemulsion, and a TLR-9 agonist. In someembodiments, the TLR4 agonist is MPLA, and the TLR9 agonist is CpG. In some embodiments, the TLR9 agonist comprises a lipophilic tail (e.g., a cholesterol molecule of a phosphatidyl moiety) attached thereto. In such embodiments, the presence of the lipophilic tail facilitates incorporation of the TLR9 agonist into the interface of the nanoemulsion. In the absence of the lipophilic tail, the TLR9 agonist remains in soluble form and is not incorporated into the lipid core.

[0065] Vaccine Compositions:

[0066] The present invention may further feature a vaccine composition comprising an adjuvant as described herein and an antigen. In some embodiments, the adjuvant comprises a lipid nanoparticle (LNP) adjuvant or a nanoemulsion adjuvant, both as described herein. The antigen may be a protein antigen, a polypeptide antigen, or an mRNA encoding the antigen. In certain embodiments, the antigen is derived from a microorganism, including a virus, bacterium, or parasite. In other embodiments, the antigen is derived from a tumor cell. Non-limiting examples of suitable antigens include the Spike protein from SARS-CoV-2, the VP1 protein from Picornavirus, the N protein from Peribunyavirus, and the influenza hemagglutinin H1 protein.

[0067] In some embodiments, the antigen is adsorbed onto a surface of the lipid bilayer. In certain embodiments, the adjuvant remains localized at the site of administration. In some embodiments, the adjuvant is maintained within the lymphatic system. In yet other embodiments, the vaccine composition reduces side effects as compared to compositions that enter systemic circulation.

[0068] In certain embodiments, the present invention provides a lipid nanoparticle-based vaccine composition comprising one or more adjuvants and an antigen. The lipid nanoparticle includes a cationic lipid that facilitates the formation of the nanoparticle and enables the adsorption of a protein antigen onto the nanoparticle surface. The LNP may incorporate monophosphoryl lipid A (MPLA), a Toll-like receptor 4 (TLR4) agonist, into the lipid bilayer structure of the nanoparticle. The nanoparticle may further comprise CpG, a Toll-like receptor 9 (TLR9) agonist, which may be encapsulated within the interior of the nanoparticle or associated externally with the nanoparticle surface. In this configuration, the lipid nanoparticle contains both antigenic and adjuvant components necessary to stimulate a robust adaptive immune response. Furthermore, the positively charged nature of the nanoparticle promotes uptake by antigen-presenting cells and facilitates retention within the lymphatic system, thereby preventing direct entry into systemic circulation without first traversing the lymphatic network.

[0069] In some embodiments, the vaccine compositions described herein may be formulated forintranasal administration. Without limiting the present invention to any particular theory or mechanism, it is believed that the LNP formulations (e.g., DOTMA LNP formulations) possess bioadhesive properties that enable adhesion to mucosal surfaces, making them suitable candidates for intranasal vaccine delivery. Intramuscular, intravenous, subcutaneous, intraperitoneal, etc. In other embodiments, the vaccine compositions may be administered via other routes, including but not limited to intramuscular, intravenous, subcutaneous, or intraperitoneal administration.

[0070] In some embodiments, the present invention features a method of inducing an immune response in a subject in need thereof, the method comprising administering a vaccine composition as described herein. In certain embodiments, the vaccine composition is administered via injection. In other embodiments, the vaccine composition is administered via inhalation.

[0071] In some embodiments, the present invention further provides a method of manufacturing an adjuvant composition comprising a lipid nanoparticle having a lipid bilayer that includes a plurality of cationic lipids and structural lipids, and at least one agonist associated with the lipid nanoparticle, wherein the agonist activates a pattern recognition receptor. In certain embodiments, the adjuvant composition is formulated with an antigen to produce a vaccine composition as described herein, which may be used to induce an immune response in a subject in need thereof.

[0072] EXAMPLE

[0073] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0074] Combination Lipid Nanoparticle (LNP) Adjuvants

[0075] Four cationic LNP combination adjuvants were formulated with either DNA (DNA lipoplex), RNA (poly l / C, RNA lipoplex), lipophylic muramyl dipeptide (MDP), or cyclic GMP / AMP (cGAMP) (Table 1). These adjuvants were extemporaneously mixed with recombinant protein antigens to produce the vaccines. The adjuvant effects of these four vaccines were compared with IVAX-1 , which was developed and well characterized by the Inventors, as well as with two benchmark adjuvants, AddaVax and Alum.

[0076] Table 1 : Adjuvant LNP formulation and dose / mouseBuffer: 10mM Tris-based / 10% Sucrose buffer, pH 7.4

[0077] Table 2: Nanoparticle Size and Zeta Potential (charge)*AII adjuvant LNPs showed desired particle size distribution and encapsulation.

[0078] In some embodiments, CpG encapsulation is assessed using the Oligreen assay, a fluorescence-based method for detecting single-stranded DNA. In certain embodiments, MPLA is incorporated into the lipid film during LNP preparation and achieves complete (100%) incorporation.

[0079] IVAX-1 : IVAX-1 is a nanoemulsion formulation based on the squalene oil-in-water emulsion AddaVax, incorporating monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotide (ODN) 1018. The adjuvant components are combined with antigens byextemporaneous mixing. IVAX-1 was selected from an adjuvant screening study and has been shown to induce a Th1-biased immune response and generate neutralizing antibody activity.

[0080] Cationic Liposome / DNA: Cationic liposomes and plasmid DNA individually exhibit limited immunostimulatory activity, their combination results in liposome-mediated potentiation of immune responsiveness, primarily through recognition of non-methylated CpG motifs present in bacterial plasmid DNA. For complex formation, cationic lipid DOTMA and helper lipid DOPE (Avanti Polar Lipids, Inc.) are combined at an equal weight ratio to form cationic liposomes. DOTMA / DOPE cationic liposomes have been reproducibly produced with an average particle size of approximately 150 nm in diameter, and can form cationic liposome / plasmid DNA complexes with similar size distributions across a range of charge ratios (0.5-2). DOTMA, which contains an ether linkage between the C18 alkyl chains and polar head group, provides enhanced stability, while DOPE facilitates DNA complexation and promotes the formation of fusogenic inverted hexagonal lipid phases. Additional helper lipids, such as cholesterol and PEG-lipids, may be incorporated to improve liposome stability and surface hydrophilicity, respectively.

[0081] Cationic Liposome / Poly(l: C): Polyinosinic-polycytidylic acid (poly(l:C)) is a synthetic analog of double-stranded RNA (dsRNA), a pathogen-associated molecular pattern (PAMP) commonly associated with viral infections. Poly(l:C) activates the immune response through two distinct pathogen recognition receptors (PRRs): endosomal poly(l:C) activates Toll-like receptor 3 (TLR3), while cytosolic poly(l:C) activates the RIG-l / MDA-5 pathway. Both signaling pathways promote Th1-biased cellular immunity through the induction of interleukin-12 (IL-12) and type I interferons (IFNs), and poly(l:C) has demonstrated potent adjuvant activity in various vaccine formulations. However, administration of soluble poly(l:C) has been associated with adverse effects due to the induction of pro-inflammatory cytokines. Complexation of poly(l:C) with cationic liposomes to form lipid nanoparticles may mitigate these side effects. In addition, the cationic liposome can potentiate the immunostimulatory effects of poly(l:C) while also protecting it from enzymatic degradation

[0082] Cationic Liposome / Lipophilic MDP: Muramyl dipeptide (MDP) is the minimal bioactive motif derived from bacterial peptidoglycan and represents the essential structural component required for adjuvant activity in vaccines. MDP is recognized by the intracellular pattern recognition receptor NOD2. Among various MDP derivatives, L18-MDP, a lipophilic derivative containing a stearoyl fatty acid moiety, exhibits enhanced activity in promoting protective immune responses against bacterial infections. Due to its limited aqueous solubility, lipophilic L18-MDP may be incorporated into the lipid bilayer of cationic liposomes during lipid film formation. The cationic liposome not only improves the solubility and incorporation efficiency ofL18-MDP but also serves as a nanoparticle delivery vehicle and adjuvant potentiator.

[0083] Cationic liposome / Sting: 2’3’-cGAMP is a cyclic dinucleotide produced in mammalian cells by cyclic GMP-AMP synthase (cGAS) in response to the presence of double-stranded DNA in the cytoplasm. Cyclic dinucleotides (CDNs), such as 2’3’-cGAMP, have been shown to enhance vaccine potency by activating innate immunity. Specifically, 2’3’-cGAMP binds directly to the endoplasmic reticulum-resident receptor STING (stimulator of interferon genes), initiating a signaling cascade that induces expression of interferon-p (IFN-P) and nuclear factor KB (NF-KB)-dependent pro-inflammatory cytokines. However, due to its anionic nature, 2’3’-cGAMP exhibits poor membrane permeability, limiting its ability to access cytosolic STING receptors. To overcome this limitation, cationic liposomes may be used to encapsulate or complex divalent 2’3’-cGAMP, thereby facilitating its cytosolic delivery. In vitro studies with antigen-presenting cells (APCs) have demonstrated that liposomal formulations significantly enhance cellular uptake of 2’3’-cGAMP and induce higher levels of pro-inflammatory gene expression compared to free drugs.

[0084] Cationic LNP-1 : IVAX-1 is a nanoemulsion-based formulation comprising Addavax combined with CpG and MPLA. Studies conducted by the Inventors demonstrated that none of the tested cationic nanoparticle formulations outperformed IVAX-1 in terms of immunogenicity and efficacy. Notably, the prior formulations evaluated did not include the combination of CpG and MPLA. The tested formulations containing cGAMP, plasmid DNA, poly(l:C), or lipophilic MDP generated immune responses in mice that were inferior to the reference standards (Addavax and IVAX-1). However, surprisingly, when the inventors replaced the Addavax component of the IVAX-1 formulation with DOTMA and incorporated CpG and MPLA into the DOTMA cationic lipid formulation, an adjuvant was produced that unexpectedly induced higher Th1-biased antibody responses than IVAX-1 , while also avoiding the induction of inflammatory cytokines at three hours post-prime and post-boost, and without significant vaccine-induced transient weight loss.

[0085] Fourteen-day post-prime IgG data were obtained to compare DOTMA lipid nanoparticle (LNP) formulations containing CpG and MPLA (FIG. 1-4). In this study, mice were immunized with a single H5N1 hemagglutinin (HA) protein antigen (A / Vietnam / 1194 / 2004), and plasma samples were analyzed using an influenza protein microarray containing 28 H5N1 HA variant antigens. Antigen alone failed to induce detectable antibody responses, whereas IVAX-1 induced broad antibody responses across all variants, as expected. The combination of IVAX-1 with lipophilic cholesterol-conjugated CpG further enhanced antibody levels. DOTMA LNP alone exhibited weak adjuvant activity; however, encapsulation of CpG within DOTMA LNP resulted in superior antibody induction compared to IVAX-1 . The addition of MPLA to the DOTMA / CpG LNPformulation yielded the highest antibody levels observed. In contrast, ionizable cationic LNPs containing CpG and MPLA induced antibody responses comparable to IVAX-1. Weight loss following vaccination, used as a sensitive indicator of reactogenicity and toxicity, revealed that buffer and H5N1 HA protein alone did not cause weight loss, while IVAX-1 induced transient weight loss, as expected. DOTMA LNP formulations, with or without CpG and MPLA, did not induce significant weight loss. In contrast, CpG and MPLA in ionizable cationic LNPs induced substantial weight loss, with a more gradual recovery.

[0086] Without wishing to limit the present invention to any particular theory or mechanism, it is believed that the reduced reactogenicity observed with the DOTMA LNP formulation, compared to the emulsion containing cholesterol-conjugated CpG (chol-CpG), may be attributable to differences in surface charge rather than CpG diffusion. For example, DOTMA LNP formulations, which possess a positive surface charge, exhibit significantly reduced reactogenicity. In some embodiments, the surface charge may influence the trafficking of nanoparticles to the draining lymph nodes in vivo, thereby impacting both immunogenicity and reactogenicity. This is further supported by the observation that ALC LNPs, which are neutral at physiological pH yet encapsulate CpG, remain highly reactogenic, indicating that restricted CpG diffusion alone does not account for reduced systemic side effects.

[0087] The DOTMA / CpG / MPLA LNP formulation demonstrated potent adjuvant activity with low reactogenicity. Mice immunized with this formulation exhibited minimal weight loss and low levels of inflammatory cytokines in the blood three hours post-immunization (FIG. 6). Vaccine-induced innate cytokine levels were analyzed at this time point to assess early reactogenicity. In addition, a single dose of the DOTMA / CpG / MPLA LNP induced a strong magnitude and breadth of antibody responses (FIGs. 2-4). Plasma samples were collected on Day 28 post-boost, and antibody titers were evaluated by ELISA to assess the adaptive immune response.

[0088] As used herein, the term “about” refers to plus or minus 10% of the referenced number.

[0089] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could bedescribed as “consisting essentially of’ or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met.

Claims

WHAT IS CLAIMED IS:1 . An adjuvant composition comprising: a) a lipid nanoparticle having a lipid bilayer comprising a plurality of cationic lipids, and a plurality of structural lipids; and b) at least one agonist associated with the lipid nanoparticle, wherein the at least one agonist activates a pattern recognition receptor.

2. The composition of claim 1 , wherein the cationic lipids comprise1 .2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA) or ([(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), or other ionizable lipids.

3. The composition of claim 1 or claim 2, wherein the structural lipids comprise 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or1 .2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

4. The composition of any one of claims 1-3, wherein the lipid bilayer further comprises phosphatidylcholines (PC), phosphatidylethanolamine (PE), cholesterol, or PEG-lipids.

5. The composition of any one of claims 1-4, wherein the lipid bilayer is a disordered polymorphic lipid bilayer.

6. The composition of any one of claims 1-5, wherein the at least one agonist is either incorporated into or encapsulated into the lipid nanoparticle.

7. The composition of any one of claims 1-6, wherein the agonist interacts covalently with the lipid bilayer.

8. The composition of any one of claims 1-6, wherein the agonist interacts non-covalently with the lipid bilayer.

9. The composition of claim 7 or claim 8, wherein the agonist interacts via ionic interactions, hydrophobic interactions, or a combination thereof.

10. The composition of any one of claims 1-9, wherein the agonist is a toll-like receptor (TLR) agonist, nucleotide oligomerization domain (NOD)-like receptor (NLR) agonist, or a cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway agonist.

11. The composition of any one of claims 1-10, wherein the agonist is a TLR4-agonist; wherein the TLR4-agonist is MPLA or an MPLA analogue.

12. The composition of claim 11 , wherein the TLR4-agonist is incorporated into the lipid nanoparticle.

13. The composition of any one of claims 1-12, wherein the agonist is a TLR9-agonist; wherein the TLR9-agonist is CpG.

14. The composition of claim 13, wherein the TLR9-agonist is encapsulated within the lipidnanoparticle.

15. The composition of any one of claims 1-14, wherein the composition comprises two different agonists associated with the lipid nanoparticle.

16. The composition of claim 15, wherein the composition comprises a TLR4-agonist and a TLR9-agonist.

17. The composition of claim 16, wherein the TLR4-agonist is incorporated into the lipid nanoparticle and the TLR9-agonist is encapsulated within the lipid nanoparticle.

18. The composition of claim 16 or claim 17, wherein the TLR4-agonist is MPLA and the TLR9 agonist is CpG.

19. The composition of any one of claims 1-14, wherein the composition comprises at least two different agonists associated with the lipid nanoparticle.

20. An adjuvant composition comprising: a) a lipid nanoparticle having a lipid bilayer comprising a plurality of cationic lipids and structural lipids; b) a toll-like receptor (TLR)-4 agonist incorporated into the lipid nanoparticle; and c) a TLR-9 agonist encapsulated within the lipid nanoparticle.

21. The composition of claim 20, wherein the cationic lipids comprise1 .2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA).

22. The composition of claim 20 or claim 21 , wherein the structural lipids comprise 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or1 .2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

23. The composition of any one of claims 20-22, wherein the lipid bilayer further comprises phosphatidylcholines (PC), phosphatidylethanolamine (PE), cholesterol, or PEG-lipids.

24. The composition of any one of claims 20-23, wherein the TLR4-agonist is MPLA or an MPLA analogue and the TLR9 agonist is CpG.

25. An adjuvant composition comprising: a) a lipid nanoparticle having a lipid bilayer comprising a plurality of ionizable cationic lipids and structural lipids; b) a toll-like receptor (TLR)-4 agonist incorporated into the lipid nanoparticle; and c) a TLR-9 agonist encapsulated within the lipid nanoparticle.

26. The composition of claim 25, wherein the ionizable cationic lipids comprise ([(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315).

27. The composition of claim 25 or claim 26, wherein the structural lipids comprise 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

28. The composition of any one of claims 25-27, wherein the lipid bilayer further comprisesphosphatidylcholines (PC), phosphatidylethanolamine (PE), cholesterol, or PEG-lipids.

29. The composition of any one of claims 25-28, wherein the TLR4-agonist is MPLA or an MPLA analogue and the TLR9 agonist is CpG.

30. An adjuvant composition comprising: a) an oil-in-water nanoemulsion comprising a squalene lipid core; and b) at least one agonist, wherein the at least one agonist activates a pattern recognition receptor.

31. The composition of claim 30, wherein the agonist is a toll-like receptor (TLR) agonist, nucleotide oligomerization domain (NOD)-like receptor (NLR) agonist, or a cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway agonist.

32. The composition of claim 30 or 31 , wherein the at least one agonist comprises a TLR-4 agonist or a TLR-9 agonist.

33. The composition of claim 32, wherein the TLR4-agonist is MPLA.

34. The composition of claim 32 or 33, wherein the TLR4-agonist is incorporated into an interface of the oil-in-water nanoemulsion.

35. The composition of claim 32, wherein the TLR9-agonist is CpG.

36. The composition of claim 32 or claim 35, wherein the TLR9-agonist further comprises a lipophilic tail attached thereto, wherein the lipophilic tail comprises a cholesterol molecule or a phosphatidyl moiety.

37. The composition of claim 36, wherein the TLR9-agonist is incorporated into an interface of oil-in-water nanoemulsion.

38. The composition of any one of claims 30-37, wherein the composition comprises two different agonists associated with the nanoemulsion.

39. The composition of claim 38, wherein the composition comprises a TLR4-agonist and a TLR9-agonist.

40. The composition of claim 39, wherein the TLR4-agonist is MPLA or MPLA analogue and the TLR9 agonist is CpG.

41. The composition of claim 39 or claim 40, wherein the TLR4-agonist is incorporated into an interface of oil-in-water nanoemulsion.

42. The composition of claim 39 or claim 40, wherein the TLR9-agonist further comprises a lipophilic tail attached thereto, wherein the lipophilic tail comprises a cholesterol molecule.

43. The composition of claim 42, wherein the TLR9-agonist is incorporated into an interface of oil-in-water nanoemulsion.

44. An adjuvant composition comprising: a) an oil-in-water nanoemulsion comprising a squalene lipid core;b) a toll-like receptor (TLR)-4 agonist incorporated into an interface of the nanoemulsion; and c) a TLR-9 agonist.

45. The composition of claim 44, wherein the TLR4-agonist is MPLA and the TLR9 agonist is CpG.

46. The composition of claim 43 or claim 44, wherein the TLR9-agonist further comprises a lipophilic tail attached thereto, wherein the lipophilic tail comprises a cholesterol molecule.

47. The composition of claim 46, wherein the TLR9-agonist is incorporated into an interface of oil-in-water nanoemulsion.

48. A vaccine composition comprising: a) an adjuvant according to any one of claims 1-47; and b) an antigen.

49. The composition of claim 48, wherein the antigen is a protein antigen, a polypeptide antigen, or an mRNA encoding the antigen.

50. The composition of claim 48 or claim 49, wherein the antigen may be derived from different microorganisms, including viruses, bacteria, or parasites.

51. The composition of claim 48 or claim 49, wherein the antigen may be derived from a tumor cell.

52. The composition of any one of claims 48-51 , wherein the antigen is absorbed on a surface of the lipid bilayer.

53. The composition of any one of claims 48-52, wherein the adjuvant remains localized at an injection site.

54. The composition of any one of claims 48-53, wherein the adjuvant is maintained in the lymphatic system.

55. The composition of any one of claims 48-55, wherein the vaccine composition reduces side effects as compared to compositions that enter systemic circulation.

56. A method to induce an immunological response in a subject in need thereof, the method comprising: administering a vaccine composition according to any one of claims 48-55.

57. The method of claim 56, wherein the vaccine composition is administered via injection.

58. The method of claim 56, wherein the vaccine composition is administered via inhalation.

59. A method of manufacturing an adjuvant composition comprising: a) a lipid nanoparticle having a lipid bilayer comprising a plurality of cationic lipids and structural lipids; and b) at least one agonist associated with the lipid nanoparticle, wherein the at least one agonist activates a pattern recognition receptor.

0. A method of inducing an immune response in a subject in need thereof, the method comprises administering a vaccine composition comprising: a) an adjuvant composition comprising: i. a lipid nanoparticle having a lipid bilayer comprising a plurality of cationic lipids and structural lipids; and ii. at least one agonist associated with the lipid nanoparticle, wherein the at least one agonist activates a pattern recognition receptor; and b) an antigen.

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