Lipid nanoparticles with integrated glycolipid adjuvant to promote tissue-specific cellular immunity

Abstract

A lipid nanoparticle (LNP) comprising a plurality of lipids and a glycolipid adjuvant integrated into a core of the LNP. The LNPs may further comprise a nucleic acid encoding an antigen, including mRNA. A method of producing the LNPs is provided, including introducing the glycolipid adjuvant into a lipid phase prior to in-line mixing with an aqueous nucleic acid phase to form glycolipid-integrated LNPs with high incorporation efficiency and preserved physicochemical properties. Also disclosed are pharmaceutical compositions comprising the LNPs and methods of use, including immunizing a subject to induce tissue-specific cellular immunity. In certain embodiments, administration of the glycolipid-integrated LNPs induces liver-resident immune responses and provides sterile protection against parasitic infection.

Description

LIPID NANOPARTICLES WITH INTEGRATED GLYCOLIPID ADJUVANT TO PROMOTE TISSUE-SPECIFIC CELLULAR IMMUNITYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Application No. 63 / 759,779, filed on February 18, 2025, the disclosure of which is hereby incorporated by reference in its entirety.STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with Government support under Grant No. 1U01AI155313 awarded by the National Institutes of Health. The Government has certain rights in the invention.STATEMENT REGARDING SEQUENCE LISTING

[0003] The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 3915-P1390WO.UW_Sequence_Listing_20260126.xml. The XML file is 4,244 bytes; was created on January 26, 2026; and is being submitted electronically via Patent Center with the filing of the Specification.BACKGROUND

[0004] Lipid nanoparticles (LNPs) hold potential as vaccines for pathogens that have historically been difficult to effectively vaccinate against, such as Plasmodium. The most clinically advanced LNPs are composed of four lipid components: an ionizable lipid, a helper lipid, sterol, and a PEGylated lipid. Selection of the specific lipids and their molar ratios determine the immunogenicity and organ-targeting of the LNPs. mRNA LNPs are considered self-adjuvanted as their ionizable lipids and mRNA modifications are intrinsically immunogenic and thus supplemental adjuvants have not yet been utilized. However, as-is mRNA LNP vaccines may be insufficient for achieving protection against complex pathogens,

[0005] Many pre-clinical studies have evaluated mRNA vaccines with intrinsic or exogenous adjuvants, demonstrating improved vaccine efficacy with potent immunostimulatory adjuvants. Adjuvants can dramatically impact the immune responseand offer several advantages for vaccination in that they may: 1) fine-tune the immune response to improve vaccine efficacy for specific diseases, 2) improve the heterogeneity of vaccine efficacy across diverse populations, and, 3) simplify the dosage and / or administration route or schedule. Yet, despite this, there is limited research on mRNA LNP-compatible adjuvants.

[0006] Prior studies have shown sterile protection from Plasmodium sporozoite (spz) challenge requires high levels of anti -sporozoite antibodies and / or liver-specific CD8+T cells that have proven difficult to achieve with subunit vaccines.

[0007] There remains a need for providing ongoing sterile protection from parasites.SUMMARY

[0008] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0009] In an aspect, the present disclosure provides a lipid nanoparticle (LNP) including a plurality of lipids and a glycolipid adjuvant, wherein the glycolipid adjuvant is structurally integrated into a core of the LNP during formation of the LNP.

[0010] In some embodiments, the plurality of lipids comprises an ionizable lipid, a helper lipid, a sterol, and a PEGylated lipid.

[0011] In some embodiments, the LNP further includes an antigenic factor.

[0012] In some embodiments, the LNP further includes a nucleic acid encoding one or more polypeptides.

[0013] In some embodiments, the one or more polypeptides comprise an antigen.

[0014] In some embodiments, the glycolipid adjuvant is present in an amount of between about 0.1 pg to about 2.5 pg glycolipid adjuvant per 10 pg nucleic acid.

[0015] In some embodiments, the glycolipid adjuvant is present in an amount of between about 0.4 µg to about 2.0 µg glycolipid adjuvant per 10 pg nucleic acid

[0016] In some embodiments, the nucleic acid is selected from the group consisting of an mRNA, a circRNA, a saRNA, and a DNA.

[0017] In some embodiments, the nucleic acid is mRNA,

[0018] In some embodiments, the glycolipid adjuvant is 7DW8-5,

[0019] In some embodiments, the ionizable lipid is selected from the group consisting of Dlin-MC3-DMA, SM-102, and ALC-0315.

[0020] In some embodiments, the helper lipid is selected from the group consisting of l-stearoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (18PG). 1.2-distearoylsn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-3-trimethylammonium- propane (DOTAP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), dimethyldioctadecyl ammonium (DDAB), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA), and sn-(3-oleoyl-2-hydroxy)-glycerol-l-phospho-sn-3'-(r,2'-dioleoyl)-glycerol (ammonium salt) (I8BMP).

[0021] In some embodiments, the sterol is a cholesterol.

[0022] In some embodiments, the PEGylated lipid is selected from the group consisting of DMG-PEG200 and ALC-0159.

[0023] In some embodiments, a molar ratio of the ionizable lipid in the lipid nanoparticle is in the range of about 30% to about 55%.

[0024] In some embodiments, a molar ratio of the helper lipid in the lipid nanoparticle is in the range of about 5% to about 10%.

[0025] In some embodiments, a molar ratio of the sterol in the lipid nanoparticle is in the range of about 38% to about 45%.

[0026] In some embodiments, a molar ratio of the PEGylated lipid in the lipid nanoparticle is in the range of about 0.05% to about 2%.

[0027] In some embodiments, the glycolipid adjuvant has a 70% or greater incorporation efficiency into the core of the LNP.

[0028] In an aspect, the present disclosure provides a composition comprising any of the LNPs described herein and a pharmaceutically acceptable carrier.

[0029] In an aspect, the present disclosure provides a method of producing a pharmaceutical composition, the method including: combining a plurality of lipidsdissolved in a water-miscible organic solvent, thereby forming a lipid phase; introducing a glycolipid into the lipid phase; and mixing the lipid phase with nucleic acids dissolved in an aqueous phase in an in-line mixing device.

[0030] In some embodiments, the in-line mixing device is selected from a group of micro-fluid mixers. 2-inlet confined impinging jet mixers, 3-inlet confined impinging jet mixers, 4-inlet confined impinging jet mixers, multi-inlet vortex mixers, and T-junction mixers

[0031] In some embodiments, the plurality of lipids comprises an ionizable lipid, a helper lipid, a sterol, and a PEGylated lipid.

[0032] In some embodiments, the nucleic acids are selected from the group consisting of an mRNA. a circRNA, a saRNA, and a DNA.

[0033] In some embodiments, a molar ratio of ionizable lipid in the lipid nanoparticle is in the range of about 30% to about 55%; a molar ratio of the helper lipid in the lipid nanoparticle is in the range of about 5% to about 10%; a molar ratio of the sterol in the lipid nanoparticle is in the range of about 38% to about 45%; a molar ratio of the PEGylated lipid in the lipid nanoparticle is in the range of about 0.05% to about 2%; and wherein the glycolipid adjuvant is present in an amount of between about 0.1 pg to about 2.5 pg glycolipid adjuvant per 10 pg nucleic acid.

[0034] In an aspect, the present disclosure provides a method of immunizing a subject against a parasite, the method comprising administering to the subject a therapeutically effective dose of any of the LNPs described herein.

[0035] In some embodiments, the LNP is administered via intravenous (IV) injection.

[0036] In some embodiments, the LNP is administered via intramuscular (IM) injection.

[0037] In some embodiments, the parasite is Plasmodium.

[0038] In some embodiments, immunizing comprises inducing liver-resident immunity leading to sterile protection against parasites.DESCRIPTION OF THE DRAWINGS

[0039] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0040] FIGURE 1 depicts a lipid nanoparticle (LNP) according to embodiments of the present disclosure;

[0041] FIGURE 2A depicts an experimental design of IVIS studies, according to embodiments of the present disclosure;

[0042] FIGURE 2B depicts IVIS imaging of BALB / cJ mice 24 hours (left) or 48 hours (right) post vaccination with FIII-7 LNPs containing 5 pg of Luciferase mRNA (mLuc) injected IV or IM, with Regions of Interest (ROI) drawn around the entire mouse body;

[0043] FIGURE 2C depicts IVIS imaging of BALB / cJ mice 24 hours (left) or 48 hours (right) post vaccination with FIII-7 LNPs containing 5 pg of Luciferase mRNA (mLuc) injected IV or IM, with Regions of Interest (ROI) drawn around the primary expression site (thigh for IM or liver for IV);

[0044] FIGURE 2D depicts IVIS imaging of BALB / cJ mice 24 hours (left) or 48 hours (right) post vaccination with FIII-7 LNPs containing 5 pg of Luciferase mRNA (mLuc) injected IV or IM, with Regions of Interest (ROI) drawn around the liver;

[0045] FIGURE 3A depicts an experimental design of IVIS studies, according to embodiments of the present disclosure;

[0046] FIGURE 3B depicts IVIS imaging of mice 24 or 48 hours post vaccination with FIII-7 LNPs containing 5 pg of Luciferase mRNA (mLuc) injected IV or IM, with Regions of Interest (ROI) drawn around the entire mouse body.

[0047] FIGURE 3C depicts IVIS imaging of mice 24 or 48 hows post vaccination with FIII-7 LNPs containing 5 pg of Luciferase mRNA (mLuc) injected IV or IM, with ROI drawn around the primary expression site (thigh for IM or liver for IV).

[0048] FIGURE 3D depicts IVIS imaging of mice 24 or 48 hours post vaccination with FIII-7 LNPs containing 5 pg of Luciferase mRNA (mLuc) injected IV or IM, with ROI drawn around the liver.

[0049] FIGURE 4A depicts an experimental design of immunogenicity studies, according to embodiments of the present disclosure;

[0050] FIGURE 4B depicts C57BL / 6J mice were primed with 5 pg FIII-7 LNPs containing 5 µg of Ovalbumin mRNA (mOVA) and spleens were harvested on Day 7 for IFNγ ELISPOT;

[0051] FIGURE 4C depicts an experimental design of immunogenicity studies, according to embodiments of the present disclosure;

[0052] FIGURE 4D depicts C57BL / 6J mice were prime-boosted with 5 pg FIII-7 mOVA LNPs and spleens were harvested on Day 56 for IFNy EL1SPOT.

[0053] FIGURE 4E depicts C57BL / 6J mice were prime-boosted with 5 pg FIII-7 mOVA LNPs and li vers were harvested on Day 56 for IFNγ ELISPOT

[0054] FIGURE 5A depicts an experimental design of ELISA studies, according to embodiments of the present disclosure;

[0055] FIGURE 5B depicts C57BL / 6J mice were prime-boosted with 5 pg FIII-7 mOVA LNPs and plasma was isolated on Day 56 for anti-0 VA IgG antibody ELISA;

[0056] FIGURE 6A depicts an adjuvant incorporation efficiency (IE) assessment of FIII-7 mLuc LNPs, where 2 pg of AF680-labeled 7DW8-5 or non-labeled 7DW8-5 was added directly into the ethanol phase during FIII-7 LNP synthesis and the resulting LNPs were concentrated to 200 pL, and the fluorescence intensity was measured by plate reader (BioTek Synergy H1), excitation at λ = 633 nm, emission at λ = 700 nm,

[0057] FIGURE 6B depicts the adjuvant IE calculated based on the results in FIGURE 6A using the following equation: IE% = (RFU of 200 pL LNPs with 2 pg AF680-labelled 7DW8-5) / (RFU of 200 µL water with 2 µg AF680-labelled 7DW8-5) x 100%;

[0058] FIGURE 6C depicts representative Z-average diameter distributions of the FIII-7 mLuc LNPs with or without 7DW8-5 by dynamic light scattering (DLS);

[0059] FIGURE 6D depicts Z-average size measurements of the FIII-7 mLuc LNPs with or without 7DW8-5 by DLS;

[0060] FIGURE 6E depicts PDI measurements of the FIII-7 mLuc LNPs with or without 7DW8-5 by DLS;

[0061] FIGURE 6F depicts zeta potential measurements of the FIII-7 mLuc LNPs with or without 7DW8-5 by DLS;

[0062] FIGURE 6G depicts mRNA encapsulation efficiency of the FIII-7 mLuc LNPs with or without 7DW8-5, assessed with the Quant-it RiboGreen assay;

[0063] FIGURE 7A depicts an experimental design of immunogenicity and protection studies, according to embodiments of the present disclosure;

[0064] FIGURE 7B depicts BALB / c mice were primed IM or IV with FIII-7 LNPs containing 5 pg of Py circumsporozoite protein mRNA (mCSP) with or without 7DW8-5, where control mice received 0.5 pg 7DW8-.5 in 9,5% trehalose injected IM or IV or nothing (naive), and serum was isolated at 5hpi for IFNy (top) or IL4 (bottom) ELISA;

[0065] FIGURE 7C depicts mice were prime-boosted IM or IV with FIII-7 LNPs containing 5 pg of mCSP with or without 7DW8-5 and then challenged four weeks later with 1x10³ Py spz injected IV and spleens and livers were harvested for ELISPOT at 44hpi.

[0066] FIGURE 7D depicts mice were prime-boosted IM or IV with FIII-7 LNPs containing 5 pg of mCSP with or without 7DW8-5 and then challenged four weeks later with 1x103Py spz injected IV and spleens and livers were harvested for RT-PCR at 44hpi. where FIII-7 protection data is displayed as the percentage of protected mice;

[0067] FIGURE 7E depicts mice were prime-boosted IM or IV with FIII-7 LNPs containing 5 pg of mCSP with or without 7DW8-5 and then challenged four weeks later with 1x103Py spz injected IV and spleens and livers were harvested for RT-PCR at 44hpi, where FIII-7 protection data is displayed as liver parasitemia;

[0068] FIGURE 7F depicts mice were primed IM with ALC-0315 or SM-102 LNPs containing 10 pg mCSP with or without 7DW8-5 and then boosted IV with 10 pg of the same LNPs and challenged as above, where livers were harvested for RT-PCR at 44hpi, and LNP protection data is displayed as the percentage of protected mice;

[0069] FIGURE 7G depicts mice were primed IM with ALC-0315 or SM-102 LNPs containing 10 pg mCSP with or without 7DW8-5 and then boosted IV with 10 pg of the same LNPs and challenged as above, where livers were harvested for RT-PCR at 44hpi, and LNP protection data is displayed as liver parasitemia:

[0070] FIGURE 8A depicts an experimental design of immunogenicity studies, according to embodiments of the present disclosure:

[0071] FIGURE 8B depicts C57BL / 6J mice were primed with 5 pg FIII-7 LNPs containing 5 pg of Ovalbumin mRNA (mOV A) with or without 7DW8-5 and spleens were harvested on Day 7 for IFNγ ELISPOT, where adjuvanted groups show N=3 C57BL / 6 mice from one experiment, and where non-adjuvanted groups are copied here from FIGURE 4A-4D for comparison;

[0072] FIGURE 8C depicts an experimental design of immunogenicity studies, according to embodiments of the present disclosure;

[0073] FIGURE 8D depicts C57BL / 6J mice were prime-boosted with 5 µg FIII-7 mOVA LNPs and spleens were harvested on Day 56 for IFNγ ELISPOT, where adjuvanted groups show N=3 C57BL / 6 mice from one experiment, and where non-adjuvanted groups are copied here from FIGURE 4A-4D for comparison;

[0074] FIGURE 8E depicts C57BL / 6J mice were prime-boosted with 5 pg FIII-7 mOVA LNPs and livers were harvested on Day 56 for IFNγ ELISPOT, where adjuvanted groups show N=3 C57BL / 6 mice from one experiment, and where non-adjuvanted groups are copied here from FIGURE 4A-4D for comparison;

[0075] FIGURE 9A depicts an experimental design of protection studies, according to embodiments of the present disclosure;

[0076] FIGURE 9B depicts BALB / cJ control mice received 1 pg 7DW8-5 in 9.5% trehalose injected IM or IV or nothing (naive) on Days 0 and 28, and mice were then challenged four weeks later with 1x103 Py spz injected IV and monitored for the presence of blood stage parasites by Giemsa-stained thin blood smear microscopy for 14 days;

[0077] FIGURE 10A depicts an experimental design of immunogenicity studies, according to embodiments of the present disclosure;

[0078] FIGURE 10B depicts C57BL / 6J mice were primed IM or IV with 5 pg ALC-0315 (left) or SM-102 (right) LNPs containing 5 pg of Ovalbumin mRNA (mOVA) with or without 7DW8-5 and spleens were harvested on Day 7 for IFNγ ELISPOT;

[0079] FIGURE 10C depicts an experimental design of immunogenicity studies, according to embodiments of the present disclosure;

[0080] FIGURE 10D depicts BALB / cJ mice were IM primed and boosted IM or IV with ALC-0315 or SM-102 LNPs containing 10 pg CSP mRNA(mCSP) with or without 7DW8-5 and then challenged one month later, and spleens and livers were harvested for ELISPOT at 44hpi;

[0081] FIGURE 10E depicts BALB / cJ mice were IM primed and boosted IM or IV with ALC-0315 or SM-102 LNPs containing 10 pg CSP mRNA (mCSP) with or without 7DW8-5 and then challenged one month later, and spleens and livers were harvested for RT-PCR at 44hpi, where LNP protection data is displayed as liver parasitemia; and

[0082] FIGURE 11 depicts a schematic illustration of a method of producing lipid nanoparticles, according to embodiments of the present disclosure.DETAILED DESCRIPTION

[0083] To address these and related challenges, the present disclosure provides, in various aspects lipid nanoparticles (LNP) including a plurality of lipids and a glycolipid adjuvant, wherein the glycolipid adjuvant is structurally integrated into a core of the LNP during formation of the LNP, a composition comprising any of the LNPs described herein and a pharmaceutically acceptable carrier, a method of producing a pharmaceuticalcomposition, the method including: combining a plurality of lipids configured to generate a lipid nanoparticle, thereby forming a lipid phase; introducing a glycolipid into the lipid phase; and mixing the lipid phase with nucleic acids in an in-line mixing device, and a method of immunizing a subject against a parasite, the method including administering to the subject a therapeutically effective dose of any of the LNPs described herein.

[0084] mRNA LNP vaccines are attractive for malaria since large-scale production has been demonstrated, they have precedent as antibody-inducing vaccines in humans, and LNPs can be selectively optimized for liver-targeted expression. A liver-targeting mRNA LNP vaccine for malaria using the glycolipid adjuvant 7DW8-5 is here described. 7DW8-5 is a synthetic glycolipid that stimulates invariant natural killer T (iNKT) cells and enhances both humoral and CD8+ T cell responses to vaccination. 7DW8-5 has previously been shown to increase efficacy of malaria vaccines in mice and non¬ human primates. Incorporation of the 7DW8-5 into mRNA LNPs does not significantly change LNP characteristics and prime-boost immunization with 7DW8-5 adjuvanted mRNA LNPs improves protection against P. yoelii spz challenge. Advantageously, incorporation of a glycolipid adjuvant according to the present disclosure does not alter LNP physicochemical properties at the effect dose range.

[0085] As used herein, the terms "ionizable cationic lipid" and "ionizable lipids" refers to ionizable lipids that are positively charged at acidic pH to condense nucleic acids into lipid nanoparticles. Ionizable cationic lipids are neutral at physiological pH to minimize toxicity. Representative ionizable cationic lipids include, but are not limited to, unsaturated ionizable lipids, including DLin-MC3-DMA, OF-02, A6, and A18-Iso5-2DCI8; multi-tail ionizable lipids, including 98N12-5, C12-200, cKK-E12, and 9A1P9; ionizable polymeric lipids, including 7C1 and G0-C14; biodegradable ionizable lipids, including L319, 304013, OF-Deg-Lin, and 306-012B; and branched tail ionizable lipids, including 306OH0 and FTT5. Other ionizable lipids suitable for use with the presently disclosed LNPs include SM-102, ALC-0315, A9, 2,2(8, 8)4C CH3, and LP01. See, for example. Han et al., An ionizable lipid toolbox for RNA delivery. Nature Communications, 12:7233 (2021), which is incorporated herein by reference in its entirety. In particular embodiments, the ionizable cationic lipid comprises Dlin-MC3-DMA, SM-102, or ALC-0315.

[0086] As used herein, "helper lipid" refers to a lipid component that facilitates the formation and structural stability of lipid nanoparticles Helper lipids may beelectrostatically neutral, cationic, anionic, or zwitterionic, depending on their charge state at the physiological pH. Representative helper lipids include, but are not limited to, neutral and zwitterionic phospholipids such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), di oleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); cationic lipids such as 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dimethyldioctadecylammonium bromide (DDAB); anionic lipids such as 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA), 1,2-distearoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (18PG), and 1,2-distearoyl-sn-glycero-3-phospho-(l'-rac-glycerol-bis(monoacylglycerol)) (18BMP); as well as synthetic variants and derivatives thereof.

[0087] As used herein, a "sterol" refers to cholesterol or a cholesterol derivative that serves as a polycyclic lipid component that modulates membrane stability, rigidity, and permeability of lipid nanoparticles, and regulates particle fluidity and structural integrity during formulation, storage, and delivery. Representative sterols include, but are not limited to, cholesterol, phytosterols, cholesterol derivatives, sitosterol, ergosterol, stigmasterol, campesterol, and synthetic sterol analogs.

[0088] As used herein, a " PEGylated lipid" refers to a lipid conjugated to polyethylene glycol (PEG) that provides a hydrophilic coating on the surface of lipid nanoparticles to reduce particle aggregation, enhance colloidal stability, extend circulation time in vivo, minimize non-specific protein binding and opsonization, and reduce immune recognition. Representative PEGylated lipids include, but are not limited to, DMG-PEG, DSPE-PEG, PEG-ceramide. ALC-0159, and variants with varying PEG molecular weights (e.g., PEG-200, PEG-500, PEG- 1000, PEG-2000, PEG-5000) and lipid anchor structures.

[0089] As used herein, a "glycolipid adjuvant" refers to a lipid component comprising one or more carbohydrate moieties that enhances the immunogenicity of the lipid nanoparticle formulation, modulates immune responses, facilitates antigen presentation, activates pattern recognition receptors, stimulates innate immune pathways, or targets specific immune cell populations to improve vaccine efficacy or therapeutic outcomes. Glycolipid adjuvants may function through various mechanisms including, but not limited to, activation of natural killer T cells, stimulation of toll-like receptors, enhancement of cytokine production, promotion of dendritic cell maturation, or facilitation of cross-presentation of antigens. Representative glycolipid adjuvants include, but are notlimited to, ionizable amino glycolipids such as 7DW8-5 and related structural analogs, monophosphoryl lipid A (MPLA) and lipid A derivatives: alpha-galactosylceramide (a-GalCer) and glycosphingolipid variants; gangliosides including GM1, GM2, and GM3; trehalose-based glycolipids such as trehalose dibehenate (TDB) and trehalose dimycolate (TDM); glucosylceramides; galactosylceramides; mannosylated lipids; synthetic glycolipid derivatives: and combinations thereof. The glycolipid adjuvant may be cationic, anionic, zwitterionic, or neutral at physiological pH, and may contain varying numbers of sugar residues, lipid tails of different chain lengths and saturation levels, and functional groups that modulate immunostimulatory activity.

[0090] A "therapeutically effective dose" refers to refers to the amount of a therapeutic agent (i. e., drug, or therapeutic agent composition) that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following:

[0091] (1 ) preventing the condition; for example, preventing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease;

[0092] (2) inhibiting the condition; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology' or symptomatology of the disease, condition or disorder: and

[0093] (3) ameliorating the condition; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology' or symptomatology' of the disease, condition or disorder (i.e., reversing the pathology’ and / or symptomatology) such as decreasing the severity of condition.

[0094] As used herein, "pharmaceutically acceptable" means suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use within the scope of sound medical judgment.

[0095] A thorough discussion of pharmaceutically acceptable excipients and salts is available in Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990),

[0096] As used herein, the term "individual," "subject." or "patient," used interchangeably, refers to any animal, including mammals, preferably mice, rats, otherrodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.

[0097] As used herein, "incorporation efficiency" refers to the percentage of a composition that is successfully integrated into the structure of a lipid nanoparticle. In some embodiments, integration may be made into the core of the lipid nanoparticle.LIPID NANOPARTICLES

[0098] In an aspect, the present disclosure provides a lipid nanoparticle (LNP) including a plurality of lipids and a glycolipid adjuvant, wherein the glycolipid adjuvant is structurally integrated into a core of the LNP during formation of the LNP.

[0099] In this regard. FIGURE 1 is a schematic illustration of a LNP 100 according to embodiments of the present disclosure. LNP 100 includes a lipid structure 110 which includes a hydrophilic component 112 and a hydrophobic component 114. In some embodiments, the lipid structure 110 is composed of a plurality of lipids, such as where the plurality' of lipids includes an ionizable lipid, a helper lipid, a sterol, and a PEGylated lipid.

[0100] Within the hydrophobic component 114 is core 120, where sub-regions of core 120 encapsulate a cargo 130. In some embodiments, the cargo 130 may be an antigenic factor, a nucleic acid, or a combination thereof.

[0101] Embedded within the lipid structure 110 is a glycolipid adjuvant 140. While FIGURE 1 depicts the glycolipid adjuvant 140 as being integrated only within lipid structure 110, it should be understood that the glycolipid adjuvant 140 may be integrated into other portions of the lipid layers of the LNP 100, including within the lipid layers forming the core 120 of the LNP 100.

[0102] In some embodiments, the plurality of lipids comprises an ionizable lipid, a helper lipid, a sterol, and a PEGylated lipid.

[0103] In some embodiments, the LNP further includes an antigenic factor

[0104] In some embodiments, the LNP further includes a nucleic acid encoding one or more polypeptides.

[0105] In some embodiments, the one or more polypeptides comprise an antigen.

[0106] In some embodiments, the glycolipid adjuvant is present in an amount of between about 0.1 pg to about 2.5 pg glycolipid adjuvant per 10 pg nucleic acid.

[0107] In some embodiments, the glycolipid adjuvant is present in an amount of between about 0.4 pg to about 2.0 pg glycolipid adjuvant per 10 pg nucleic acid.

[0108] In some embodiments, the nucleic acid is selected from the group consisting of an mRNA, a circRNA, a saRN A. and a DNA.

[0109] In some embodiments, the nucleic acid is mRNA.

[0110] In some embodiments, the glycolipid adjuvant is 7DW8-5,[0111| In some embodiments, the ionizable lipid is selected from the group consisting of Dlin-MC3-DMA, SM-102, and ALC-0315.

[0112] In some embodiments, the helper lipid is selected from the group consisting of 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG), 1,2- distearoylsn-glycero-3-phosphocholine (DSPC), 1.2-dioleoyl-3-trimethylammonium- propane (DOT AP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), dimethyldioctadecyl ammonium (DDAB), 1.2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA), and sn- (3-oleoyl-2-hydroxy)-glycerol-l-phospho-sn-3'-(l',2'-dioleoyl)-glycerol (ammonium salt) (18BMP).

[0113] In some embodiments, the sterol is a cholesterol.

[0114] In some embodiments, the PEGylated lipid is selected from the group consisting of DMG-PEG200 and ALC-0159.

[0115] In some embodiments, a molar ratio of the ionizable lipid in the lipid nanoparticle is in the range of about 30% to about 55%.

[0116] In some embodiments, a molar ratio of the helper lipid in the lipid nanoparticle is in the range of about 5% to about 10%.

[0117] In some embodiments, a molar ratio of the sterol in the lipid nanoparticle is in the range of about 38% to about 45%.

[0118] In some embodiments, a molar ratio of the PEGylated lipid in the lipid nanoparticle is in the range of about 0.05% to about 2%.

[0119] In some embodiments, the glycolipid adjuvant has a 70% or greater incorporation efficiency into the core of the LNP.

[0120] In an aspect, the present disclosure provides a composition comprising any of the LNPs described herein and a pharmaceutically acceptable earner.METHODS OF PRODUCING NANOPARTICLES AND METHODS OF USE

[0121] In an aspect, the present disclosure provides a method of producing a pharmaceutical composition, the method including: combining a plurality of lipids dissolved in a water-miscible organic solvent, thereby forming a lipid phase; introducing a glycolipid into the lipid phase; and mixing the lipid phase with nucleic acids dissolved in an aqueous phase in an in-line mixing device.

[0122] Materials & Methods:

[0123] Mice-.

[0124] Female BALB / cJ or C57BL / 6J mice were purchased at 4-8 weeks old (Jackson Laboratories, Barr Harbor, ME). Mice were housed at the University of Washington in an Institutional Animal Care and Use Committee-approved animal facility and were used under an approved protocol (4317-01 to S. C. M.).

[0125] mRNA:

[0126] mRNA encoding firefly luciferase (mLuc, L-7602) and ovalbumin (mOVA, #L-7610) were purchased from TriLink BioTechnologies (San Diego, CA). mRNA encoding full-length Plasmodium yoelii (Py) CSP protein without the major repeat region (SEQ ID No. I) was custom synthesized at TriLink utilizing the same CleanCap® technology as the aforementioned commercial mRNAs, as described in Watson, F N., et al., Ultra-low volume intradermal administration of radiation-attenuated sporozoites with the glycolipid adjuvant 7DW8-5 completely protects mice against malaria. Res Sq, 2023 (" Watson"), the contents of which are incorporated by reference herein. The full-length Py CSP sequence is depicted in TABLE 1 in SEQ ID No 2, with the major repeat region that is omitted from SEQ ID No 1 depicted as SEQ ID No. 3.

[0127] TABLE 1; CSP Protein Sequence ListingsSEQ ID No. Listing1 MKKCTILVVASLLLVDSLLPGYGQNKSVQAQRNLNELCYNEEND NKLYHVLNSKNGKIYNRNIVNRLLGDALNGKPEEKKDDPPKDGN KDDLPKEEK1WDLPKEEKKDDPPKDPKKDDPPKEAQNKLNQPVV ADENVDQPRPQPDGNNNNNNNNGNNNEDSYVPSAEQILEFVKQIS SQLTEEWSQCSVTCGSGVRVRKRKNVNKQPENLTLEDIDTEICKM DKCSSIFNIVSNSLGFVILLVLVFFN2 MKKCTILVVASLLLVDSLLPGYGQNKSVQAQRNLNELCYNEEND NKLYHVLNSKNGKIYNRNIVNRLLGDALNGKPEEKKDDPPKDGN KDDLPKEEKKDDLPKEEKKDDPPKDPKKDDPPKEAQNKLNQPVV ADENVDQGPGAPQGPGAPQGPGAPQGPGAPQEPPQQPPQQPPQQP PQQPRPQPDGNNH^JNNNWsTGNNNEDSYVPSAEQILEFVKQISSQLT EEWSQCSVTCGSGVRVRKRKNVNKQPENLTLEDIDTEICKMDKCSSIFNIVSNSLGFVILLVLVFFN3 GPGAPQGPGAPQGPGAPQGPGAPQEPPQQPPQQPPQQPPQQ

[0128] 7DW8-5:

[0129] 7DW8-5 and Alexa Fluor 680-labeled 7DW8-5 (as described by Li, X., et al., Colocalization of a CDld-Binding Glycolipid with a Radiation-Attenuated Sporozoite Vaccine in Lymph Node-Resident Dendritic Cells for a Robust Adjuvant Effect. J Immunol, 2015. 195(6): p 2710-21) (AF680; Molecular Probes, Carlsbad, CA) were prepared as described in Watson, the contents of which are incorporated herein by reference. Mice received 0.5 pg 7DW8-5 per 5 pg mRNA via intravenous (IV) or intramuscular (IM) injection.

[0130] Lipid nanoparticles (LNPs):

[0131] Lipids were purchased, and LNPs were synthesized as described in Zhu, Y., et al., Multi-step screening of DNA / lipid nanoparticles and co-delivery with siRNA toenhance and prolong gene expression, Nat Commun, 2022. 13(1): p. 4282 (" Zhu I"), the contents of which are incorporated herein by reference. Briefly, a lipid phase was prepared in 100% ethanol by combining four lipids at a predetermined molar ratio: an ionizable lipid (DLin-MC3-DMA, MedKoo Biosciences; SM-102 or ALC-0315, Broadpharm), a helper lipid (18PG or DSPC, Avanti Polar Lipids), cholesterol (Sigma- Aldrich), and a PEGylated lipid (DMG-PEG2000, Avanti Polar Lipids; ALC-0159, Broadpharm) (TABLE 2).

[0132] TABLE 2. Composition details of three exemplary LNP formulations. LNP formulation FIII-7 SM-102 ALC-0315 Ionizable lipid DLin-MC3-DMA SM-102 ALC-0315 Helper lipid 18PG DSPC DSPCSterol Cholesterol Cholesterol Cholesterol PEGylated lipid DMG-PEG 2000 DMG-PEG 2000 ALC-0159 Lipid molar ratio (%) 5455: 5.45: 39.92: 0.08 50: 10: 38.5: 1.5 46.3: 9.4: 42.7: 1.6N / P ratio 8 6 6

[0133] For 7DW8-5-adjuvanted LNPs, the glycolipid was added as a fifth lipid component to the lipid phase (1 pg 7DW8-5 per 10 pg mRNA). The mRNA phase was prepared by diluting mRN A in 25 mM magnesium acetate buffer (pH 4.0, Fisher). All mRNAs were stored at ~80°C and were allowed to thaw on ice before use. The mRNA and lipid phases were mixed at a 3:1 volume ratio in a flash nanocomplexation device using syringe pumps (i.e., an "in-line mixing device") and then dialyzed against deionized (DI) water in a 100 kDa molecular weight cut-off cassette (Fisher) at 4°C for 24 hours. The final LNP solution was ultrafiltered using a 100-kDa MWCO centrifugal filter (Amicon, #UFC510096) to the desired concentration. Size, polydispersity index, and zeta potentials of LNPs were measured using dynamic light scattering (DLS) (ZetaPALS, Brookhaven Instruments). Diameters are reported as the intensity mean average. Encapsulation efficiency of the mRNA LNPs was assessed with the Quant-it RiboGreen assay (Fisher, R11490) as described by Zhu, Y., et al., Screening for lipid nanoparticles that modulate the immune activity of helper T cells towards enhanced antitumour activity Nat Biomed Eng, 2024. 8(5): p. 544-560 (" Zhu II"), the contents of which are incorporated by reference herein. LNPs were stored in 9.5% (w / v) trehalose solution at -80°C until use.

[0134] In some embodiments, the in-line mixing device is selected from a group of micro-fluid mixers, 2 -inlet confined impinging jet mixers, 3-inlet confined impingingjet mixers, 4-inIet confined impinging jet mixers, multi-inlet vortex mixers, and T-junction mixers.

[0135] In some embodiments, the plurality of lipids comprises an ionizable lipid, a helper lipid, a sterol, and a PEGylated lipid.

[0136] In some embodiments, the nucleic acids are selected from the group consisting of an mRNA, a circRNA, a saRNA, and a DNA.

[0137] In some embodiments, a molar ratio of ionizable lipid in the lipid nanoparticle is in the range of about 30% to about 55%; a molar ratio of the helper lipid in the lipid nanoparticle is in the range of about 5% to about 10%; a molar ratio of the sterol in the lipid nanoparticle is in the range of about 38% to about 45%; a molar ratio of the PEGylated lipid in the lipid nanoparticle is in the range of about 0.05% to about 2%; and wherein the glycolipid adjuvant is present in an amount of between about 0.1 pg to about 2.5 pg glycolipid adjuvant per 10 pg nucleic acid.

[0138] IVIS:

[0139] Live in vivo imaging of Luc protein expression following mLuc LNP administration was performed as described by Olsen, T. M., et al, Prime-and-Trap Malaria Vaccination To Generate Protective CD8 - Liver-Resident Memory T Cells. 'lire Journal of Immunology, 2018. 201(7): p. 1984-1993 (" Olsen''), the contents of which are incorporated by reference herein Briefly, bioluminescence was evaluated by intraperitoneal administration of 50 mg / mL D-luciferin (Gold Biotechnology) followed by isoflurane anesthesia and In Vivo Imaging System (IVIS) imaging. IVIS images were evaluated using Living Image 3,0 software (Perkin-Elmer) with regions of interest (RO I) placed around the whole body, liver, or thigh.

[0140] Liver leukocyte and splenocyte ELISPOT

[0141] Liver leukocytes and splenocytes were isolated as described by Olsen. Briefly, non-perfused livers were mashed through 100 pm filters and centrifuged through a 35% Percoll gradient (GE Healthcare) to isolate leukocytes. Spleens were similarly mashed through a filter. All cells were subject to ammonium-chlori de-potassium lysis, and final pellets were resuspended in RPMI 1640 supplemented with glutamine and 1 % fetal bovine serum.

[0142] OVA (SIINFEKL) and PyCSP (SYVPS AEQI) peptides were synthesized by Genemed Synthesis (San Francisco, CA) and reconstituted in DMSO. Mouse IFNy ELISPOT (eBioscience, San Diego, CA) was conducted by stimulating cells with peptide(or DMSO control) at I pg / mL for ~18 hours at 37°C and developed following manufacturer guidelines as described by Watson and Olsen. Tire number of spot-forming units (SFU) was determined using an ImmunoSpot 5.1 Analyzer (Cellular Technology Limited). SFU were normalized to DMSO wells, and SFU per million cells were reported.

[0143] ELISA:

[0144] Serum and plasma were isolated from mice and frozen at ~~80°C prior to testing. Interferon-y (IFNy) and Interleukin 4 (IL4) serum cytokines were measured with commercial ELISA kits according to manufacturer's instructions (BioLegend, #430801 and #431104). Anti-OVA Immunoglobulin G (IgG) plasma antibody titers were measured with a commercial ELISA kit according to manufacturer's instructions (Chondrex. #3011).

[0145] Plasmodium yoelii challenge sporozoites:

[0146] Mice were challenged IV with Ixl 03wild-type infectious Py spz that were either freshly -dissected (University of Washington Tnsectaiy, Seattle, WA) or cryopreserved (Sanaria Inc, Rockville, MD). Challenged mice were sacrificed at 44 hours post infection for liver Plasmodium 18S RT-PCR analysis.

[0147] Parasite burden reverse transcription polymerase chain reaction (RT- PCR):[0148| Liver parasitemia was calculated as described by Watson. Briefly, livers were bead beaten in NucliSENS lysis buffer (bioMerieux). total nucleic acids were extracted, and RT-PCR was conducted using the SensiFAST™ Probe Lo-ROX Kit (Bioline) using pnmers / probes specific for mouse GAPDH mRNA and pan-Plasmodium 18S rRNA. Plasmodium 18S rRNA copy numbers per liver were determined using a standard curve generated from a custom lot of quantified Armored RNA encoding full-length Plasmodium 18S rRNA (zksuragen, Austin, TX). Mice were considered sterilely protected if no cycle threshold (CT) was generated during 40 cycles of RT-PCR.

[0149] Statistics:

[0150] Data were analyzed with non-parametric Mann- Whitney test or Kruskal-Wallis test with Dunn's multiple comparisons unless otherwise stated in the figure legend Error bars represent the standard deviation (SD) of the mean with individual mouse samples shown unless otherwise stated in the figure legend. P values >0.05 were considered nonsignificant. GraphPad Prism 9.1.2 Software (San Diego, CA) was used for calculations,

[0151] Results and Discussion:

[0152] A DNA LNP formulation " FIII-7" was identified to selectively target the liver following I V administration. As mRNA technology is more advanced, an mRNA Fill- 7 LNP vaccine administered via the more translational IM route was investigated. To assess protein expression after vaccination, mice were vaccinated IV or IM with luciferase mRNA (mLuc) LNPs, and bioluminescence was evaluated using IVIS at 24-and-48 hours postinjection (hpi) (FIGURE 2A-2D).

[0153] FIGURE 2A depicts experimental design of IVIS studies. FIGURE 2B-2D depict I VIS imaging of mice 24 (left) or 48 (right) hours post vaccination with FIII-7 LNPs containing 5 pg of Luciferase mRNA (mLuc) injected IV or IM. Regions of Interest (ROD were drawn around the entire mouse body (FIGURE 2B), the primary expression site (thigh for IM or liver for IV) (FIGURE 2C), or the liver (FIGURE 2D). Error bars represent the 95% CI of the median of N=7-10 BALB / cJ mice across at least two independent experiments. IVIS data was analyzed with Kruskal-Wallis test and Dunn's multiple comparisons test, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns p>0.05. hpi=hours post injection. IM=mtramuscular. lV=intravenous.

[0154] Accordingly, in an aspect, the present disclosure provides a method of immunizing a subject against a parasite, the method comprising administering to the subject a therapeutically effective dose of any of the LNPs described herein.

[0155] In some embodiments, the LNP is administered via intravenous (IV) injection.

[0156] In some embodiments, the LNP is administered via intramuscular (IM) injection

[0157] In some embodiments, the parasite is Plasmodium.[0158| In some embodiments, immunizing comprises inducing liver-resident immunity leading to sterile protection against parasites

[0159] IV injection was found to have induced high liver expression at 24 hpi, which significantly declined by 48 hpi. Following IM injection, expression was primarily at the injection site with very' low expression in the liver. Expression levels at the IM injection site did not significantly decline from 24 to 48 hpi (FIGURE 3A-3D).

[0160] FIGURE 3A depicts an experimental design of IVIS studies. FIGURES 3B-3D depict IVIS imaging of mice 24 or 48 hours post vaccination with FIII-7 LNPs containing 5 pg of Luciferase mRNA (mLuc) injected IV or IM. Regions of Interest (ROI) were drawn around the entire mouse body (FIGURE 3B), the primary expression site (thighfor IM or liver for IV) (FIGURE 3C). or the liver (FIGURE 3D). Error bars represent the 95% CI of the median of N=7-10 BALB / cJ mice across at least two independent experiments IVIS data was analyzed with Mann-Whitney Tests, **p<0.0L *p<0.05. ns p>0.05. hpi==hours post injection. IM=intramuscular. IV=intraveneous.

[0161] Thus, the biodistribution of protein expression differs between IV- and IM-injected mRNA FIII-7 LNP vaccines.

[0162] Next, whether IV and IM mRNA FIII-7 LNPs could effectively prime and boost CD8+T cells and induce antibodies was determined. Mice were immunized IV or IM with ovalbumin mRNA (mOVA) LNPs, and cellular responses were evaluated at seven days by ELISPOT (FIGURE 4A) and at 56 days (FIGURE 4C).

[0163] In this regard. FIGURE 4A and 4C depict experimental design of immunogenicity' studies. C57BL / 6J mice were primed with 5 µg FIII-7 LNPs containing 5 pg of Ovalbumin mRNA (mOVA) and spleens were harvested on Day 7 for IFNγ ELISPOT (FIGURE 4B) or mice were prime-boosted with 5 pg FIII-7 mOVA LNPs (FIGURE 4C) and spleens (FIGURE 4D) or livers (FIGURE 4E) were harvested on Day 56 for IFNγ ELISPOT. Error bars represent the 95% CI of the median of the mean of N=8 C57BL / 6J mice across two independent experiments. ELISPOT data was analyzed with Mann-Whitney Test, ns p>0.05. IM=intramuscular. IV=intravenous.

[0164] Surprisingly, both immunization routes induced similar numbers of antigen-specific CD8+T cells in the spleen at seven days (FIGURE 4B). The same trend was observed in the spleens and livers of primed and boosted mice at a memory' timepoint (FIGURE 4C-4E).

[0165] Interestingly, only IV injection reliably induced anti-0 V A IgG antibodies (FIGURE 5A-5B).

[0166] In this regard, FIGURE 5A depicts an experimental design of ELISA studies for IV administered FIII-7 mOVA to induce anti-OVA IgG antibodies. FIGURE 5B depicts C57BL / 6J mice were prime-boosted with 5 pg FIII-7 mOVA LNPs and plasma was isolated on Day 56 for anti-OVA IgG antibody ELISA. The median is shown for N=10 mice from two independent experiments. ELISA data was analyzed with Mann- Whitney Test, ***p<0.001. IM=intramuscular. IV=intraveneous.

[0167] Thus, despite inducing lower liver expression, both IV- and IM-injecled mRNA FIII-7 LNPs can effectively prime in the periphery and induce CD8+memory T cells

[0168] A glycolipid-adjuvanted mRNA LNP vaccine was then tested. Since 7DW8-5 is a lipid, adjuvanted LNPs were constructed by directly adding the glycolipid into the lipid phase during LNP production. The incorporation of 7DW8-5 into the LNP is advantageous as it facilitates co-delivery of antigen-encoding mRNA and adjuvant to the same antigen-presenting cells. To assess incorporation efficiency, an AF680-labeled 7DW8-5 was used to find that >75% of the adjuvant was incorporated into mLuc LNPs (FIGURES 6A-6B).

[0169] In this regard, FIGURES 6A and 6B depict adjuvant incorporation efficiency (IE) assessments of FIII-7 mLuc LNPs. 2 ug of AF680-labeled 7DW8-5 or non-labeled 7DW8-5 was added directly into the ethanol phase during FIII-7 LNP synthesis and the resulting LNPs were concentrated to 200 pL. The fluorescence intensity was measured by plate reader (BioTek Synergy H1), excitation at X = 633 nm, emission at = 700 nm (FIGURE 6A), and the adjuvant IE was calculated (FIGURE 6B) using the following equation: IE% = (RFU of 200 pL LNPs with 2 ug AF680-labelled 7DW8-5) / (RFU of 200 pL water with 2 pg AF680-labelled 7DW8-5) x 100%. Next, the Z-average diameter distributions, Z-average size, poly dispersity index (PDI), zeta potential, and mRNA encapsulation efficiency of mLuc LNPs with or without incorporated 7DW8-5 were compared and found no significant differences (FIGURE 6C-6G). In this regard, FIGURE 6C depicts representative Z-average diameter distributions of the FIII-7 mLuc LNPs with or without 7DW8-5 by dynamic light scattering (DLS). FIGURES 6D-6F depict Z-average size (FIGURE 6D), PDI (FIGURE 6E), and zeta potential (FIGURE 6F) measurements of the FIII-7 mLuc LNPs with or without 7DW8-5 by DLS. FIGURE 6G depicts mRNA encapsulation efficiency of the FIII-7 mLuc LNPs with or without 7DW8-5, assessed with the Quant-it RiboGreen assay. Error bars represent the SD of the mean of N=6 from two independent experiments. Fluorescent intensity data was analyzed with one-way ANOVA and Tukey’s multiple comparison tests. All other data was analyzed with an unpaired / -test, ****p < 0.0001, ns p>0.05.

[0170] Thus, 7DW8-5 can be incorporated into mRNA FIII-7 LNPs without altering the physical properties or size characteristics

[0171] Next, whether 7DW8-5-adjuvanted mRNA FIII-7 LNPs would result in typical cytokine induction was determined. IV 7DW8-5 induces systemic IFNy and IL4 responses, while IM 7DW8-5 does not. To test cytokines responses, mice were primed with Py circumsporozoite protein mRNA (mCSP) LNPs with or without 7DW8-5, and serumwas collected five hpi for ELISA (FIGURE 7 A). Only FIII-7 LNPs with 7DW8-5 were found to induce cytokines after IV but not IM injection (FIGURE 7B). Further, LNPs without adjuvant did not induce detectable levels of IFNy or IL4.

[0172] In this regard, FIGURE 7A depicts an experimental design of immunogenicity and protection studies. FIGURE 7B depicts BALB / cJ mice were primed IM or IV with FIII-7 LNPs containing 5 pg of Py circumsporozoite protein mRNA (mCSP) with or without 7DW8-5. Control mice received I pg 7DW8-5 in 9.5% trehalose injected IM or IV or nothing (naive). Serum was isolated at 5hpi for IFNy (FIGURE 7B, top) or IL4 (FIGURE 7B, bottom) ELISA. Error bars represent the 95% CI of the median of N=4-6 mice across two independent experiments. ELISA data was analyzed with Mann- Whitney tests.

[0173] Next, whether mCSP FIII-7 LNPs with or without 7DW8-5 could protect mice against Py spz challenge was evaluated. Mice were prime-boosted IV or IM and challenged four weeks later (FIGURE 7A). Despite the induction of strong immune responses by unadjuvanted LNPs (FIGURE 7C and FIGURE 8A-8E), there was no sterile protection in any of the groups receiving unadjuvanted LNPs regardless of the vaccination route (FIGURE 7D and 7E). In this regard, FIGURE 7C-7E depict results when mice were prime-boosted IM or IV with FIII-7 LNPs containing 5 pg of mCSP with or without 7DW8-5 and then challenged four weeks later with 1x103 Py spz injected IV and spleens and livers were harvested for ELISPOT (FIGURE 7C) or RT-PCR (FIGURES 7D-7E) at 44hpi. FIII-7 protection data is displayed as the percentage of protected mice (FIGURE 7D) and as liver parasitemia (FIGURE 7E).

[0174] FIGURES 7F-7G depict mice were primed IM with ALC-0315 or SM-102 LNPs containing 10 pg mCSP with or without 7DW8-5 and then boosted IV with 10 pg of the same LNPs and challenged as above. Livers were harvested for RT-PCR at 44hpi. LNP protection data is displayed as the percentage of protected mice (FIGURE 7F) and as liver parasitemia (FIGURE 7G). The median is shown for N=6-15 mice across 2-3 independent experiments, RT-PCR data was analyzed with Kruskal-Wallis test, Protection data was analyzed with Fisher Exact tests, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns p>0.05. Open circles=cryopreserved challenge PyWT sporozoites. hpr=hours post injection.

[0175] Similarly, FIGURES 8A and 8C depict an experimental design of immunogenicity studies. C57BL / 6J mice were primed with 5 pg FIII-7 LNPs containing 5g of Ovalbumin mRNA (mOVA) with or without 7DW8-5 and spleens were harvested on Day 7 for IFNγ ELISPOT (FIGURE 8B) or mice were prime-boosted with 5 pg FIII-7 mOVA LNPs (FIGURE 8C) and spleens (FIGURE 8D) or livers (FIGURE 8E) were harvested on Day 56 for IFNγ ELISPOT. Error bars represent the 95% CI of the median. Adjuvanted groups show N=3 C57BL / 6 mice from one experiment. Non-adjuvanted groups are copied here from FIGURE 3 A-3D for comparison. ELISPOT data was analyzed with Kruskal-Wallis test and Dunn's multiple comparisons test, ns p>0.05.

[0176] Thus, sterile protection was found only in groups that received 7DW8-5 adjuvanted LNPs by either route, and adjuvant alone did not induce any protection (FIGURE 9A-9B).

[0177] In this regard. FIGURE 9A depicts an experimental design of protection studies. FIGURE 9B depicts BALB / cJ control mice received I pg 7DW8-5 in 9.5% trehalose injected IM or IV or nothing (nai ve) on Days 0 and 28. Mice were then challenged four weeks later with 1x103 Py spz injected IV and monitored for the presence of blood stage parasites by Giemsa-stained thin blood smear microscopy for 14 days. Data is from N=4-10 mice across two independent experiments, ns p>0.05. hpi=hours post injection rhe IV -administered vaccine showed higher rates of sterile protection than the IM- administered vaccine.

[0178] Lastly, whether 7DW8-5 could enhance other LNP compositions as an adjuvant platform-enabling technology was tested. LNP compositions were selected based on two FDA-approved formulations: SM-102 and ALC-0315 (formulations used in the Modema and Pfizer-BioNTech COVID-19 vaccines, respectively) (TABLE 2). mCSP LNPs with or without 7DW8-5 were constructed and mice were primed IM, boosted IM or IV, and challenged as before. While SM-102 LNPs did not show adjuvant-induced enhancement of protection, 7DW8-5-containing ALC-0315 LNPs did increase protection after IV boosting (FIGURE 7F-7G and FIGURE 10A-10E).

[0179] Similarly, FIGURE 10A-10E depict ALC-0315 and SM-102 giycolipid-incorporating LNPs are immunogenic in the spleen.

[0180] In this regard, FIGURES 10A and 10C depict an experimental design of immunogenicity studies. In FIGURE 10B, C57BL / 6J mice were primed IM or IV with 5 pg ALC-0315 (FIGURE 10B, left) or SM-102 (FIGURE 10B, right) LNPs containing 5 pg of Ovalbumin mRNA (mOVA) with or without 7DW8-5 and spleens were harvested on Day 7 for IFNγ ELISPOT. Error bars represent the 95% CI of the median of N=6-7C57BL / 6J mice from two independent experiments. In FIGURE 10C. BALB / cJ mice were IM primed and boosted IM or IV with ALC-0315 or SM-102 LNPs containing 10 pg CSP mRNA (mCSP) with or without 7DW8-5 and then challenged one month later. Spleens and livers were harvested for ELISPOT (FIGURE 10D) or RT-PCR (FIGURE 10E) at 44hpi. LNP protection data is displayed as liver parasitemia (FIGURE 10E). Error bars represent the 95% CI of the median of N=4-10 BALB / cJ mice from 1-3 experiments. Data was analyzed with Kruskal-Wallis test and Dunn's multiple comparisons test, ns p>0.05, *p<0.05. hpi=hours post injection. Open circles=cryopreserved PyWT challenge sporozoites. IM=intramuscular. IV=intraveneous.

[0181] Taken together, these data demonstrate a novel glycolipid-adjuvanted LNP by direct addition of glycolipids as a fifth lipid in LNP manufacturing. FII-7 and ALC-0315 adjuvanted-LNPs conferred more protection against spz challenge than unadjuvanted LNPs.EXAMPLE PRODUCTION PROCESS

[0182] FIGURE 11 provides a schematic view of a process 200 of producing LNPs with a glycolipid adjuvant according to embodiments of the present disclosure.

[0183] Process 200 begins with providing a plurality of lipids 210, where the plurality of lipids is configured to generate a LNP, thereby forming a lipid phase. In this regard, the plurality of lipids 210 may include an ionizable lipid 215, a helper lipid 216. a sterol 217, and a PEGylated lipid 218. Process 200 further includes providing a glycolipid 240. In some embodiments, the plurality of lipids 210, ionizable lipid 215, helper lipid 216, sterol 217, PEGylated lipid 218, and glycolipid 240 are examples of any of the plurality of lipids, ionizable lipid, helper lipid, sterol. PEGylated lipid, and glycolipids described elsewhere herein.

[0184] In process 200, the plurality of lipids 210 are combined in a manner configured to generate a LNP (such as LNP 100', which in some embodiments is an example of LNP 100 described further herein). This combining of the plurality of lipids 210 thus forms a lipid phase Next, in process 200, the glycolipid 240 is introduced into the lipid phase. When the plurality of lipids 210 and the glycolipid 240 are thus combined, the lipid phase is then mixed with cargo 230 (such as a nucleic acid) in an in-line mixing device 250. In some embodiments, the in-line mixing device 250 is selected from a group of microfluid mixers, 2-inlet confined impinging jet mixers, 3-inlet confined impinging jet mixers, 4-inlet confined impinging jet mixers, multi-inlet vortex mixers, and T-junction mixers. Insome embodiments, the nucleic acid of the cargo 230 is selected from the group consisting of mRNA. circRNA, saRNA, and DNA

[0185] The detailed description set forth above in connection with the appended drawings, where like numerals reference like elements, are intended as a description of various embodiments of the present disclosure and are not intended to represent the only embodiments. Each embodiment described in this disclosure is provided as a representative example or illustration and should not be construed as preferred or advantageous over other embodiments. The representative examples provided herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Similarly, any steps described herein may be interchangeable with other steps, or combinations of steps, in order to achieve the same or substantially similar result. Generally, the embodiments disclosed herein are non-limiting, and the inventors contemplate that other embodiments within the scope of this disclosure may include structures and functionalities from more than one specific embodiment shown in the figures and described in the specification. That is, the present disclosure includes embodiments that combine features from different embodiments.

[0186] In the foregoing description, specific details are set forth to provide a thorough understanding of exemplary embodiments of the present disclosure. It will be apparent to one skilled in the art, however, that the embodiments disclosed herein may be practiced without embodying all the specific details. In some instances, well-known process steps have not been described in detail in order not to unnecessarily obscure various aspects of the present disclosure. Further, it will be appreciated that embodiments of the present disclosure may employ any combination of features described herein.

[0187] In the detailed description herein, references to "one embodiment," "an embodiment." "an example embodiment," "some embodiments," "one or more embodiments," etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. In addition, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described. After reading the description, it will be apparent to one skilled in the relevant art(s) how to implement thedisclosure in alternative embodiments. Thus, it will be appreciated that embodiments of the present disclosure may employ any combination of features described herein. All such combinations or sub-combinations of features are within the scope of the present disclosure.

[0188] In view of the limitations of the processing techniques available in the field, the terms "approximately", "substantially", and "about" reflect a certain inability (or uncertainty) to precisely control the exact dimensions of certain features and measurements described herein. Depending on the level of precision that can be achieved using the commercially available processing and measurement tools available at the time, the terms "approximately", "substantially", and "about" may be used to mean within ±5% of a target value for some features. The terms "approximately", "substantially", and "about" may include the target value.

[0189] Throughout this specification, terms of art may be used. These terms are to take on their ordinary meaning in the art from which they come, unless specifically defined herein or the context of their use would clearly suggest otherwise.

[0190] The drawings in the FIGURES are not to scale. Similar elements are generally denoted by similar references in the FIGURES. For the purposes of this disclosure, the same or similar elements may bear the same references. Furthermore, the presence of reference numbers or letters in the drawings cannot be considered limiting, even when such numbers or letters are indicated in the claims.

[0191] Terms such as "a," "an," "the." and "said" are used to indicate the presence of one or more elements and components. The terms "comprise," "include," "have," "contain." and their variants are used to be open ended and may include or encompass additional elements, components, etc., in addition to the listed elements, components, etc., unless otherwise specified. The terms "first," "second," etc may be used as differentiating identifiers of individual or respective components among a group thereof, rather than as a descriptor of a number of the components, unless clearly indicated otherwise.

[0192] Although relative terms such as "on," "below," "upper." "lower," "top," "bottom," "right," and "left" may be used to describe the relative spatial relationships of certain structural features, these terms are used for convenience only, as a direction in the examples. Thus, if a structure is turned upside down, the "upper" component will become a "low^er" component. When a structure or feature is described as being "on" (or formed on) another structure or feature, the structure can be positioned directly on (i.e, contacting)the other structure, without any other structures or features intervening between the structure and the other structure. When a structure or feature is described as being "over" (or formed over) another structure or feature, the structure can be positioned over the other structure, with or without other structures or features intervening between them.

[0193] When two components are described as being "coupled to" each other, the components can be electrically coupled to each other, with or without other components being electrically coupled and intervening between them. When two components are described as being "directly coupled to" each other, the components can be electrically coupled to each other, without other components being electrically coupled between them.

[0194] The present application may also reference quantities and numbers. Unless specifically stated, such quantities and numbers are not to be considered restrictive, but exemplary of the possible quantities or numbers associated with the present application. Also in this regard, the present application may use the term "plurality" to reference a quantity or number. In this regard, the term "plurality" is meant to be any number that is more than one, for example, two, three, four, five, etc. The term "based upon" means "based at least partially upon."

[0195] The principles, representative embodiments, and modes of operation of the present disclosure have been described in the foregoing description. However, aspects of the present disclosure, which are intended to be protected, are not to be construed as limited to the particular embodiments disclosed. Further, the embodiments described herein are to be regarded as illustrative rather than restrictive. It will be appreciated that variations and changes may be made by others, and equivalents employed, without departing from the spirit of the present disclosure. Accordingly, it is expressly intended that all such variations, changes, and equivalents fall within the spirit and scope of the present disclosure as claimed

[0196] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.N ON-LIMITIN G EMBODIMENTS

[0197] While general features of the disclosure are described and shown and particular features of the disclosure are set forth in the claims, the following non-limiting embodiments relate to features, and combinations of features, that are explicitly envisioned as being part of the disclosure. The following non-limiting embodiments contain elementsthat are modular and can be combined with each other in any number, order, or combination to form a new non-limiting embodiment, which can itself be further combined with other non-limiting embodiments.

[0198] Embodiment 1. A lipid nanoparticle (LNP) including a plurality of lipids and a glycolipid adjuvant, wherein the glycolipid adjuvant is structurally integrated into a core of the LNP during formation of the LNP.

[0199] Embodiment 2. The LNP of Embodiment I or any other Embodiment, wherein the plurality of lipids comprises an ionizable lipid, a helper lipid, a sterol, and a PEGylated lipid.

[0200] Embodiment 3. The LNP of Embodiments 1-2 or any other Embodiment, further including an antigenic factor.

[0201] Embodiment 4. The LNP of Embodiments 1-3 or any other Embodiment, further including a nucleic acid encoding one or more polypeptides.

[0202] Embodiment 5. The LNP of Embodiments 1-4 or any other Embodiment, wherein the one or more polypeptides comprise an antigen.

[0203] Embodiment 6. The LNP of Embodiments 1-5 or any other Embodiment, wherein the glycolipid adjuvant is present in an amount of between about 0.1 pg to about 2.5 pg glycolipid adjuvant per 10 pg nucleic acid.

[0204] Embodiment 7. The LNP of Embodiments 1-6 or any other Embodiment, wherein the glycolipid adjuvant is present in an amount of between about 0.4 pg to about 2,0 pg glycolipid adjuvant per 10 pg nucleic acid.

[0205] Embodiment 8. The LNP of Embodiments 1-7 or any other Embodiment, wherein the nucleic acid is selected from the group consisting of an mRNA, a circRNA, a saRNA, and a DNA.

[0206] Embodiment 9. The LNP of Embodiments 1-8 or any other Embodiment, wherein the nucleic acid is mRNA.

[0207] Embodiment 10. The LNP of Embodiments 1-9 or any other Embodiment, wherein the glycolipid adjuvant is 7DW8-5,OH

[0208] Embodiment 11. The LNP of Embodiments 1-10 or any other Embodiment, wherein the ionizable lipid is selected from the group consisting of Dlin-MC3-DMA, SM-102, and ALC-0315.

[0209] Embodiment 12. The LNP of Embodiments 1-11 or any other Embodiment, wherein the helper lipid is selected from the group consisting of 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-( 1 '-rac-gly cerol) ( 18PG), 1,2-distearoylsn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), dimethyldioctadecyl ammonium (DDAB), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), L2-dimyristoyl-sn-glycero-3-phosphate (14PA), and sn-(3-oleoyl-2-hydroxy)-glycerol-l-phospho-sn-3'-(T,2'-dioleoyl)-gly cerol (ammonium salt) (18BMP).

[0210] Embodiment 13. The LNP of Embodiments 1-12 or any other Embodiment, wherein the sterol is a cholesterol.

[0211] Embodiment 14. The LNP of Embodiments 1-13 or any other Embodiment, wherein the PEGylated lipid is selected from the group consisting of DMG-PEG200 and ALC-0159.

[0212] Embodiment 15. The LNP of Embodiments 1-14 or any other Embodiment, wherein a molar ratio of the ionizable lipid in the lipid nanoparticle is in the range of about 30% to about 55%.

[0213] Embodiment 16. The LNP of Embodiments 1-15 or any other Embodiment, wherein a molar ratio of the helper lipid in the lipid nanoparticle is in the range of about 5% to about 10%.

[0214] Embodiment 17. The LNP of Embodiments 1-16 or any other Embodiment, wherein a molar ratio of the sterol in the lipid nanoparticle is in the range of about 38% to about 45%.

[0215] Embodiment 18. The LNP of Embodiments 1-17 or any other Embodiment, wherein a molar ratio of the PEGylated lipid in the lipid nanoparticle is in the range of about 0.05% to about 2%.

[0216] Embodiment 19. The LNP of Embodiments 1-18 or any other Embodiment, wherein the glycolipid adjuvant has a 70% or greater incorporation efficiency into the core of the LNP.

[0217] Embodiment 20. A composition comprising the LNP of Embodiments 1-19 or any other Embodiment and a pharmaceutically acceptable carrier.

[0218] Embodiment 21. A method of producing a pharmaceutical composition, the method including: combining a plurality of lipids dissolved in a water- miscible organic solvent, thereby forming a lipid phase; introducing a glycolipid into the lipid phase; and mixing the lipid phase with nucleic acids dissolved in an aqueous phase in an in-line mixing device

[0219] Embodiment 22. The method of Embodiment 21 or any other Embodiment, wherein the in-line mixing device is selected from a group of micro-fluid mixers. 2-inlet confined impinging jet mixers, 3-inlet confined impinging jet mixers, 4-inlet confined impinging jet mixers, multi-inlet vortex mixers, and T-junction mixers.

[0220] Embodiment 23. The method of Embodiments 21-22 or any other Embodiment, wherein the plurality of lipids compnses an ionizable lipid, a helper lipid, a sterol, and a PEGylated lipid.

[0221] Embodiment 24. The method of Embodiments 21-23 or any other Embodiment, wherein the nucleic acids are selected from the group consisting of an mRNA, a circRNA, a saRNA, and a DNA.

[0222] Embodiment 25. The method of Embodiments 21-24 or any other Embodiment, wherein: a molar ratio of ionizable lipid in the lipid nanoparticle is in the range of about 30% to about 55%; a molar ratio of the helper lipid in the lipid nanoparticle is in the range of about 5% to about 10%; a molar ratio of the sterol m the lipid nanoparticle is in the range of about 38% to about 45%; a molar ratio of the PEGylated lipid in the lipid nanoparticle is in the range of about 0.05% to about 2%; and wherein the glycolipid adjuvant is present in an amount of between about 0.1 pg to about 2.5 pg glycolipid adjuvant per 10 pg nucleic acid

[0223] Embodiment 26. A method of immunizing a subject against a parasite, the method comprising administering to the subject a therapeutically effective dose of the LNP of Embodiments 1—19 or any other Embodiment.

[0224] Embodiment 27. The method of Embodiment 26 or any other Embodiment, wherein the LNP is administered via intravenous (IV) injection.

[0225] Embodiment 28. The method of Embodiments 26-27 or any other Embodiment, wherein the LNP is administered via intramuscular (IM) injection.

[0226] Embodiment 29. The method of Embodiments 26-28 or any other Embodiment, wherein the parasite is Plasmodium.

[0227] Embodiment 30. The method of Embodiments 26-29 or any other Embodiment, wherein immunizing comprises inducing liver-resident immunity leading to sterile protection against parasites.

[0228] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMSWhat is claimed is:

1. A lipid nanoparticle (LNP) comprising:a plurality of lipids; anda glycolipid adjuvant, wherein the glycolipid adjuvant is structurally integrated into a core of the LNP during formation of the LNP.

2. The LNP of claim 1. wherein the plurality of lipids comprises an ionizable lipid, a helper lipid, a sterol, and a PEGylated lipid.

3. The LNP of claim 1, further comprising an antigenic factor.

4. The LNP of claim 1, further comprising a nucleic acid encoding one or more polypeptides.

5. The LNP of claim 4, wherein the one or more polypeptides comprise an antigen.

6. The LNP of claim 4, wherein the glycolipid adjuvant is present in an amount of between about 0.1 pg to about 2.5 pg glycolipid adjuvant per 10 ug nucleic acid.

7. The LNP of claim 6, wherein the glycolipid adjuvant is present in an amount of between about 0.4 pg to about 2.0 pg glycolipid adjuvant per 10 ug nucleic acid.

8. The LNP of claim 4, wherein the nucleic acid is selected from the group consisting of an mRNA, a circRNA, an saRNA. and a DNA.

9. The LNP of claim 8, wherein the nucleic acid is mRNA.

10. The LNP of claim 1, wherein the glycolipid adjuvant is 7DW8-5,11. The LNP of claim 2, wherein the ionizable lipid is selected from the group consisting of Dlin-MC3-DMA, SM-102, and ALC-0315.

12. The LNP of claim 2, wherein the helper lipid is selected from the group consisting of 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG). 1,2-distearoylsn-glycero-3-phosphocholine (DSPC), 1,2-dioleoy 1-3 -tri methylammoniumpropane (DOT AP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), dimethyldioctadecyl ammonium (DDAB), 1,2-dioleoy l-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA), and sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1',2'-dioleoyl)-glycerol (ammonium salt) (18BMP).

13. The LNP of claim 2, wherein the sterol is a cholesterol.

14. The LNP of claim 2, wherein the PEGylated lipid is selected from the group consisting of DMG-PEG200 and ALC-0159.

15. The LNP of claim 1. wherein the glycolipid adjuvant has a 70% or greater incorporation efficiency into the core of the LNP.

16. A composition comprising the LNP of claim 1 and a pharmaceutically acceptable carrier.

17. A method of producing a pharmaceutical composition, the method comprising:combining a plurality of lipids dissolved in a water-miscible organic solvent, thereby forming a lipid phase;introducing a glycolipid into the lipid phase: andmixing the lipid phase with nucleic acids dissolved in an aqueous phase in an inline mixing device.

18. A method of immunizing a subject against a parasite, the method comprising administering to the subject a therapeutically effective dose of the LNP of claim 1.

19. The method of claim 18, wherein the parasite is Plasmodium.

20. The method of claim 18, wherein immunizing comprises inducing liver-resident immunity leading to sterile protection against parasites.

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