Immunogenic vaccine composition incorporating a saponin

Abstract

Provided herein are lipid-based nanoparticle compositions, and methods of making and using thereof. The compositions include nanostructured lipid carriers (NLC), liposomes, lipid nanoparticles (LNPs), solid lipid nanoparticles (SLNs), oil-in-water emulsions, cationic lipid–nucleic acid complexes, cationic nanoemulsions (CNE), charge-altering releasable transporters (CARTs), or polymeric nanoparticles, and further comprise a saponin adjuvant, and optionally a sterol and / or a bioactive agent. The bioactive agent can be self-amplifying RNA. The compositions are capable of delivery of a biomolecule to a cell for the generation of an immune response, for example, for vaccine, therapeutic, allergy desensitization, or diagnostic uses. Compositions and methods related to making the compositions and using the compositions for stimulating an immune response are also provided.

Description

Immunogenic Vaccine Composition Incorporating a SaponinFIELD

[0001] The present disclosure relates generally to the fields of pharmaceutical and vaccine formulations.BACKGROUND

[0002] Nucleic acid immunization is an attractive strategy for the rapid development of vaccines to existing or emerging infectious disease threats. Nucleic acid vaccine candidates are easily generated by common synthetic methods and can be constructed within weeks of the emergence of a new infectious disease. In addition, because the biophysical characteristics of a nucleic acid vaccine are independent of the expressed antigen, the new vaccine requires minimal antigen-specific process development for manufacturing. Plasmid DNA vaccines are currently in development for select infectious diseases; however, thus far, no DNA based vaccines have been approved for use in humans due to the associated complications (McKay, Cope et al. 2014, Tregoning and Kinnear 2014).

[0003] Delivery of antigens using RNA-based platforms has been proposed as a promising alternative compared to DNA based platforms. The transient nature of RNA is desirable for antigen delivery; the risk of long-term vaccine persistence is reduced relative to DNA, and nuclear translocation of the delivered vaccine is not required for protein production. However, the relative instability of RNA and the limited expression from a single mRNA transcript has made large scale distribution and use of these vaccines difficult in the field and for commercial development. Abundant research on methods to improve RNA stability through modification of RNA structure has provided several solutions to this problem (Tavernier, Andries et al. 2011 , Youn and Chung 2015). In particular, self-amplifying RNA (saRNA) based vaccines have demonstrated a potential mechanism to improve the magnitude and duration of antigen expression (Reviewed in (Vander Veen, Harris et al. 2012, Ljungberg and Liljestrom 2015)).

[0004] To enable a robust immune response, formulations such as liposomes and oil-in-water emulsions are typically employed to enhance the delivery of RNA into cells (Geall, Verma et al. 2012, Ulmer, Mason et al. 2012, Brito, Chan et al. 2014, Bogers, Oostermeijer et al. 2015, Brito, Kommareddy et al. 2015, Geall and Ulmer 2015). These formulations may also be used to enhance the delivery of drugs or other therapeutics into cells. However, liposomes or oil-in-water emulsions such as cationic lipid emulsions (CNE) can be structurally unstable in physiological environments, increasing the likelihood of toxicity from acute exposure to individual components. The toxic potential iof such carriers can also compound or confound toxicity concerns commonly associated with cationic phospholipids used for RNA adsorption (Bertholet et al. 2010). Additionally, there is limited understanding of how the physicochemical makeup of oil-in-water emulsions (e.g., size, surface charge, chemical nature of excipients, and their relative ratios) affects RNA binding, delivery, and ultimately antigen expression.

[0005] Thus, there is a need for a formulation platform that is both physically and chemically suited to serve as a versatile, stable, and safe system for the delivery of bioactive agents including nucleic acids to cells.BRIEF SUMMARY

[0006] The present inventors have developed formulations that are surprisingly effective at inducing an immune response to a bioactive agent. Accordingly, provided herein, inter alia, are such formulations (also referred to herein as compositions) and their methods of use. In some embodiments, the formulations comprise lipid-based nanoparticle formulations. In some embodiments, the formulations comprise nanostructured lipid carriers (NLC), liposomes, lipid nanoparticles (LNPs), solid lipid nanoparticles (SLNs), oil-in-water emulsions, cationic lipid-nucleic acid complexes, cationic nanoemulsions (CNE), charge-altering releasable transporters (CARTs), or polymeric nanoparticles. In some embodiments, the formulations of the disclosure comprise an adjuvant. In some embodiments, the adjuvant comprises a saponin.

[0007] In some embodiments, the formulations comprise nanostructured lipid carrier (NLC)- based formulations. It will be understood by the skilled practitioner that a NLC is made up of NLC particles. NLCs are described in Beloqui et al., Nanomedicine. NBM 2016; 12: 143-161. Exemplary NLC particles of the present invention comprise (a) an oil core comprising a liquid phase lipid and a solid phase lipid, (b) a cationic lipid, (c) a hydrophobic surfactant (preferably a sorbitan ester (e.g., sorbitan monoester, diester, or triester), (e) a hydrophilic surfactant, and (f) a saponin. In some embodiments, an NLC of the disclosure can further comprise a sterol. Exemplary sterols are described herein. Exemplary compositions are stable and are capable of inducing an immune response to bioactive agents. The immune response can be, for example, for the generation of an immune response, for vaccination against a disease or pathogen, for desensitization to an allergen, and / or for treatment of other disease and health conditions in a subject.

[0008] These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain aspects of this invention. All references cited herein, including patent applications and patent publications are herein incorporated by reference in theirentirety, as if each individual reference is specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 shows an exemplary QS-21 adjuvanted saRNA vaccine delivery formulation of the disclosure.

[0010] FIGS. 2A-2C demonstrate humoral immune responses 21 days following vaccination. FIG. 2A shows the results of a SARS-CoV-2 spike binding IgG ELISA assay. FIG. 2B shows the results of SARS-CoV-2 pseudovirus neutralizing antibody assay. NLCs adjuvanted with three different saponin concentrations are compared to a NLC without saponin adjuvanting and a negative control containing SEAP RNA. Dosing of 2 pig QS-21 significantly increases the SARS-CoV-2 spike binding IgG titers induced in vaccinated mouse sera, as well as the neutralizing antibody titers in vaccinated mouse sera after a single low RNA vaccine dose. FIG. 2C is a repeat of the neutralizing antibody assay using both the original Wuhan SARS-CoV-2 strain and the delta strain of SARS-CoV-2. Three different doses of QS-21 adjuvant are compared to an NLC that lacks QS-21 but is otherwise identical.

[0011] FIGS. 3A-3C demonstrate T cell responses 21 days following vaccination. Incorporation of QS-21 into cholesterol-contai ni ng NLCs drives substantially higher CD8 T cell responses 3 weeks post-vaccination even with a low 1 pig RNA vaccine dose. CD4 T cell responses were affected only minimally, indicating that the adjuvant is skewing the vaccine response more towards the highly- desirable CD8 T cell response. Statistical significance shown is based on 1 way ANOVA.

[0012] FIGS. 4A and 4B depict neutralizing antibody titers demonstrating durable humoral immunity four months after vaccination. FIG. 4A shows neutralizing titers of serum neutralizing antibodies for five different NLC formulations comparing multiple different adjuvants and a vector control (SEAP). Various NLCs were complexed with RNA encoding AAHI-SC2 saRNA and neutralizing antibodies and assayed 4 months after a single 1 pig IM injection. FIG. 4B shows titers of serum neutralizing antibodies in the same mice, using serum collected 6 weeks and 4 months after a single 1 pig IM injection. Data was analyzed by an ordinary two-way ANOVA with test followed by Dunnett's multiple comparisons test comparing all groups to the standard saRNA / NLC vaccine responses, on log-normalized antibody titer data.

[0013] FIGS. 5A-5C demonstrate durable cellular immunity with CD8 T cell data 4 months post vaccination. Five different NLC formulations are compared to a vector control. Four months after a single dose of vaccine, after T cell responses have retracted to memory levels, the QS-21 adjuvanted saRNA / NLC vaccinated mice show clearly higher polyfunctional CD8+ T cell responses than the normal unadjuvanted saRNA / NLC vaccine. This suggests that the addition of QS-21 may wellincrease the durability of vaccine induced cellular immunity in addition to enhancing the magnitude of the initial responses.

[0014] FIGS. 6A-6D show that QS-21 enhances the immunostimulatory properties of saRNA- NLC vaccines as demonstrated in vitro in human PBMCs. Human PBMCs were isolated from two donors and plated at 2 x 105cells / well in a microtiter plate. The PBMCs were then stimulated with two dilutions of NLC, NLC+saRNA, and NLC+saRNA+QS-21 to examine the immunostimulatory contributions of each agent, incubated for 18 hours, and supernatants harvested for chemokine / cytokine assay.

[0015] FIGS. 7A and 7B demonstrate that appropriately dosed cholesterol / QS-21 -formulated NLCs significantly enhance neutralizing antibody and CD8+ T cell responses after a single low-dose saRNA vaccination in C57BL / 6 mice.

[0016] FIGS. 8A and 8B demonstrate cholesterol / QS-21-formulated NLC saRNA vaccine immunogenicity enhancement also improves immune durability.

[0017] FIGS. 9A and 9B demonstrate QS-21 -adjuvanted saRNA-qNLC vaccine protects mice against lethal Zika virus challenge. Male and female mice were vaccinated by a single 1-pig dose of cholesterol / QS-21 NLC formulated (2 pig QS-21) Zika saRNA vaccine or vector control saRNA-qNLC. Three weeks post-vaccination, mice were transiently immunocompromised by injection with IFNAR- blocking monoclonal antibody and challenged with 106PFU of mouse-adapted Zika strain Dakar.

[0018] FIGS. 10A and 10B demonstrate humoral immune responses 21 days following vaccination. FIG. 10A shows the results of a SARS-CoV-2 spike binding IgG ELISA assay. FIG. 10B shows the results of SARS-CoV-2 pseudovirus neutralizing antibody assay. Two LNP platforms, one CNE platform, and three NLCs - one adjuvanted with 1 pig QS-21 + cholesterol, one with no adjuvant, and one with alpha-tocopherol - are compared to a negative control NLC containing SEAP RNA. Adjuvanting with alpha-tocopherol decreased the SARS-CoV-2 spike binding IgG titers induced in vaccinated mouse sera, as well as the neutralizing antibody titers in vaccinated mouse sera, to levels similar to the negative control. However, adjuvanting with QS-21 + cholesterol resulted in SARS-CoV- 2 spike binding IgG titers similar to other formulations and the highest neutralizing antibody titers.DETAILED DESCRIPTION

[0019] Provided herein are compositions for delivering a bioactive agent to a cell and methods of such delivery. NLCs have a core comprising of a combination of liquid-phase and solid-phase lipids. In contrast to solid lipid nanoparticles (LNPs), which have a completely solid crystalline core, the mixed phase core in NLCs provides more versatility to incorporate active molecules of various structures. Without being bound by theory, it is believed that the addition of solid lipids to the composition yieldsNLC cores with structural integrity and stability. In the NLCs of the present invention, the mixed phase oil core is emulsified with a mixture of surfactants (typically a sorbitan ester and a hydrophilic surfactant) and a cationic component (typically a cationic lipid or phospholipid). Whereas typically, bioactive agents, such as small molecule drugs, have been incorporated in the oil core of the NLCs, the present inventors have synthesized NLCs that can also interact with bioactive agents such as negatively charged molecules (e.g., RNA) at or near their surface (i.e., the bioactive agent is not encapsulated by the NLC). The present inventors have discovered that inclusion of a saponin results in a surprisingly advantageous immune response as compared to prior NLC formulations and compared to formulations that use other adjuvants. It was not known in advance which, if any, adjuvant would beneficially interact with an NLC formulation complexed to RNA. The ability of the described NLCs to deliver nucleic acid to a cell, and the ability of a nucleic acid bioactive agent and NLC to be manufactured and stored separately and mixed just prior to use, permit the use of these NLCs in a wide variety of applications, including as a rapid response nucleic acid platform technology for the development of multiple prophylactic or therapeutic treatments. The rapid response nucleic acid delivery platform is based on a flexible system utilizing NLC compositions to deliver nucleic acids (e.g., self-amplifying RNA (saRNA)) to drive RNA replication and / or protein expression (e.g., leading to robust and rapid immune responses to diverse viral, bacterial, or parasitic antigens). The NLC compositions of the present invention can be manufactured and stockpiled for extended time periods. The stockpiled NLC vehicle can then be combined with nucleic acid (e.g., synthetic saRNA expressing a protective antigen) during an emerging disease outbreak or other public health event.

[0020] In addition to providing NLCs for combination with a bioactive agent, also provided are those NLCs once combined. Accordingly, in some aspects, the bioactive agent is associated with the NLC. The bioactive agent can be delivered to a subject in a time of need by administration of the NLC- bioactive agent composition.

[0021] I. Definitions

[0022] The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0023] In the present description, the terms "about” and "consisting essentially of” mean ± 20% of the indicated range, value, or structure, unless otherwise indicated.

[0024] The use of the alternative {e.g., "or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0025] As used herein, the terms "include,” "have” and "comprise” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[0026] As used herein and in the appended claims, the singular forms "a,” "an,” and "the” include plural reference unless the context clearly indicates otherwise.

[0027] The term "macromolecule" as used herein refers to large molecules exemplified by, but not limited to, peptides, proteins, oligonucleotides and polynucleotides of biological or synthetic origin.

[0028] The term "alkyl” means a straight chain or branched, noncyclic or cyclic, unsaturated or saturated aliphatic hydrocarbon containing the indicated number of carbon atoms. Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms.

[0029] The terms "polypeptide”, "peptide”, and "protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified nucleotides or amino acids, and it may be interrupted by non-nucleotides or non-amino acids. The terms also encompass a nucleotide or amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polynucleotides or polypeptides containing one or more analogs of a nucleotide or an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0030] The term "isolated” means the molecule has been removed from its natural environment.

[0031] "Purified” means that the molecule has been increased in purity, such that it exists in a form that is more pure than exists in its natural environment and / or when initially synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.

[0032] A "polynucleotide” or "nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, include DNA and RNA. The nucleotides can be, for example, deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer.

[0033] "Oligonucleotide," as used herein, generally refers to short, generally single stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.

[0034] An "individual” or a "subject" is any mammal. Mammals include, but are not limited to humans, primates, farm animals, sport animals, pets (such as cats, dogs, horses), and rodents.

[0035] A “repl icon” as used herein includes any genetic element, for example, a plasmid, cosmid, bacmid, phage, or virus that is capable of replication largely under its own control. A rephcon may be either RNA or DNA and may be single or double stranded.

[0036] Vitamin E refers to both tocopherols (TCPs) and tocotrienols and can be naturally occurring or synthetic.

[0037] Monoacylglycerols are esters of the trihydric alcohol glycerol in which one of the hydroxyl groups is esterified with a long chain fatty acid.

[0038] Lauroyl polyoxylglycerides are a mixture of monoesters, diesters, and triesters of glycerol and monoesters and diesters of polyethylene glycols with a mean relative molecular weight typically between about 300 and about 1500.

[0039] C apric / capry lie triglyceride is a mixed triester of glycerin and caprylic and capric acids.

[0040] The term liquid phase lipid refers to a lipid that, prior to mixing with any other component, is liquid at ambient temperature.

[0041] The term solid phase lipid refers to a lipid that, prior to mixing with any other component, is solid at ambient temperature.

[0042] Ambient temperature is between 15°C and 25°C.

[0043] Glycerolipid is a fatty molecule composed of glycerol linked esterically to a fatty acid. Glycerolipids include triglycerides and diglycerides.

[0044] The term "sorbitan ester” as used herein refers to an ester of sorbitan. Sorbitan is as shown in Formula A

[0046] Formula A

[0047] Particularly preferred sorbitan esters are sorbitan alkyl esters, wherein the alkyl is a Ci- C30 alkyl group, preferably a saturated or unsaturated C1-C20 alkyl group, more preferably a saturated or unsaturated C10-C20 alkyl group.

[0048] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, recombinant DNA, biochemistry, and chemistry, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al., U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); B. Perbal, A Practical Guide to Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

[0049] II. Introduction

[0050] The development of appropriate adjuvants for RNA vaccines is a challenging enterprise. Immune stimulation by RNA vaccines is often far from ideal. Indeed, mRNA vaccines typically rely on immune stealth provided by their carefully engineered RNA structures, modified nucleosides, and immune-silent delivery systems to avoid stimulating innate immune responses which rapidly and effectively shut down antigen expression (K. Kariko, et al., Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Therlb, 1833-40 (2008) and result in poor clinical immunogenicity (J. Cohen, What went wrong with CureVac's highly anticipated new mRNA vaccine for COVID-19? Scienceinsider (2021) (October 12, 2022). Thus, adjuvanting standard modified mRNA vaccines is difficult or impossible. Selfamplifying RNA vaccines, however, allow for greater flexibility in the addition of adjuvanting compounds because natural amplification of alphavirus replicase-based saRNAs allows for greatly decreased sensitivity to immune stimulation. Due to this natural amplification, saRNA vaccines retain antigen expression in the presence of innate immune stimulation. The replication of the saRNAs robustly stimulates innate immune responses via endosomal receptors (e.g., TLR3) and cytosolic receptors (e.g., RIG-I, MDA5, etc.) (see, e.g., A. K. Blakney, S. Ip, A. J. Geall, An Update on Self- Amplifying mRNA Vaccine Development. Vaccines 9, K.-J. Kallen, et al., A novel, disruptive vaccination technology. Hum. Vaccines Immunother. 9, 2263-2276 (2013) 97 (2021); K.-J. Kallen, et al., A novel, disruptive vaccination technology. Hum. Vaccines Immunother K.-J. Kallen, et al., A novel, disruptive vaccination technology. Hum. Vaccines Immunother. 9, 2263-2276 (2013). 9, 2263-2276 (2013); N. Pardi, M. J. Hogan, F. W. Porter, D. Weissman, mRNA vaccines - a new era in vaccinology. Nat Rev Drug Discov 17, 261-279 (2018), a form of.stimulation typically considered to lead to establishment of strong and durable immune memory. Additionally, the nsp2 replicase protein of Venezuelan equine encephalitis virus (VEEV) and other alphaviruses used for saRNA replicon construction has interferon-antagonistic functions (see, e.g., J. D. Simmons, etal., Venezuelan Equine Encephalitis Virus Disrupts STAT 1 Signaling by Distinct Mechanisms Independent of Host Shutoff. J. Virol. 83, 10571-10581 (2009); L. Breakwell, et al., Semliki Forest Virus Nonstructural Protein 2 Is Involved in Suppression of the Type I Interferon Response. J. Virol. 81, 8677-8684 (2007); N. Bhalla, et al.). Host translation shutoff mediated by non-structural protein 2 is a critical factor in the antiviral state resistance of Venezuelan equine encephalitis virus {Virology 496, 147-165 (2016), further allowing for antigen expression despite innate immune stimulation. While type I interferons do stillrestrict saRNA replication and antigen expression, the interplay between saRNA replication, innate stimulation, and recruitment of immune cells to the site of injection mimics a typical virus-host interaction and allows for flexibility in immunogenic RNA vaccine design and formulation. The unique mechanism by which saRNA vaccines induce immune responses, however, still requires careful selection of vaccine adjuvants to enhance vaccine immunogenicity while also maintaining RNA-based antigen expression.

[0051] The instant disclosure provides a vaccine platform comprising lipid-based nanoparticle compositions and formulations and an adjuvant comprising a saponin that shows significantly enhanced immunogenicity relative to non-adjuvanted saRNA. In some embodiments, the formulations comprise nanostructured lipid carriers (NLC), liposomes, lipid nanoparticles (LNPs), solid lipid nanoparticles (SLNs), oil-in-water emulsions, cationic lipid-nucleic acid complexes, cationic nanoemulsions (CNE), charge-altering releasable transporters (CARTs), or polymeric nanoparticles. In some embodiments, the compositions and / or formulations comprise nanostructured lipid carrier (NLC)-based formulations. Further, the vaccine platform can be applied to polypeptide or peptide antigens and epitopes as well as RNAs that encode protein or polypeptide or peptide antigens and epitopes.

[0052] In some embodiments, the saRNA vaccine platform comprises a VEEV-based vaccine saRNA, complexed on the outside of a squalene-containing nanostructured lipid carrier (NLC) delivery particle that allows for ready vaccine lyophilization and long-term thermostability (see, e.g., A. Gerhardt, et al., A flexible, thermostable nanostructured lipid carrier platform for RNA vaccine delivery. Mol. Ther. Methods Clin. Dev. 25, 205-214 (2022); Voigt, E.A., Gerhardt, A., Hanson, D. et a / . A selfamplifying RNA vaccine against COVID-19 with long-term room-temperature stability, npj Vaccines 7, 136 (2022)). In some embodiments, the vaccine platform comprises an inflammasome-stimulating saponin. In some embodiments, the saponin is QS-21. In some embodiments, the vaccine platform comprises a unique combination of squalene, saponin, and saRNA that that significantly enhances both neutralizing antibody and important CD8+ T cell responses in preclinical mouse models.

[0053] III. Nanostructured Lipid Carriers

[0054] The present disclosure provides, inter alia, NLCs for delivery of a bioactive agent to a cell. In some embodiments, the NLC compositions are made up of NLC particles comprising (a) an oil core comprising a liquid phase lipid and a solid phase lipid, (b) a cationic component (preferably, a cationic lipid or phospholipid,) (c) a hydrophobic surfactant, preferably a sorbitan ester (e.g., sorbitan monoester, diester, or triester), (d) a surfactant (preferably, a hydrophilic surfactant), and (e) a saponin. In some embodiments, the NLC compositions further comprise a sterol. If present, the sterol component is added to promote complexation with QS-21 based on the inventors' earlier workdemonstrating that sterol-containing liposomes ameliorated the hemolytic / cytolytic activity of QS-21. In some embodiments, the NLC compositions do not comprise a sterol. The NLCs of the present invention typically comprise an unstructured or amorphous solid lipid matrix made up of a mixture of blended solid and liquid lipids dispersed in an aqueous phase. One or more of the surfactants can be present in the oil phase, the aqueous phase, or at the interface between the oil and aqueous phase. In certain aspects the sorbitan ester and the cationic lipid are present at the interface between the oil and aqueous phase. An exemplary QS-21 adjuvanted saRNA NLC formulation is shown in FIG. 1.

[0055] The present inventors have found that the claimed NLCs are particularly effective at delivering protein-encoding nucleic acid, such as RNA. In addition, it has been found that by manipulating certain components of the NLC, the levels of expression of the encoded protein can be increased. Surprisingly, exemplary NLCs are not only capable of effectively delivering RNA, they are also able to improve the immune response to the encoded proteins. Importantly, the data provided herein show that the NLCs of the disclosure can increase the innate immune response, as measured by type I interferon (IFN) and chemokine expression (e.g., MCP-1 and MIP-1 p ), while at the same time antigen expression remains sufficient in vivo for robust vaccine responses. This result is unexpected, because type I IFNs restrict saRNA replication, which would be expected to reduce antigen expression in vivo and result in a weak vaccine response. Without being bound by theory, the strong stimulation of antigen presenting cell (APC)-recruiting chemokines such as MIP-1 p is hypothesized to be the mechanism behind the observed in vivo enhancement of neutralizing antibody and CD8+T cell responses.

[0056] A. Solid-Phase and Liquid-Phase Lipids

[0057] NLCs are composed of a blend of solid and liquid lipids. The liquid and solid lipids to be used in the NLCs can be any lipid capable of forming an unstructured or amorphous solid lipid matrix and forming a stable composition. The weight ratio of solid to liquid can vary widely, for example from 0.1 :99.9 to 99.9:0.1. In some exemplary embodiments, the solid lipids are mixed with liquid lipids in a solidJiquid lipid weight ratio of from about 70:30 to about 99.9:0.1 or from about 1 : 10 to about 1 :30. In some aspects, the solid lipids are mixed with liquid lipids in a solidJiquid lipid weight of about 1 :16.

[0058] The total oil core component (solid lipid + liquid oil) of the NLC-based composition or formulation is typically present in an amount from about 0.2% to about 50% (w / v). For example, the NLC may comprise from about 0.2% to about 50% (w / v) oil core component, 0.2% to about 40% (w / v) oil core component, from about 0.2% to about 30% (w / v) oil core component, from about 0.2% to about 20% (w / v) oil core component, from about 0.2% to about 15% (w / v) oil core component, from about 0.2% to about 10% (w / v) oil core component, from about 0.2% to about 9% (w / v) oil core component, from about 0.2% to about 8% (w / v) oil core component, from about 0.2% to about 7%(w / v) oil core component, from about 0.2% to about 6% (w / v) oil core component, from about 0.2% to about 5% (w / v) oil core component, from about 0.2% to about 4.3% (w / v) oil core component, from about 0.3% to about 20% (w / v) oil core component, from about 0.4% to about 20% (w / v) oil core component, from about 0.5% to about 20% (w / v) oil core component, from about 1 % to about 20% (w / v) oil core component, from about 2% to about 20% (w / v) oil core component, from about 3% to about 20% (w / v) oil core component, from about 4% to about 20% (w / v) oil core component, from about 5% to about 20% (w / v) oil core component, about 0.5% (w / v) oil core component, about 1 % (w / v) oil core component, about 1.5% (w / v) oil core component, about 2% (w / v) oil core component, about 2.5% (w / v) oil core component, about 3% (w / v) oil core component, about 3.5% (w / v) oil core component, about 4% (w / v) oil core component, about 4.3% (w / v) oil core component, about 5% (w / v) oil core component, or about 10% (w / v) oil core component or any other amount or range described herein for the oil core component. Higher or lower w / v percentages are contemplated herein, particularly when considering diluted or concentrated formulations.

[0059] The oil core of the NLC comprises a liquid phase lipid. Preferably, although not necessarily, the liquid phase lipid is a metabolizable, non-toxic oil; more preferably one of about 6 to about 30 carbon atoms including, but not limited to, alkanes, alkenes, alkynes, and their corresponding acids and alcohols, the ethers and esters thereof, and mixtures thereof. The oil may be, for example, any vegetable oil, fish oil, animal oil or synthetically prepared oil that can be administered to a subject. In some aspects, the liquid phase lipid will be non-metabolizable.

[0060] The oil can be, for example, any long chain alkane, alkene or alkyne, or an acid or alcohol derivative thereof either as the free acid, its salt, or an ester such as a mono-, or di or triester, such as the triglycerides and esters of 1 ,2-propanediol or similar poly-hydroxy alcohols. Alcohols may be acylated employing a mono or poly-functional acid, for example acetic acid, propanoic acid, citric acid, or the like. Ethers derived from long chain alcohols which are oils and meet the other criteria set forth herein may also be used.

[0061] The individual alkane, alkene, or alkyne moiety and its acid or alcohol derivatives will generally have from about 6 to about 40 or from 6 to about 30 carbon atoms. The moiety may have a straight or branched chain structure. It may be fully saturated or have one or more double or triple bonds. Where mono or poly ester or ether-based oils are employed, the limitation of about 6 to about 40 carbons applies to the individual fatty acid or fatty alcohol moieties, not the total carbon count.

[0062] Any suitable oils from an animal, fish, or vegetable source may be used. Sources for vegetable oils include nuts, seeds, and grains, and suitable oils include, for example, peanut oil, soybean oil, coconut oil, and olive oil, and the like. Other suitable seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil, and theoil of other cereal grains such as wheat, oats, rye, rice, teff, triticale, and the like may also be used. The technology for obtaining vegetable oils is well developed and well known. The compositions of these and other similar oils may be found in, for example, the Merck Index, and source materials on foods, nutrition and food technology.

[0063] Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Naturally occurring or synthetic terpenoids, also referred to as isoprenoids, can be used herein as a liquid phase lipid. Squalene, a branched, unsaturated terpenoid, is particularly preferred herein. A major source of squalene is shark liver oil, although plant oils (primarily vegetable oils), including amaranth seed, rice bran, wheat germ, and olive oils, are also suitable sources. Squalane, the saturated analog to squalene, is also preferred. Oils, including fish oils such as squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art. Oils to be used herein may also be made using synthetic means, including genetic engineering (e.g., oils made from bioengineered yeast, including squalene).

[0064] Exemplary liquid phase lipids that can be used in the present invention include, for example, castor oil, coconut oil, corn oil, cottonseed oil, evening primrose oil, fish oil, grapeseed oil, jojoba oil, lard oil, linseed oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, squalene, squalane, sunflower oil, wheatgerm oil, mineral oil, capric / caprylic triglyceride (e.g., Myglyol®810, Myglyol®812, Labrafac™), , lauroyl polyoxylglycerides (e.g., Gelucire®44 / 14), monoacylglycerols (e.g., Myverol 18-99 K), soy lecithin (e.g., Epikuron ™200), farnesene, or a combination thereof.

[0065] The liquid phase lipid can include for example, squalene, sunflower oil, soybean oil, olive oil, grapeseed oil, squalane, capric / caprylic triglyceride, or a combination thereof.

[0066] The liquid phase lipid can include for example, squalene, squalene, capric / caprylic triglyceride, or a combination thereof.

[0067] The liquid phase lipid can include for example, capric / caprylic triglyceride, lauroyl polyoxylglycerides, monoacylglycerols, soy lecithin, squalene, or squalane, or a combination thereof.

[0068] The liquid phase lipid can include for example, squalene, squalene, or farnesene, or a combination thereof.

[0069] The oil core of the NLC comprises a solid phase lipid. A wide variety of solid phase lipids can be used, including for example, glycerolipids. Exemplary solid phase lipids include, for example, glyceryl palmitostearate (Precitol ATO®5), glycerylmonostearate, glyceryl dibehenate (Compritol®888 ATO), cetyl palmitate (Crodamol™ CP), stearic acid, tripalmitin, or a microcrystallinetriglyceride. Exemplary microcrystalline triglycerides include those sold under the trade name Dynasan® (e.g., trimyristin (Dynasan®114) or tristearin (Dynasan®118) or tripalmitin (Dynasan®116)).

[0070] The solid phase lipid can be, for example, a microcrystalline triglyceride, for example, one selected from trimyristin (Dynasan®114) or tristearin (Dynasan®118).

[0071] Preferably, the solid phase lipid of the oil core is solid at ambient temperature. When indoors, ambient temperature is typically between 15°C and 25°C.

[0072] In any of the embodiments provided herein, the solid phase lipid can be a glycerolipid, for example, a microcrystalline triglyceride.

[0073] In any of the embodiments provided herein, the liquid phase lipid can be synthetic or naturally-occurring squalene.

[0074] B. Sterol Component

[0075] In some embodiments, the NLCs described herein comprise a sterol component, such as cholesterol. Without being bound by theory, the sterol component may associate with the saponin and decrease reactogenicity of the saponin. The sterol may be cholesterol, synthetic cholesterol, semisynthetic cholesterol, 3p-[N— (N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol (DC Cholesterol), phytosterol, or a cholesterol analogue. Cholesterol analogues include naturally-occurring and synthetic sterols with structures similar to cholesterol. Some non-limiting examples of cholesterol analogues are provided in Patel, S., Ashwanikumar, N., Robinson, E. et al. Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA. Nat Commun 11 , 983 (2020). In some embodiments, the NLCs described herein do not comprise a sterol component.

[0076] With respect to weight per volume, an exemplary NLC-based composition or formulation may comprise, for example, from about 0.05 % to about 5% or to about 10% w / v sterol component.

[0077] C. Cationic Lipid

[0078] The NLCs described herein comprise a cationic lipid. The cationic lipid is useful for interacting with negatively charged bioactive agents on the surface on the NLC. Any cationic lipid capable of interacting with negatively charged bioactive agents that will not disturb the stability of the NLC and can be administered to a subject can be used. Generally, the cationic lipid contains a nitrogen atom that is positively charged under physiological conditions. Suitable cationic lipids include, benzalkonium chloride (BAK), benzethonium chloride, cetrimide (which contains tetradecyltrimethylammonium bromide and possibly small amounts of dodecyltrimethylammonium bromide and hexadecyltri methyl ammonium bromide), cetylpyridinium chloride (CPC), cetyl trimethylammonium chloride (CTAC), primary amines, secondary amines, tertiary amines, includingbut not limited to N,N',N'-polyoxyethylene (10)-N-tallow-1 ,3-diaminopropane, other quaternary amine salts, including but not limited to dodecyltrimethylammonium bromide, hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammonium bromide, benzyldimethyldodecylammonium chloride, benzyldimethylhexadecyl-ammonium chloride, benzyltrimethylammonium methoxide, cetyldimethylethylammonium bromide, dimethyldioctadecyl ammonium bromide (DDAB), methylbenzethonium chloride, decamethonium chloride, methyl mixed trialkyl ammonium chloride, methyl trioctylammonium chloride, N, N-dimethyl-N-[2 (2-methyl-4-(1 , 1 ,3,3tetramethylbutyl)-phenoxy]- ethoxy)ethyl]-benzenemetha-naminium chloride (DEBDA), dialkyldimethylammonium salts, [1-(2,3- dioleyloxy)-propyl]-N,N,N, trimethylammonium chloride, 1 ,2-diacyl-3-(trimethylammonio) propane (acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3(dimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1 ,2-dioleoyl-3-(4'-trimethyl- ammonio)butanoyl-sn-glycerol, 1 ,2-dioleoyl 3-succinyl-sn-glycerol choline ester, cholesteryl (4- trimethylammonio) butanoate), N-alkyl pyridinium salts (e.g. cetylpyridinium bromide and cetylpyridinium chloride), N-alkylpiperidinium salts, dicationic bolaform electrolytes (C12Me6; C12Bu6), dialkylglycetylphosphorylcholine, lysolecithin, L-o dioleoylphosphatidylethanolamine, cholesterol hemisuccinate choline ester, lipopolyamines, including but not limited to dioctadecylamidoglycylspermine (DOGS), dipalmitoyl phosphatidylethanol-amidospermine (DPPES), lipopoly-L (or D)-lysine (LPLL, LPDL), poly (L (or D)-lysine conjugated to N- glutarylphosphatidylethanolamine, didodecyl glutamate ester with pendant amino group (C12GluPhCnN+), ditetradecyl glutamate ester with pendant amino group (014GluCnN+), cationic derivatives of cholesterol, including but not limited to cholesteryl-3p- oxysuccinamidoethylenetrimethylammonium salt, cholesteryl-3p- oxysuccinamidoethylenedimethylamine, cholesteryl-3p-carboxyamidoethylenetrimethylammonium salt, cholesteryl-3p-carboxyamidoethylenedimethylamine, and 3y-[N— (N',N- dimethylaminoetanecarbomoyl]cholesterol) (DC-Cholesterol), 1,2-dioleoyloxy-3- (trimethylammonio)propane (DOTAP), dimethyldioctadecylammonium (DDA), 1 ,2-Dimyristoyl-3- TrimethylAmmoniumPropane (DMTAP), dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), and combination thereof.

[0079] Other cationic lipids suitable for use in the invention include, e.g., the cationic lipids described in U.S. Patent Publications 2008 / 0085870 (published Apr. 10, 2008) and 2008 / 0057080 (published Mar. 6, 2008).

[0080] Other cationic lipids suitable for use in the invention include, e.g., Lipids E0001-E0118 or E0119-E0180 as disclosed in Table 6 (pages 112-139) of WO 2011 / 076807 (which also discloses methods of making, and method of using these cationic lipids). Additional suitable cationic lipidsinclude N-[1 -(2,3-dioleyloxy)propyl]-N,N, N-trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N- dimethylammonium chloride (DODAC), 1 , 2-diol eoy l-sn-glycero-3-ethy I phosphochol I ne (DOEPC), 1 ,2- dioleoyl-3-dimethylammonium-propane (DODAP), 1 , 2-di 11 noley loxy-3-di methyl ami nopropane (DLinDMA).

[0081] The NLCs may comprise one or any combination of two or more of the cationic lipids described herein.

[0082] In exemplary embodiments, the cationic lipid is selected from the group consisting of 1 ,2- dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 313-[N— (N',N'-Dimethylaminoethane)- carbamoyl]Cholesterol (DC Cholesterol), dimethyldioctadecylammonium (DDA), 1 ,2-Dimyristoyl-3- TrimethylAmmoniumPropane (DMTAP), dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), Lipids E0001-E0118 or E0119-E0180 as disclosed in Table 6 (pages 112-139) of WO 2011 / 076807, and combinations thereof.

[0083] In other exemplary embodiments, the cationic lipid is selected from the group consisting of 1 ,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 313-[N— (N',N'-Dimethylaminoethane)- carbamoyl]Cholesterol (DC Cholesterol), dimethyldioctadecylammonium (DDA), 1 ,2-Dimyristoyl-3- TrimethylAmmoniumPropane (DMTAP), dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1 ,2- dioleoy I -sn-g lycero-3-ethy Iphosphochol i ne (DOEPC), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), Lipids E0001-E0118 or E0119- E0180 as disclosed in Table 6 (pages 112-139) of WO 2011 / 076807, and combinations thereof.

[0084] Exemplary cationic lipids are selected from the following: 1 ,2-dioleoyloxy-3- (trimethylammonio)propane (DOTAP), 3p-[N— (N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol (DC Cholesterol), dimethyldioctadecylammonium (DDA), 1 ,2-Dimyristoyl-3- TrimethylAmmoniumPropane (DMTAP), dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1 ,2- dioleoy I -sn-g lycero-3-ethy Iphosphochol i ne (DOEPC), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1 ,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), or a combination thereof. Additional suitable cationic lipids may be known by one of skill in the art.

[0085] In certain embodiments, the NLC-based composition or formulation comprises from about 0.5 mg / ml to about 50 mg / ml of the cationic component (e.g., the cationic lipid). In certain embodiments, the cationic lipid is DOTAP. The NLC may comprise, for example, from about 0.5 mg / ml to about 25 mg / ml or 30 mg / ml DOTAP or any other amount or range described herein for DOTAP.

[0086] In certain embodiments, the cationic lipid is DC Cholesterol. In certain aspects, the NLC may comprise DC Cholesterol at from about 0.1 mg / ml to about 5 mg / ml DC Cholesterol. In certain embodiments, the cationic lipid is DDA. The NLC may comprise, for example, from about 0.1 mg / ml to about 5 mg / ml DDA. In certain embodiments, the cationic lipid is DOTMA. The NLC may comprise, for example, from about 0.5 mg / ml to about 25 or 30 mg / ml DOTMA. In certain embodiments, the cationic lipid is DOEPC. The NLC may comprise, for example, from about 0.5 mg / ml to about 25 mg / ml DOEPC. In certain embodiments, the cationic lipid is DSTAP. The NLC may comprise, for example, from about 0.5 mg / ml to about 50 mg / ml DSTAP. In certain embodiments, the cationic lipid is DODAC. The NLC may comprise, for example, from about 0.5 mg / ml to about 50 mg / ml DODAC. In certain embodiments, the cationic lipid is DODAP. The NLC may comprise, for example, from about 0.5 mg / ml to about 50 mg / ml DODAP.

[0087] With respect to weight per volume, an exemplary NLC-based composition or formulation may comprise, for example, from about 0.05 % to about 5% or to about 10% w / v cationic component (e.g., cationic lipid such as DOTAP), from about 0.2% to about 10% w / v cationic component (e.g., cationic lipid such as DOTAP), from about 0.2% to about 5% w / v cationic component (e.g., cationic lipid such as DOTAP), from about 0.2% to about 2% w / v cationic component (e.g., cationic lipid such as DOTAP), from about 2% to 10% w / v cationic component (e.g., cationic lipid such as DOTAP), from about 2% to about 5% w / v cationic component (e.g., cationic lipid such as DOTAP), from about 1% to about 5% w / v cationic component (e.g., cationic lipid such as DOTAP), from about 3% to about 5% w / v cationic component (e.g., cationic lipid such as DOTAP), or from about 3% to about 4% w / v cationic component (e.g., cationic lipid such as DOTAP) or any other amount or range described herein for the cationic component. Higher or lower w / v percentages are contemplated herein, particularly when considering diluted or concentrated formulations.

[0088] In some cases, it may be desirable to use a cationic lipid that is soluble in the oil core. For example, DOTAP DOEPC, DODAC, and DOTMA are soluble in squalene or squalane. In other cases, it may be desirable to use a cationic lipid that is not soluble in the oil core. For example, DDA and DSTAP are not soluble in squalene. It is within the knowledge in the art to determine whether a particular lipid is soluble or insoluble in the oil and choose an appropriate oil and lipid combination accordingly. For example, solubility can be predicted based on the structures of the lipid and oil (e.g., the solubility of a lipid may be determined by the structure of its tail). For example, lipids having one or two unsaturated fatty acid chains (e.g., oleoyl tails), such as DOTAP, DOEPC, DODAC, DOTMA, are soluble in squalene or squalane; whereas lipids having saturated fatty acid chains (e.g., stearoyl tails) are not soluble in squalene. Alternatively, solubility can be determined according to the quantity of the lipid that dissolves in a given quantity of the oil to form a saturated solution).

[0089] The NLC may comprise additional lipids (i.e., neutral and anionic lipids) in combination with the cationic lipid so long as the net surface charge of the NLC prior to mixing with the bioactive agent is positive. Methods of measuring surface charge of a NLC are known in the art and include for example, as measured by Dynamic Light Scattering (DLS), Photon Correlation Spectroscopy (PCS), or gel electrophoresis.

[0090] D. Sorbitan Monoester

[0091] The present inventors discovered that a sorbitan ester when added to the NLC can act to enhance the effectiveness of the NLC in delivering the bioactive agent to a cell and / or in eliciting antibodies to an antigen in a subject where the bioactive agent is an antigen or encodes antigen and the composition is administered to a subject. In particular, it was discovered that the immune response to encoded proteins in the bioactive nucleic acid can be modulated by selection of sorbitan ester used in the NLC. It was surprisingly discovered that use of a sorbitan monoester was particularly effective at enhancing the effectiveness of the NLC. In some aspects, the acyl chain of the sorbitan monoester is saturated. In addition, without being bound by theory, it was surprisingly discovered that the sorbitan ester, and in particular, sorbitan monoester, acts in combination with the solid lipid (e.g., microcrystalline triglycerides) to enhance the effectiveness of the adjuvant activity of the NLC (e.g., in eliciting antibodies to an antigen in a subject where the bioactive agent is an antigen or encodes antigen and the composition is administered to a subject).

[0092] Exemplary sorbitan monoesters are commercially available under the tradenames SPAN® or ARLACEL®. An exemplary sorbitan monoester for use herein can be represented as a compound of Formula I or a stereoisomer thereof (including, but not limited to, Formula la, lb, Ic, or Id) wherein R is a saturated or unsaturated C1-C30 alkyl group, preferably a saturated or unsaturated C1-C20 alkyl group, more preferably a saturated or unsaturated C10-C20 alkyl group. In exemplary embodiments, the alkyl group is non-cyclic. Exemplary sorbitan monoesters also include positional isomers of Formulas I, la, lb, Ic, or Id (e.g., one of the hydroxy functional groups is replaced by an ester functional group -e.g., an alkyl ester wherein the alkyl is a saturated or unsaturated C1-C30 alkyl group, preferably a saturated or unsaturated C1-C20 alkyl group, more preferably a saturated or unsaturated C10-C20 alkyl group and R is OH). The skilled artisan will appreciate that exemplary sorbitan monoesters may be salt forms (e.g., pharmaceutically acceptable salts) of Formulas I, la, lb, Ic, Id and stereoisomers or positional isomers thereof.Formula IFormula Ic Formula Id

[0093] Particularly preferred sorbitan monoesters in this regard are sorbitan monostearate (also knowns as Span®60 and shown below) and sorbitan monooleate (also known as Span®80 and shown below), although other sorbitan monoesters can be used (including, but not limited to, sorbitan monolaurate (Span®20), sorbitan monopalmitate (Span®40)). Exemplary sorbitan monostearate is represented by Formula II or Ila or a salt form thereof and exemplary sorbitan monooleate is represented by Formula III or Illa or a salt form thereof.Formula Illa

[0094] In addition to providing sorbitan monoesters as a component of a NLC, also contemplated is the substitution of the sorbitan monoester for an alternative hydrophobic surfactant, including alternative sorbitan-based non-ionic surfactants. Accordingly, also provided herein are NLC particles comprising an oil core comprising a liquid phase lipid and a solid phase lipid, a cationic component (preferably a cationic lipid or phospholipid), a hydrophobic surfactant (e.g., non-ionic surfactantsincluding sorbitan-based non-ionic surfactants) and a hydrophilic surfactant. Sorbitan-based non-ionic surfactants include sorbitan esters other than sorbitan monoesters, for example sorbitan diesters and sorbitan triesters, such as for example, sorbitan trioleate (SPAN85™) and sorbitan tristearate (SPAN65™). Generally, the non-ionic surfactant (including sorbitan-based non-ionic surfactant) will have a hydrophilic-lipophilic balance (HLB) number between 1.8 to 8.6. All of the embodiments provided herein for the NLCs comprising a sorbitan monoester are applicable and contemplated for the NLCs comprising an alternative hydrophobic surfactant in place of the sorbitan monoester, e.g., NLCs comprising a sorbitan diester or triester in place of the sorbitan monoester. The sorbitan diester and triester or other hydrophobic surfactant can be present in the same concentrations as the sorbitan monoester. In some aspects, the acyl chains of the sorbitan diester or triester will be saturated.

[0095] Generally, the sorbitan esters (e.g., sorbitan monoesters) have a hydrophile-lipophile balance (HLB) value from 1 to 9. In some embodiments, the sorbitan esters (e.g., sorbitan monoesters) have an HLB value from 1 to 5. In some embodiments, the hydrophobic surfactant has a HLB value from about 4 to 5.

[0096] An exemplary sorbitan diester for use herein can be represented as a compound of Formula IV below or a stereoisomer thereof (e.g., wherein R is a saturated or unsaturated C1-C30 alkyl group, preferably a saturated or unsaturated C1-C20 alkyl group, more preferably a saturated or unsaturated C10-C20 alkyl group and at least one of R1 is H while the other is -C(=O)Y wherein Y is a saturated or unsaturated C1-C30 alkyl group, preferably a saturated or unsaturated C1-C20 alkyl group, more preferably a saturated or unsaturated C10-C20 alkyl group). In exemplary embodiments, the alkyl group is non-cyclic. Exemplary sorbitan diesters also include positional isomers of Formulas IV. The skilled artisan will appreciate that exemplary sorbitan diesters may be salt forms (e.g., pharmaceutically acceptable salts) of Formula IV and stereoisomers or positional isomers thereof.

[0097] As exemplary sorbitan triester for use herein can be represented as a compound of Formula V below or a stereoisomer thereof (including, but not limited to, Formula Va, Vb, or Vc) wherein R is a saturated or unsaturated C1-C30 alkyl group, preferably a saturated or unsaturated C1-C20 alkyl group, more preferably a saturated or unsaturated C10-C20 alkyl group and R1 is- C(=O)Y wherein Y can be the same or different in each instance and is a saturated or unsaturated C1-C30 alkyl group, preferably a saturated or unsaturated C1-C20 alkyl group, more preferably asaturated or unsaturated C10-C20 alkyl group. In exemplary embodiments, the alkyl group is non- cyclic. Exemplary sorbitan triesters also include positional isomers of Formulas V, Va, Vb, or Vo (e.g., the hydroxy functional group is replaced by an ester functional group (e.g., an alkyl ester wherein the alkyl is a saturated or unsaturated C1-C30 alkyl group, preferably a saturated or unsaturated C1-C20 alkyl group, more preferably a saturated or unsaturated C10-C20 alkyl group) and one of the alkyl esters (e.g., a ring alkyl ester or non-ring alkyl ester) is replaced by a hydroxy functional group). The skilled artisan will appreciate that exemplary sorbitan triesters may be salt forms (e.g., pharmaceutically acceptable salts) of Formulas V, Va, Vb, or Vc and stereoisomers or positional isomers thereof.Formula Va Formula Vb Formula Vc

[0098] With respect to stereoisomers, the skilled artisan will understand that the sorbitan esters may have chiral centers and may occur, for example, as racemates, racemic mixtures, and as individual enantiomers and diastereomers.

[0099] In embodiments wherein the sorbitan-based non-ionic surfactants is a sorbitan ester, typically, the NLC-based composition or formulation typically contains, for example, from about 0.1 % to about 15% sorbitan ester (w / v), 0.1% to about 10% sorbitan ester (w / v), from 0.1 % to about 5% sorbitan ester (w / v), about 0. 1 % to about 4 % sorbitan ester (w / v), about 0. 1 % to about 4% sorbitan ester (w / v), about 0. 1 % to about 2.5% sorbitan ester (w / v), about 0. 1 % to about 2% sorbitan ester (w / v), 0.1% to about 1.5% sorbitan ester (w / v), 0.1 % to about 1 % sorbitan ester (w / v), 0.1 % to about 0.5% sorbitan ester (w / v), 0.3% to about 2.5% sorbitan ester (w / v), about 0.3% to about 2% sorbitan ester (w / v), 0.3% to about 1.5% sorbitan ester (w / v), 0.3% to about 1 % sorbitan ester (w / v), 0.3% to about 0.5% sorbitan ester (w / v) or any other amount or range described herein for a sorbitan ester, including from about 0.25 % to about 15% sorbitan ester. In some aspects, the NLC-based compositions contain about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 2%, about 3% or about 4% (w / v) sorbitanester. Higher or lower w / v percentages are contemplated herein, particularly when considering diluted or concentrated formulations.

[0100] Accordingly, when the sorbitan ester is a sorbitan monoester (e.g., SPAN60™, SPAN80™), the NLC-based composition or formulation typically contains, for example, from about 0.1 % to about 15% sorbitan monoester (w / v), 0.1 % to about 10% sorbitan monoester (w / v), from 0.1% to about 5% sorbitan monoester (w / v), about 0. 1 % to about 4 % sorbitan monoester (w / v), about 0. 1% to about 4% sorbitan monoester (w / v), about 0. 1 % to about 2.5% sorbitan monoester (w / v), about 0. 1 % to about 2% sorbitan monoester (w / v), 0.1 % to about 1.5% sorbitan monoester (w / v), 0.1 % to about 1 % sorbitan monoester (w / v), 0.1 % to about 0.5% sorbitan monoester (w / v), 0.3% to about 2.5% sorbitan monoester (w / v), about 0.3% to about 2% sorbitan monoester (w / v), 0.3% to about 1.5% sorbitan monoester (w / v), 0.3% to about 1 % sorbitan monoester (w / v), 0.3% to about 0.5% sorbitan monoester (w / v) or any other amount or range described herein for sorbitan monoester, including from about 0.25 % to about 15% sorbitan monoester. In some aspects, the NLC-based composition or formulation contains about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1 %, about 2%, about 3% or about 4% (w / v) sorbitan monoester. Higher or lower w / v percentages are contemplated herein, particularly when considering diluted or concentrated formulations.

[0101] Accordingly, when the sorbitan ester is a sorbitan diester, the NLC-based composition or formulation typically contain, for example, from about 0.1 % to about 15% sorbitan diester (w / v), 0.1 % to about 10% sorbitan diester (w / v), from 0.1 % to about 5% sorbitan diester (w / v), about 0. 1% to about 4 % sorbitan diester (w / v), about 0. 1 % to about 4% sorbitan diester (w / v), about 0. 1 % to about 2.5% sorbitan diester (w / v), about 0. 1% to about 2% sorbitan diester (w / v), 0.1% to about 1.5% sorbitan diester (w / v), 0.1 % to about 1 % sorbitan diester (w / v), 0.1 % to about 0.5% sorbitan diester (w / v), 0.3% to about 2.5% sorbitan diester (w / v), about 0.3% to about 2% sorbitan diester (w / v), 0.3% to about 1.5% sorbitan diester (w / v), 0.3% to about 1 % sorbitan diester (w / v), 0.3% to about 0.5% sorbitan diester (w / v) or any other amount or range described herein for sorbitan diester, including from about 0.25 % to about 15% sorbitan diester. In some aspects, the NLC-based composition or formulation contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1 %, about 2%, about 3% or about 4% (w / v) sorbitan diester. Higher or lower w / v percentages are contemplated herein, particularly when considering diluted or concentrated formulations.

[0102] Accordingly, when the sorbitan ester is a sorbitan triester (e.g., SPAN85™ or SPAN65™), the NLC-based composition or formulation typically contain, for example, from about 0.1 % to about 15% sorbitan triester (w / v), 0.1 % to about 10% sorbitan triester (w / v), from 0.1 % to about 5% sorbitantriester (w / v), about 0. 1% to about 4 % sorbitan triester (w / v), about 0. 1 % to about 4% sorbitan triester (w / v), about 0. 1 % to about 2.5% sorbitan triester (w / v), about 0. 1 % to about 2% sorbitan triester (w / v), 0.1 % to about 1.5% sorbitan triester (w / v), 0.1% to about 1 % sorbitan triester (w / v), 0.1 % to about 0.5% sorbitan triester (w / v), 0.3% to about 2.5% sorbitan triester (w / v), about 0.3% to about 2% sorbitan triester (w / v), 0.3% to about 1 .5% sorbitan triester (w / v), 0.3% to about 1 % sorbitan triester (w / v), 0.3% to about 0.5% sorbitan triester (w / v) or any other amount or range described herein for sorbitan triester, including from about 0.25 % to about 15% sorbitan triester. In some aspects, the NLC-based composition or formulation contains about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 2%, about 3% or about 4% (w / v) sorbitan triester. Higher or lower w / v percentages are contemplated herein, particularly when considering diluted or concentrated formulations.

[0103] In exemplary embodiments, the sorbitan ester (e.g., sorbitan monoester, diester or triester) is present in an amount sufficient to increase the ability of the composition to facilitate delivery and / or expression of the bioactive agent (e.g., RNA) as compared to a comparable composition lacking the sorbitan ester (e.g., sorbitan monoester, diester, or triester respectively). In embodiments where the composition is administered to the subject in an effective amount, the composition may elicit antibody titers to the antigen equal to or greater than the antibody titers elicited when a comparable composition lacking the sorbitan ester is administered to the subject or when the bioactive agent is administered to the subject without the NLC. In some embodiments, the composition induces an immune response (e.g., neutralizing antibody titers) in the subject at a higher level than the immune response induced in the subject by a comparable composition lacking the sorbitan ester. Immune response may be, for example, innate, cellular or antibody responses. Neutralizing antibody titers may be determined by any assay known to one of skill in the art, including, without limitation, a plaque reduction neutralization titer analysis (Ratnam, S et al. J. Clin. Microbiol (2011), 33 (4): 811-815; Timiryazeva, T et al. Am J Trap Med Hyg (2013), 88(5): 962-970).

[0104] E. Surfactants

[0105] The NLCs described herein comprise a surfactant, in addition to the sorbitan-based nonionic surfactants (e.g., sorbitan ester). There are a number of surfactants specifically designed for and commonly used in biological applications. Such surfactants are divided into four basic types and can be used in the present invention: anionic, cationic, zwitterionic, and nonionic. A particularly useful group of surfactants are the hydrophilic non-ionic surfactants and, in particular, polyoxyethylene sorbitan monoesters and polyoxyethylene sorbitan triesters. These materials are referred to as polysorbates and are commercially available under the mark TWEEN® and are useful for preparing the NLCs. TWEEN® surfactants generally have a HLB value falling between 9.6 to 16.7. TWEEN®surfactants are commercially available. Other non-ionic surfactants which can be used are, for example, polyoxyethylene fatty acid ethers derived from lauryl, acetyl, stearyl, and oleyl alcohols, polyoxyethylene fatty acids made by the reaction of ethylene oxide with a long-chain fatty acid, polyoxyethylene, polyol fatty acid esters, polyoxyethylene ether, polyoxypropylene fatty ethers, bee's wax derivatives containing polyoxyethylene, polyoxyethylene lanolin derivative, polyoxyethylene fatty glycerides, glycerol fatty acid esters, or other polyoxyethylene fatty acid, alcohol, or ether derivatives of long-chain fatty acids of 12-22 carbon atoms.

[0106] In some embodiments, it is preferable to choose a non-ionic surfactant which has an HLB value in the range of about 7 to 16. This value may be obtained through the use of a single non-ionic surfactant such as a TWEEN® surfactant or may be achieved by the use of a blend of surfactants. In certain embodiments, the NLC comprises a single non-ionic surfactant, most particularly a TWEEN® surfactant, as the emulsion stabilizing non-ionic surfactant. In an exemplary embodiment, the emulsion comprises TWEEN® 80, otherwise known as polysorbate 80.

[0107] The NLC-based composition or formulation contains can contain, for example, from about 0.01% to about 15% surfactant (w / v), from about 0.01 % to about 10% surfactant (w / v) from about 0.01% to about 5% surfactant (w / v), about 0.01 % to about 2.5% surfactant, about 0.01 % to about 2% surfactant, 0.01 % to about 1.5% surfactant, 0.01 % to about 1% surfactant, 0.01 % to about 0.5% surfactant, 0.05% to about 0.5% surfactant, 0.08% to about 0.5% surfactant, about 0.08% surfactant, about 0.5% surfactant, about 0.6% surfactant, about 0.7% surfactant, about 0.8% surfactant, about 0.9% surfactant, or about 1 % surfactant, or about 2%, about 3%, about 4 % surfactant or any other amount or range described herein for surfactant. Higher or lower w / v percentages are contemplated herein, particularly when considering diluted or concentrated formulations.

[0108] Additional components can be included in the NLCs of the present invention including, for examples, components that promote NLC formation, improve the complex formation between the negatively charged molecules and the cationic particles, facilitate appropriate release of the negatively charged molecules (such as an RNA molecule), and / or increase the stability of the negatively charged molecule (e.g., to prevent degradation of an RNA molecule).

[0109] The aqueous phase (continuous phase) of the NLCs is typically a buffered salt solution (e.g., saline) or water. The buffered salt solution is typically an aqueous solution that comprises a salt (e.g., NaCI), a buffer (e.g., a citrate buffer), and can further comprise, for example, an osmolality adjusting agent (e.g., a saccharide), a polymer, a surfactant, or a combination thereof. If the emulsions are formulated for parenteral administration, it is preferable to make up final buffered solutions so that the tonicity, i.e., osmolality, is essentially the same as normal physiological fluids, to prevent undesired post-administration consequences, such as post-administration swelling or rapid absorption of thecomposition. It is also preferable to buffer the aqueous phase to maintain a pH compatible with normal physiological conditions. Also, in certain instances, it may be desirable to maintain the pH at a particular level to ensure the stability of certain components of the NLC. For example, it may be desirable to prepare a NLC that is isotonic (i.e., the same permeable solute, e.g., salt, concentration as the normal cells of the body and the blood) and isosmotic. To control tonicity, the NLC may comprise a physiological salt, such as a sodium salt. In some aspects, sodium chloride (NaCI), for example, may be used at about 0.9% (w / v) (physiological saline). Other salts that may be present include, for example, potassium chloride, potassium dihydrogen phosphate, disodium phosphate, magnesium chloride, calcium chloride, and the like. Non-ionic tonicifying agents can also be used to control tonicity. Monosaccharides classified as aldoses such as glucose, mannose, arabinose, and ribose, as well as those classified as ketoses such as fructose, sorbose, and xylulose can be used as non-ionic tonicifying agents in the present invention. Disaccharides such as sucrose, maltose, trehalose, and lactose can also be used. In addition, alditols (acyclic polyhydroxy alcohols, also referred to as sugar alcohols) such as glycerol, mannitol, xylitol, and sorbitol are non-ionic tonicifying agents that can be useful in the present invention. Non-ionic tonicity modifying agents can be present, for example, at a concentration of from about 0.1% to about 10% or about 1% to about 10%, depending upon the agent that is used.

[0110] The aqueous phase may be buffered. Any physiologically acceptable buffer may be used herein, such as water, citrate buffers, phosphate buffers, acetate buffers, tris buffers, bicarbonate buffers, carbonate buffers, succinate buffers, or the like. The pH of the aqueous component will preferably be between 4.0-8.0 or from about 4.5 to about 6.8. In another exemplary embodiment, the aqueous phase is, or the buffer is prepared using, RNase-free water or DEPC treated water. In some cases, high salt in the buffer might interfere with complexation of negatively charged molecule to the emulsion particle and therefore is avoided. In other cases, a certain amount of salt in the buffer may be included.

[0111] In an exemplary embodiment, the buffer is 10 mM citrate buffer e.g., (sodium citrate) with a pH between about 5.0 and 8.0. In another exemplary embodiment, the aqueous phase is, or the buffer is prepared using, RNase-free water or DEPC treated water. In other exemplary embodiments, the compositions of the present invention do not comprise a citrate buffer.

[0112] The aqueous phase may also comprise additional components such as molecules that change the osmolarity of the aqueous phase or molecules that stabilize the negatively charged molecule after complexation. Preferably, the osmolarity of the aqueous phase is adjusted using a non- ionic tonicifying agent, such as a sugar (e.g., trehalose, sucrose, dextrose, fructose, reduced palatinose, etc.), a sugar alcohol (such as mannitol, sorbitol, xylitol, erythritol, lactitol, maltitol, glycerol,etc.), or combinations thereof. If desired, a nonionic polymer (e.g., a poly(alkyl glycol) such as polyethylene glycol, polypropylene glycol, or polybutlyene glycol) or nonionic surfactant can be used.

[0113] G. Oil: Surfactant Ratios

[0114] Exemplary NLCs are composed of a hydrophobic core containing the liquid oil and solid lipid, and surfactants (also known as emulsifiers or emulsifying agents) that make up the interface separating the hydrophobic phase - liquid oil and solid lipid, collectively referred to here as oil - from the aqueous phase. Since surfactants typically reside on the surface of NLC particles, their amount dictates the total available surface area. On the other hand, the oil resides in the core and primarily contributes to the total available volume. Increasing the surfactant to oil ratio consequently increases the surface area (SA) to volume ratio (V); thus, for a fixed volume of material, increasing the SAA / ratio translates to reducing NLC particle diameter. Instead of, or in addition to, describing exemplary NLC compositions in terms of the w / v percentages of various components, NLC compositions can be described by the molar ratios of various components. In some aspects, exemplary NLCs of the present invention have an oil to surfactant molar ratio of from about 0.05 to about 12 or from about 0.05 to about 9 or from about .05 to about 8 or from about 0.05 to about 1 or from about 0.1 to about 1. The present inventors have demonstrated that by reducing the oil to surfactant molar ratio, smaller NLC particles can be synthesized. In addition, by reducing the amount of oil in the NLCs, potential toxicity of the formulations can be reduced. In other aspects, exemplary NLCs of the present invention have an oil to surfactant molar ratio of from about 0.5 to about 12, from about 0.5 to about 9, from 1 to about 9, from about 2 to about 9, from about 3 to about 9, from about 4 to about 9, from about 4.5 to about 9, or from about 4.5 or about 5 to about 7. Exemplary formulations have an oil to surfactant molar ratio of about 0.5, about 1 , about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 7, about 8, about 9, about 10, about 11 , or about 12. As used herein, the oil to surfactant molar ratio is determined by (I) adding the moles of lipid that make up the oil core (solid phase lipid and liquid phase lipid) to arrive at a value for moles of oil core lipid (II) adding the moles of the cationic component (e.g., DOTAP), hydrophobic surfactant (e.g., sorbitan ester) and hydrophilic surfactant (tween 80) to arrive at a value for moles surfactant, and (ill) dividing moles of oil core lipid by moles of surfactant.

[0115] H. Hydrophilic Surfactant: Cationic component Ratios

[0116] The present inventors have discovered that the ratio of hydrophilic surfactant to cationic component can impact the ability of the NLC to have a protective effect from RNAase degradation and can impact the immunogenicity of the formulations. The Tween:DOTAP ratios may be about 0.6 for obtaining consistent results for delivery and expression of RNA bioactive agents whereas Tween:DOTAP ratios at about 2.0 and higher are not as optimal for obtaining such consistency.Accordingly, exemplary NLCs of the present invention have a hydrophilic surfactanbcationic component (e.g., cationic lipid) ratio of from about 0.2 to about 1 .5, from about 0.2 to about 1 or from about 0.5 to about 1 . When Tween and DOTAP are in the composition, exemplary NLCs of the present invention have a tween:DOTAP ratio of from about 0.2 to about 1 .5, from about 0.2 to about 1 or from about 0.5 to about 1 . As used herein, the hydrophilic surfactanbcationic component ratio is determined by (i) adding the moles of hydrophilic surfactant to arrive at a value for moles of hydrophilic surfactant (ii) adding the moles of the cationic component to arrive at a value for moles of cationic component, and (iii) dividing moles of hydrophilic surfactant by moles of cationic component.

[0117] I. Loading Capacities

[0118] The present inventors have discovered the loading capacity of the NLC formulations can be manipulated by modulating the ratio of hydrophilic surfactant to cationic component and the amount of oil present in the formulations thereby reducing the average NLC particle size. Exemplary NLC formulations have loading capacity for RNA of at least about 10 pig / ml RNA, at least about 20 pig / ml RNA, at least about 50 pig / ml RNA, at least about 100 pig / ml RNA, at least about 200 pig / ml RNA, at least about 300 pig / ml, or at least about 400 pig / ml RNA. NLC formulations having an average particle size of from 20 nm to about 110 nm, from about 20 nm to about 80 nm, from about 20 nm to about 70 nm, or from about 20 nm to about 60 nm typically have increased loading capacity.

[0119] A. Exemplary Nanostructured Carriers and their w / v percentages

[0120] In select exemplary embodiments, the NLC composition comprises from about 0.2% to about 40% w / v liquid phase lipid, from about 0.02% to about 10% w / v solid phase lipid, from about 0.2% to about 10 % w / v cationic lipid, from about 0.25% to about 5% w / v hydrophobic surfactant (e.g., sorbitan ester), and from about 0.2% to about 10% w / v, from about 0.2% to about 5% w / v, from about 0.5% to about 5% w / v or from about 0.5% to about 10% w / v hydrophilic surfactant. This NLC composition is referred to herein as formulation A. In any aspect of formulation A, the hydrophilic surfactant can be present at 0.2% to about 10% w / v, 0.2% to about 5% w / v, 0.5% to about 5% w / v or from about 0.5% to about 10% w / v.

[0121] In select exemplary embodiments, the NLC composition comprises from about 0.2% to about 40% w / v liquid phase lipid, from about 0.1% to about 10% w / v solid phase lipid, from about 0.2% to about 10 % w / v cationic lipid, from about 0.25% to about 5% w / v hydrophobic surfactant (e.g., sorbitan ester), and from about 0.2% to about 10% w / v, from about 0.2% to about 5% w / v, from about 0.5% to about 5% w / v or from about 0.5% to about 10% w / v hydrophilic surfactant. This NLC composition is referred to herein as formulation B. In any aspect of formulation B, the hydrophilic surfactant can be present at 0.2% to about 10% w / v, 0.2% to about 5% w / v, 0.5% to about 5% w / v or from about 0.5% to about 10% w / v.

[0122] In select exemplary embodiments, the NLC composition comprises from about 0.2% to about 1% w / v liquid phase lipid, from about 0.02% to about 1 % w / v solid phase lipid, from about 2% to about 10 % w / v cationic lipid, from about 2% to about 5% w / v sorbitan ester, and from about 2% to about 5% w / v hydrophilic surfactant. This NLC composition is referred to herein as formulation C.

[0123] In select exemplary embodiments, the NLC composition comprises from about 2% to about 40% w / v liquid phase lipid, from about 0.1% to about 10% w / v solid phase lipid, from about 0.2% to about 10 % w / v cationic lipid, from about 0.25% to about 5% w / v hydrophobic surfactant (e.g., sorbitan ester), and from about 0.2% to about 10% w / v, from about 0.2% to about 5% w / v, from about 0.5% to about 5% w / v or from about 0.5% to about 10% w / v hydrophilic surfactant. This NLC composition is referred to herein as formulation D. In any aspect of formulation D the hydrophilic surfactant can be present at 0.2% to about 10% w / v, 0.2% to about 5% w / v, about 0.5% to about 5% w / v or from about 0.5% to about 10% w / v hydrophilic surfactant

[0124] In select exemplary embodiments, the NLC composition comprises from about 2% to about 10% w / v liquid phase lipid, from about 0.1% to about 10% w / v solid phase lipid, from about 0.2% to about 10 % w / v cationic lipid, from about 0.25% to about 5% w / v sorbitan ester, and from about 0.2% to about 10 % or from about 0.2% to about 5 % w / v hydrophilic surfactant. This NLC composition is referred to herein as formulation E. In any aspect of formulation E, the hydrophilic surfactant can be present at 0.2% to about 10 % or from about 0.2% to about 5 % w / v.

[0125] In select exemplary embodiments, the NLC composition comprises from about 2% to about 10% w / v liquid phase lipid, from about 0.1 % to about 3% w / v solid phase lipid, from about 1 % to about 5 % w / v cationic lipid, from about 1 % to about 5% w / v sorbitan ester, and from about 1 % to about 5 % w / v hydrophilic surfactant. This NLC composition is referred to herein as formulation F.

[0126] In select exemplary embodiments, the NLC composition comprises from about 2% to about 5% w / v liquid phase lipid, from about 0.1 % to about 2% w / v solid phase lipid, from about 2% to about 5 % w / v cationic lipid, from about 2% to about 5% w / v sorbitan ester, and from about 2% to about 5 % w / v hydrophilic surfactant. This NLC composition is referred to herein as formulation G.

[0127] In select exemplary embodiments, the NLC composition comprises from about 2% to about 10% w / v liquid phase lipid, from about 0.1 % to about 3% w / v solid phase lipid, from about 0.2% to about 2 % w / v cationic lipid, from about 0.25% to about 2% w / v sorbitan ester, and from about 0.2% to about 5% w / v or from about 0.5% to about 5% w / v hydrophilic surfactant. This NLC composition is referred to herein as formulation H. In any aspect of formulation H, the hydrophilic surfactant can be present at about 0.2% to about 5% w / v or from about 0.5% to about 5% w / v.

[0128] The skilled artisan will understand that any of the NLC compositions / formulations described herein, can be diluted or concentrated for use in the present invention. For example, whenmixed with a bioactive agent for delivery, the formulation may be diluted in the mixing process. The NLC compositions / formulations can be diluted for example, 1 :2. All of the NLC compositions / formulations described herein can be diluted, for example, from about 2 to about 500 fold, preferably from about 2 to about 100 fold. Typically, but not always, dilution occurs when mixing the formulation with a bioactive agent (e.g., RNA or DNA) for delivery. They may be diluted, for example, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 15 fold, about 20 fold, about 25 fold, about 30 fold, about 100 fold, or about 500 fold. As will be understood by the skilled practitioner, a diluted formulation of the present invention will have a decreased concentration of the liquid phase lipid, solid phase lipid, cationic component, hydrophobic surfactant (e.g., sorbitan ester), and surfactant (e.g., hydrophilic surfactant), however, the ratio of liquid phase lipid to solid phase lipid to cationic component to hydrophobic surfactant (e.g., sorbitan ester) to surfactant will remain the same. The present invention provides not only formulations A through W but diluted versions of the formulations A through W. In some cases, it is the diluted formulations that are associated with (e.g., complexed to) the bioactive agent. For example, a particularly preferred formulation is formulation S or formulation T diluted 2 fold. Such diluted formulation S comprises about 2 % w / v liquid phase lipid, about 0.13% w / v solid phase lipid, about 0.2% w / v cationic lipid, about 0.25% w / v sorbitan ester, and about 0.25% w / v hydrophilic surfactant. Such diluted formulation T comprises about 1.88 % w / v liquid phase lipid, about 0.13 % w / v solid phase lipid, about 1.5% w / v cationic lipid, about 1.85% w / v sorbitan ester, and about 1.85% w / v hydrophilic surfactant.

[0129] Alternatively, the compositions / formulations, may be concentrated, for example from about 2 to about 30 fold, preferably from about 2 to about 20 fold. They may be concentrated, for example, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 15 old, about 20 fold, about 25 fold or about 30 fold. Accordingly, the present invention provides not only formulations A through W but concentrated versions of the formulations A through U.

[0130] For any of NLC formulations of the present invention including formulations A through V, and included diluted and concentrated formulations of formulations A through V, the following and any combination of the following can apply: (I) the sorbitan ester is a sorbitan monoester, diester, or triester; (II) the sorbitan ester is a sorbitan monoester selected from sorbitan monostearate or sorbitan monooleate or sorbitan monolaurate or a sorbitan triester selected from sorbitan trioleate or sorbitan tristearate; (ill) the liquid phase lipid is squalene; (iv) the solid phase lipid is a glycerolipid; (v) the solid phase lipid is microcrystalline triglyceride; (vi) The solid phase lipid is trimyristin; (vii) The cationic lipid is DOTAP; (viii) the hydrophilic surfactant is polysorbate 80 (also referred to as Tween 80); (lx) thesorbitan ester is a monoester, the liquid phase lipid is squalene; and the solid phase lipid is a glycerolipid; (x) the sorbitan ester is a monoester; the liquid phase lipid is squalene; the solid phase lipid is a glycerolipid; the cationic lipid is DOTAP; and the hydrophilic surfactant is polysorbate 80; (xi) the sorbitan ester is sorbitan monostearate or sorbitan monooleate or sorbitan monolaurate; the liquid phase lipid is squalene; the solid phase lipid is trimyristin; the cationic lipid is DOTAP; and the hydrophilic surfactant is polysorbate 80; (xii) the sorbitan ester is a triester, the liquid phase lipid is squalene; and the solid phase lipid is a glycerolipid; (xiii) the sorbitan ester is a triester; the liquid phase lipid is squalene; the solid phase lipid is a glycerolipid; the cationic lipid is DOTAP; and the hydrophilic surfactant is polysorbate 80; (xiv) the sorbitan ester is sorbitan trioleate or sorbitan tristearate; the liquid phase lipid is squalene; the solid phase lipid is trimyristin; the cationic lipid is DOTAP; and the hydrophilic surfactant is polysorbate 80.

[0131] The present invention also provides formulations A through Q, including those indicated above wherein the oil to surfactant ratio is from about 0.05 to about 12 or from about 0.05 to about 9 or from about .05 to about 8 or from about 0.05 to about 1 or from about 0.1 to about 1 . Also provided are formulations A through Q, including those indicated above in paragraph

[0154] , wherein the oil to surfactant molar ratio of from about 0.5 to about 12, from about 1 to about 9, from about 2 to about 9, from about 3 to about 9, from about 4 to about 9, from about 4.5 to about 9, or from about 4.5 or about 5 to about 7.

[0132] The present invention provides formulations A through Q, including those indicated above, wherein the hydrophilic SurfactanbCationic component (e.g., cationic lipid) ratio is from about 0.2 to about 1 or from about 0.5 to about 1 .

[0133] Accordingly, some exemplary NLC compositions are formulations A, B, or Q diluted or concentrated, with an oil to surfactant molar ratio of about 0.5 to about 12, from about 1 to about 9, from about 2 to about 9, from about 3 to about 9, from about 4 to about 9, from about 4.5 to about 9, or from about 4.5 or about 5 to about 7 and a hydrophilic SurfactanbCationic component (e.g., cationic lipid) ratio from about 0.2 to about 1 .5.

[0134] Accordingly, some exemplary NLC compositions are formulations A, B, or Q, diluted or concentrated, with an oil to surfactant molar ratio of about 0.5 to about 12, from about 1 to about 9, from about 2 to about 9, from about 3 to about 9, from about 4 to about 9, from about 4.5 to about 9, or from about 4.5 or about 5 to about 7 and a hydrophilic SurfactanbCationic component (e.g., cationic lipid) ratio from about 0.2 to about 1 .

[0135] Some exemplary NLC compositions are formulations A, B, or Q, diluted or concentrated, with an oil to surfactant molar ratio of about 0.5 to about 12, from about 1 to about 9, from about 2 to about 9, from about 3 to about 9, from about 4 to about 9, from about 4.5 to about 9, or from about4.5 or about 5 to about 7 and a hydrophilic SurfactanbCationic component (e.g., cationic lipid) ratio from about 0.5 to about 1.

[0136] Some exemplary NLC compositions are formulations A, B, or Q, diluted or concentrated, with an oil to surfactant molar ratio of 0.05 to about 12 or from about 0.05 to about 9 or from about .05 to about 8 or from about 0.05 to about 1 or from about 0.1 to about 1 and a hydrophilic SurfactanbCationic component (e.g., cationic lipid) ratio from about 0.5 to about 1 .5.

[0137] Some exemplary NLC compositions are formulations A, B, or Q, diluted or concentrated, with an oil to surfactant molar ratio of 0.05 to about 12 or from about 0.05 to about 9 or from about .05 to about 8 or from about 0.05 to about 1 or from about 0.1 to about 1 and a hydrophilic SurfactanbCationic component (e.g., cationic lipid) ratio from about 0.5 to about 1.

[0138] The present invention provides formulations A through W wherein the average diameter of the NLC particles is from about 40 nm or 50 nm to about 80 nm, from about 40 nm or 50 nm to about 70 nm, from about 40 nm or 50 nm to about 60 nm.

[0139] B. Physiochemical Characteristics of the Nanostructured Lipid Carriers

[0140] I. Size

[0141] The size of the NLC particles can be assessed by known techniques in the art, including but not limited to, x-ray and laser diffraction, dynamic light scattering (DLS), CryoEM, or Malvern Zetasize. In some embodiments, the size of the NLC refers to the Z-average diameter.

[0142] The NLC particles have an average diameter (i.e., the number average diameter) of 1 micrometer or less. It is particularly desirable that the average particle size (i.e., the number average diameter) of the NLC is about 900 nm or less, about 800 nm or less, about 700 nm or less, about 600 nm or less, about 500 nm or less, about 400 nm or less, 300 nm or less, 200 nm or less, 100 nm or less or 80 nm or less, for example, from about 50 nm to about 900 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 50 nm to about 175 nm, from about 50 nm to about 150 nm, from about 50 nm to about 125 nm, from about 50 nm to about 100 nm, from about 50 nm to about 80 nm, from about 40 nm to about 80 nm, from about 20 nm to about 80 nm, from about 40 nm to about 80 nm, or from about 40 nm to about 60 nm. It will be understood by the skilled practitioner that a NLC is made up of NLC particles. The average particle size refers to the average diameter of the particles that make up the NLC. The average diameter of the NLC particles is typically about 40 nm, is about 60 nm, is about 80 nm, is about 85 nm, is about 90 nm, is about 95 nm, is about 100 nm, is about 105 nm, is about 110 nm, is about 115 nm, is about 120 nm, is about 125 nm, is about 130 nm, is about 135 nm, is about 140 nm, is about 145 nm, is about 150 nm, is about 155 nm, is about 160 nm, isabout 165 nm, is about 170 nm, is about 175 nm, is about 180 nm, is about 185 nm, is about 190 nm, is about 195 nm, or is about 200 nm.

[0143] In some aspects, the average diameter of the NLC particles is from about 20 nm to about 200 nm, from about 20 nm to about 150 nm, from about 20 nm to about 110 nm, from about 20 nm to about 80 nm, from about 20 nm to about 70 nm, from about 20 nm to about 60 nm.

[0144] In some aspects, the average diameter of the NLC particles is from about 50 nm to about 200 nm, from about 50 nm to about 150 nm, from about 50 nm to about 110 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm.

[0145] In some aspects, the average diameter of the NLC particles is from about 40 nm to about 80 or from about 40 nm to about 60 nm.

[0146] An exemplary NLC of the present invention is capable of being filtered through at least a 0.45 micron filter. In an exemplary embodiment, the NLC is capable of being filtered through a 0.20 or 0.22 micron filter.

[0147] 2. Stability

[0148] Exemplary NLCs provided herein are stable, allowing for ease of use, manufacturability, transportability, and storage. The physiochemical characteristics of the NLC, including, but not limited to its size, is maintained over time, at various temperatures, and under various conditions.

[0149] The evolution of particle size over a function of time provides colloidal stability information. An exemplary stable NLC composition is one whose particles retain substantially the same z-average diameter size over a time period (e.g., a 30 day or 7 day time period) at different temperatures typically but not limited to 37, 25 or 5 degrees Celsius. By retaining substantially the same z-average diameter size, it is meant that a particle remains within 20%, 15%, 10%, or 5% of its original size over a 30 day time period. A particularly stable NLC composition is one whose particles retain substantially the same z-average diameter size over a 30 day period at 4 degrees Celsius, 25 degrees Celsius or even 37 degrees Celsius.

[0150] The stability of the NLC can be measured by techniques familiar to those of skill in the art. In some embodiments, the stability is observed visually. Visual inspection can include inspection for particulates, flocculence, or aggregates. Typically, colloidal stability is determined by the particle size of the NLC, such as by measuring the z-average diameter and optionally expressed as change in size over time, or at various temperatures, or under certain conditions. In some embodiments, the stability is determined by assessing the increase in particle size. In some embodiments, stability is determined by measurement of the polydispersity index (PDI), for example with the use of the dynamic light scattering (DLS) technique. In other embodiments, stability is determined by measurement of the zeta potential with the use of the DLS technique.

[0151] In some embodiments, the Z-average diameter of the NLC increases less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 12%, less than 10%, less than 7%, less than 5%, less than 3%, or less than 1 % over the time period assayed.

[0152] In some embodiments the polydispersity index of the NLC is maintained at about 0.5, at about 0.4, at about 0.3, at about 0.2, at about 0.1 or at from about 0.1 to about 0.5, at from about 0.1 to about 0.4, at from about 0.1 to about 0.3, at from about 0.1 to about 0.2, at from about 0.2 to about 0.4, or at from about 0.2 to about 0.3. In some preferred aspects, the polydispersity index is greater than 0.1 , greater than 0.15, or greater than 0.2.

[0153] Exemplary NLC-based compositions of the present invention are stable for greater than 6 months at 25 degrees Celsius (e.g., retain substantially the same z-average diameter size).

[0154] IV. Lipid-based Nanoparticles

[0155] In some embodiments, the compositions and formulations of the disclosure comprise lipid-based nanoparticles. For example, the composition can comprise a nanostructured lipid carrier (NLC), a liposome, a lipid nanoparticle (LNP), a solid lipid nanoparticie (SLN), an oil-in-water emulsion, a cationic nanoemulsion (CNE), a charge-altering releasable transporter (CART), or a polymeric nanoparticie.

[0156] Lipid Nanoparticles

[0157] In some embodiments, compositions of this disclosure may use lipid nanoparticles (LNP) as an artificial RNA delivery system for ribonucleic acid (RNA) polynucleotide encoding a replication- competent viral genome. LNPs are one example of lipid-based nanoparticles. RNA polynucleotides of this disclosure may be complexed or combined with LNP either on the outside or inside of the particle. LNPs are spherical vesicles made of ionizable lipids, which are positively charged at low pH (enabling RNA complexation) and neutral at physiological pH (reducing potential toxic effects, as compared with positively charged lipids, such as liposomes). LNPs typically comprise an ionizable lipid, a PEGylated lipid, a phospholipid, and cholesterol. Owing to their size and properties, lipid nanoparticles are taken up by cells via endocytosis, and without being bound by theory it is believed that the ionizability of the lipids at low pH enables endosomal escape, which allows release of the cargo into the cytoplasm.

[0158] In addition, LNPs usually may contain any or all of a helper lipid to promote cell binding, cholesterol to fill the gaps between the lipids, and a polyethylene glycol (PEG) to reduce opsonization by serum proteins and reticuloendothelial clearance. The relative amounts of ionizable lipid, helper lipid, cholesterol, and PEG can affect the efficacy of lipid nanoparticles and may be optimized for a given application and administration route. Moreover, lipid type, size, and surface charge impact the behavior of lipid nanoparticles in vivo.

[0159] Lipid nanoparticle (LNP) delivery systems are described in L. A. Jackson et al., An mRNA Vaccine against SARS-CoV-2 - Preliminary Report. N Engl J Med 383, 1920-1931 (2020); Y. Y. Tam, S. Chen, P. R. Cullis, Advances in Lipid Nanoparticles for siRNA Delivery. Pharmaceutics 5, 498-507 (2013); Y. Zhao and L. Huang, Lipid nanoparticles for gene delivery. Adv Genet 88, 13-36 (2014); A. M. Reichmuth et al., mRNA vaccine delivery using lipid nanoparticles. Therapeutic Delivery 7, 319- 334 (2016); K. Bahl et al. , Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses. Mol Ther 25, 1316-1327 (2017). LNP formulations may contain cationic and ionizable lipids with RNA associated with either the interior or exterior of the particle. (A. K. Blakney et al., Inside out: optimization of lipid nanoparticle formulations for exterior complexation and in vivo delivery of saRNA. Gene Ther 26, 363-372 (2019).

[0160] Cationic Nanoemulsions

[0161] In some embodiments, compositions of this disclosure comprise cationic nanoemulsions (CNE) as an artificial RNA delivery system for ribonucleic acid (RNA) polynucleotide encoding a replication-competent viral genome. CNEs are one example of lipid-based nanoparticles. CNE consists of a dispersion of an oil phase stabilized by an aqueous phase containing the cationic lipid. These nanoemulsions present a droplet size distribution of about 200 nm and are used to formulate RNA vaccines. (L. A. Brito et al., A cationic nanoemulsion for the delivery of next-generation RNA vaccines. Mol Ther 22, 2118-2129 (2014).

[0162] Charge-Altering Releasable T ransporters

[0163] In some embodiments, compositions of this disclosure comprise charge-altering releasable transporters (CARTs). CARTs are one example of lipid-based nanoparticles. CARTs are single component amphiphilic diblock oligomers containing a sequence of lipid monomers and a sequence of cationic monomers that provide an alternative delivery vehicle RNA besides lipid particles. In some embodiments, compositions of this disclosure may use amphiphilic diblock oligomers containing a sequence of lipid monomers and a sequence of cationic monomers as an artificial RNA delivery system for ribonucleic acid (RNA) polynucleotide encoding a replication- competent viral genome. CARTs electrostatically encapsulate mRNA (or other coformulated nucleotides like CpG) and deliver the genetic cargo into cells. A unique feature of CARTs is their ability to undergo a charge-altering rearrangement to produce neutral diketopiperazine small molecules (DKPs). This transformation facilitates the release of mRNA and eliminates any toxic issues associated with persistent cations. The CART technology is described in Ole A.W. Haabeth et al., An mRNA SARS-CoV-2 vaccine employing Charge-Altering Releasable Transporters with a TLR-9 agonist induces neutralizing antibodies and T cell memory, (2021) bioRxiv 2021.04.14.439891.

[0164] Polymeric Nanoparticles

[0165] In some embodiments, compositions of this disclosure comprise polymeric nanoparticles. Polymeric nanoparticles are one example of lipid-based nanoparticles. In some embodiments, polymeric nanoparticles can be used as an artificial RNA delivery system for ribonucleic acid (RNA) polynucleotide encoding a replication-competent viral genome. Polymeric nanoparticles comprise linear PBAE polymers and branched hybrid PBAE (bhP-BAE), and are described in Kim, H., Kirtane, A.R., Kim, N.Y. et al. Gastrointestinal Delivery of an mRNA Vaccine Using Immunostimulatory Polymeric Nanoparticles. AAPS J 25, 81 (2023). https: / / doi.org / 10.1208 / s12248-023-00844-z.

[0166] V. Bioactive Agents

[0167] In some exemplary embodiments, to deliver a bioactive agent, the formulations of the present invention are mixed or otherwise formulated with one or more bioactive agents. The term "bioactive agent” as used herein refers to any material to be delivered by the formulations of the present disclosure and can include without limitation macromolecules, peptides, proteins, peptidomimetics, nucleic acids, oligonucleotides, deoxyribonucleotides, plasmid DNA, circular DNA, linear DNA, single-stranded DNA, modified DNA, antisense DNA, ribonucleotides, mRNA, chemically modified RNA, non-coding RNA, miRNA, siRNA, tRNA, ribosomal RNA, RNA ribozymes, replicon RNA, RNA aptamers, DNA aptamers, double-stranded RNA, base-substituted RNA, inosine- containing RNA, adjuvants including TLR agonists (for example TLR2, TLR3, TLR4, TLR 7, TLR8, and TLR9 agonists), Rig-I agonists, saponins, carbohydrates, carbohydrate polymers, conjugated carbohydrates, whole viral particles, virus-like particles, viral fragments, and cellular fragments. Nonlimiting exemplary adjuvants include double-stranded RNA, RIBOXXOL, poly(l:C), and Hiltonol® (poly-ICLC). Hiltonol® (poly-ICLC) is a synthetic complex of carboxymethylcellulose, polyinosinic- polycytidylic acid double-stranded RNA, and poly-L-lysine. RIBOXXOL is an annealed 50 bp RNA duplex (Riboxx GmbH). Any bioactive agent that can be delivered safely to a cell can be mixed with a NLC of the present invention. When negatively charged molecules are to be delivered, in some embodiments, the cationic NLC surface can interact with negatively charged bioactive agents thereby anchoring the molecules to the NLC.

[0168] Exemplary negatively charged molecules to be used as bioactive agents include, for example, peptide-containing antigens, nucleic acid molecules (e.g., RNA or DNA) that encode one or more peptide-containing antigens, negatively charged polysaccharides, negatively charged small molecules, and negatively charged immunological adjuvants. Negatively charged immunological adjuvants include, for example, immunostimulatory oligonucleotides (e.g., CpG oligonucleotides), single-stranded RNAs, small molecule immune potentiators (SMIPs), and the like. Negatively charged small molecules include, for example, phosphonate, fluorophosphonate, and the like.

[0169] Current adjuvants are largely Th2 biased, such as alum. In some embodiments, for vaccines against cancer and infectious disease targets (e.g., tuberculosis, several viral diseases, etc.) as well as allergy, adjuvants that promote a Th 1 bias are an unmet need. In this regard, as described herein, the present inventors have demonstrated formulations promoting a Th1 bias for TLR3 agonists, for example. Such formulations promote IFN gamma production and downregulate IL-5 and are suitable for various uses in which a Th 1 bias is desired.

[0170] One or more bioactive agents may be associated with the formulations of the present invention. One of skill in the art would understand that various combinations of bioactive agents may be associated with the formulations such as, but not limited to, multiple RNAs, multiple DNAs, one or more RNAs of a defined sequence and one or more proteins, one or more DNAs and one or more proteins, and one or more RNAs and one or more DNAs. In some aspects, one bioactive agent can be present in the oil core of an NLC particle while the other is associated with the surface of the NLC particle. For example, a nucleic acid may be associated with the NLC particle surface whereas a biologically active small molecule may be present within the oil core of the NLC particle.

[0171] In an exemplary embodiment, the negatively charged bioactive agent is complexed with an NLC particle by association with cationic surface. The association of the negatively charged bioactive agent with the NLC particle surface may be a non-covalent or a reversible covalent interaction.

[0172] In another embodiment, a hydrophobic bioactive agent such as a Toll-like receptor ligand (e.g., TLR4 ligand) can be incorporated in the oily core or at the interface of the NLC particle.A. RNA Molecules

[0173] In embodiments where the bioactive agent is a RNA molecule, the RNA molecule may encode proteins of various types, including, without limitation, antigens, antibodies, toxins, growth factors, cytokines, and hormones. RNA molecules used herein may also represent non-coding RNAs, including, without limitation, siRNA, miRNA, CRISPR guide RNA, ribozyme RNA, hairpins, RNA aptamers, RNA agonists, and immunomodulatory RNAs.

[0174] In an exemplary embodiment, the negatively charged RNA molecule is complexed with the NLC particle by association with the cationic surface. The association of the RNA molecule with the NLC particle surface may be a non-covalent or reversible covalent interaction.

[0175] In exemplary embodiments, the bioactive agent is a self-amplifying RNA molecule. Selfamplifying RNA molecules are well known in the art and can be produced by using replication elements derived from viruses (e.g., alphavirus, flavivirus, picornavirus), and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest. A self-amplifying RNA molecule is typically a (+)-strand molecule which can be directly translated after delivery to a cell, andthis translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as co-linear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen. The overall results of this sequence of transcriptions is an amplification in the number of the introduced replicon RNAs and thereby the encoded antigen becomes a major polypeptide product of the cells.

[0176] Advantageously, the cell's translational machinery is used by self-amplifying RNA molecules to generate a significant increase of encoded gene products, such as proteins or antigens, which can accumulate in the cells or be secreted from the cells. Self-amplifying RNA molecules may, for example, stimulate toll-like receptors (TLR) 3, 7 and 8 and non TLR pathways (e.g., RIG-I, MD-5) by the products of RNA replication and amplification, and translation which may induce apoptosis of the transfected cell.

[0177] The self-amplifying RNA can, for example, contain at least one or more genes selected from the group consisting of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins, and also comprise 5'and 3'-end cis-active replication sequences, and if desired, heterologous sequences that encode a desired amino acid sequences (e.g., an antigen of interest). A subgenomic promoter that directs expression of the heterologous sequence can be included in the self-amplifying RNA. If desired, the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions, with or without a ribosomal skipping peptide sequence in the selfamplifying RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0178] In certain embodiments, the self-amplifying RNA molecule is not encapsulated in a viruslike particle. Self-amplifying RNA molecules of the invention can be designed so that the selfamplifying RNA molecule cannot induce production of infectious viral particles. This can be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary for the production of viral particles in the self-amplifying RNA. For example, when the self-amplifying RNA molecule is based on an alpha virus, such as Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE), one or more genes encoding viral structural proteins, such as capsid (C) and / or envelope (E) glycoproteins, can be omitted.

[0179] If desired, self-amplifying RNA molecules of the invention can also be designed to induce production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single round of subsequent infection.

[0180] One suitable system for achieving self-replication in this manner is to use an alphavirusbased replicon. Alphaviruses comprise a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Thirty-one species have been classified within the alphavirus genus, including, Sindbis virus, Semliki Forest virus, Ross River virus, chikungunya virus, and Venezuelan equine encephalitis virus. As such, the self-amplifying RNA of the invention may incorporate an RNA replicase derived from Semliki Forest virus (SFV), Sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), Ross-River virus (RRV), eastern equine encephalitis virus, chikungunya virus, or other viruses belonging to the alphavirus genus.

[0181] An alphavirus-based “replicon” expression vector can be used in the invention. Replicon vectors may be utilized in several formats, including DNA, RNA, and recombinant replicon particles. Such replicon vectors have been derived from alphaviruses that include, for example, Sindbis virus (Xiong et al. (1989) Science 243:1188-1191; Dubensky et al., (1996) J. Virol. 70:508-519; Hariharan et al. (1998) J. Virol. 72:950-958; Polo et al. (1999) PNAS 96:4598-4603), Semliki Forest virus (Liljestrom (1991) Bio / Technology 9:1356-1361 ; Berglund et al. (1998) Nat. Biotech. 16:562-565), and Venezuelan equine encephalitis virus (Pushko et al. (1997) Virology 239:389-401). Alphaviruses- derived replicons are generally quite similar in overall characteristics (e.g., structure, replication), individual alphaviruses may exhibit some particular property (e.g., interferon sensitivity, and disease profile) that is unique. Therefore, chimeric alphavirus replicons made from divergent virus families may also be useful.

[0182] Alphavirus-based RNA replicons are typically (+)-stranded RNAs which lead to translation of a replicase (or replicase-transcriptase) after delivery to a cell. The replicase is translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic (-)-strand copies of the (+)-strand delivered RNA. These (-)-strand transcripts can themselves be transcribed to give further copies of the (+)-stranded parent RNA and also to give a subgenomic transcript which encodes the antigen. Translation of the subgenomic transcript thus leads to in situ expression of the antigen by the infected cell. Suitable alphavirus replicons can use a replicase from a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc.

[0183] An RNA replicon can comprise, for example, an RNA genome from a picornavirus, togavirus (e.g., alphaviruses such as, for example, Sindbis virus, Semliki Forest virus, Venezuelan equine encephalitis virus, or Ross River virus), flavivirus (e.g., yellow fever virus), coronavirus, paramyxovirus, which has been modified by the replacement of one or more structural protein genes with a selected heterologous nucleic acid sequence encoding a product of interest.

[0184] In some aspects, a replicon will encode (i) a RNA-dependent RNA polymerase which can transcribe RNA from the replicon and (ii) an antigen. The polymerase can be, for example, analphavirus replicase e.g. comprising one or more of alphavirus proteins nsP1 , nsP2, nsP3 and nsP4. Whereas natural alphavirus genomes encode structural virion proteins in addition to the non-structural replicase polyprotein, it is preferred that the replicon does not encode alphavirus structural proteins. Thus, a replicon can lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA-containing virions. The inability to produce these virions means that, unlike a wildtype alphavirus, the preferred replicon cannot perpetuate itself in infectious form. The alphavirus structural proteins which are necessary for perpetuation in wild-type viruses are absent from the preferred replicon and their place is taken by gene(s) encoding the antigen of interest, such that the subgenomic transcript encodes the antigen rather than the structural alphavirus virion proteins.

[0185] A replicon useful with the invention can, for example, have two open reading frames. In one example, the first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes an antigen. In some embodiments the RNA may have additional (e.g. downstream) open reading frames e.g. to encode additional antigens or to encode accessory polypeptides.

[0186] A replicon can, for example, have a 5' cap (e.g. a 7-methylguanosine), which often can enhance in vivo translation of the RNA. In some embodiments the 5' sequence of the replicon may need to be selected to ensure compatibility with the encoded replicase.

[0187] A replicon may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3' end.

[0188] Replicons can have various lengths, but they are typically 5000-25000 nucleotides long e.g. 8000-15000 nucleotides, or 9000-12000 nucleotides.

[0189] The replicon can conveniently be prepared by in vitro transcription (IVT). IVT can use a (cDNA) template created and propagated in plasmid form in bacteria or created synthetically (for example by gene synthesis and / or polymerase chain-reaction (PCR) engineering methods). For instance, a DNA-dependent RNA polymerase (such as the bacteriophage T7, T3 or SP6 RNA polymerases) can be used to transcribe the replicon from a DNA template. Appropriate capping and poly-A addition reactions can be used as required (although the replicon's poly-A is usually encoded within the DNA template). These RNA polymerases can have stringent requirements for the transcribed 5' nucleotide(s) and in some embodiments these requirements must be matched with the requirements of the encoded replicase, to ensure that the IVT-transcribed RNA can function efficiently as a substrate for its self-encoded replicase. Specific examples include Sindbis-virus-based plasmids (pSIN) such as pSINCP, described, for example, in U.S. Pat. Nos. 5,814,482 and 6,015,686, as well as in International Publication Nos. WO 97 / 38087, WO 99 / 18226 and WO 02 / 26209. The construction of such replicons, in general, is described in U.S. Pat. Nos. 5,814,482 and 6,015,686.

[0190] In other aspects, the self-amplifying RNA molecule is derived from or based on a virus other than an alphavirus, preferably, a positive-stranded RNA virus, a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus. Suitable wild-type alphavirus sequences are well-known and are available from sequence depositories, such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable alphaviruses include Aura (ATCC VR- 368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR- 1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR- 372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR- 67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCC VR-1250 ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis (ATCC VR-70, ATCC VR-1251 , ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375).

[0191] In other aspects, the self-amplifying RNA molecule is derived from or based on a replication competent virus (e.g., an oncolytic virus). An oncolytic virus preferentially infects and lyses (breaks down) cancer cells. As the infected cancer cells are destroyed, new infectious virus particles or virions are released, which can infect and destroy further cancer cells. Thus, oncolytic viruses not only cause direct destruction of cancer cells, but also stimulate host anti-cancer immune responses. In some embodiments, the oncolytic virus may encode a tumoror viral-associated antigen, neoantigen, and / or peptides. Suitable oncolytic viruses are known in the art and are available from sequence depositories, such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable oncolytic viruses include, but are not limited to, poxvirus, adenovirus, adeno-associated virus, reovirus, retrovirus, senecavirus, measles, herpes simplex virus, Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), mumps,, influenza, Parvovirus, human hanta virus, myxoma virus, cytomegalovirus (CMV), lentivirus, coxsackievirus, echoviruses, Seneca Valley virus, Sindbis virus, JX-594, p53 expressing viruses, ONYX-15, Delta24, Telemelysin, Telomelysin-GFP, and vaccinia, and the like, and recombinant variants thereof. In some embodiments, the oncolytic virus is genetically engineered for tumour selectivity. In other embodiments, the oncolytic virus is naturally occurring. Naturally occurring oncolytic viruses include, but are not limited to, reovirus and senecavirus.

[0192] The self-amplifying RNA molecules of the invention are typically larger than other types of RNA (e.g. mRNA) that have been prepared using modified nucleotides. Typically, the selfamplifying RNA molecules of the invention contain at least about 3 kb. For example, the self-amplifying RNA can contain at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb or more than 12 kb. In certain examples, the self-amplifying RNA is about 4 kb to about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb to about 12 kb, about 11 kb to about 12 kb, about 5 kb to about 11 kb, about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb.

[0193] The self-amplifying RNA molecules of the invention may comprise one or more types of modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine).

[0194] The self-amplifying RNA molecule may encode a single heterologous polypeptide antigen or, optionally, two or more heterologous polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The heterologous polypeptides generated from the self-amplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences.

[0195] The self-amplifying RNA of the invention may encode one or more polypeptides. These polypeptides may consist of binding proteins, enzymes, cytokines, chemokines, hormones or other functional proteins. Alternatively, these polypeptides may consist of antigens that contain a range of epitopes, preferably epitopes capable of eliciting either a helper T-cell response or a cytotoxic T-cell response or both.

[0196] The self-amplifying RNA molecules described herein may be engineered to express multiple nucleotide sequences, from two or more open reading frames, thereby allowing coexpression of proteins, such as a two or more antibody sequences or two or more antigens together with cytokines or other immunomodulators, which can enhance the generation of an immune response. Such a self-amplifying RNA molecule might be particularly useful, for example, in the production of various gene products (e.g., proteins) at the same time, for example, as a two different single chain antibody sequences, heavy and light chain antibody sequences or multiple antigens to create a bivalent or multivalent vaccine.

[0197] The self-amplifying RNA molecules of the invention can be prepared using any suitable method. Several suitable methods are known in the art for producing RNA molecules that contain modified nucleotides. For example, a self-amplifying RNA molecule that contains modified nucleotides can be prepared by transcribing (e.g., in vitro transcription) a DNA that encodes the self-amplifying RNA molecule using a suitable DNA-dependent RNA polymerase, such as T7 phage RNA polymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, and the like, or mutants of these polymerases which allow efficient incorporation of modified nucleotides into RNA molecules. The transcription reaction will contain nucleotides and modified nucleotides, and other components that support the activity of the selected polymerase, such as a suitable buffer, and suitable salts. The incorporation of nucleotide analogs into a self-amplifying RNA may be engineered, for example, to alter the stability of such RNA molecules, to increase resistance against RNases, to establish replication after introduction into appropriate host cells ("infectivity” of the RNA), and / or to induce or reduce innate and adaptive immune responses.

[0198] Suitable synthetic methods can be used alone, or in combination with one or more other methods (e.g., recombinant DNA or RNA technology), to produce a self-amplifying RNA molecule of the invention. Suitable methods for de novo synthesis are well-known in the art and can be adapted for particular applications. Exemplary methods include, for example, chemical synthesis using suitable protecting groups such as GEM, the p-cyanoethyl phosphoramidite method; and the nucleoside H- phosphonate method. These chemistries can be performed or adapted for use with automated nucleic acid synthesizers that are commercially available. Additional suitable synthetic methods are disclosed in Uhlmann et al. (1990) Chem Rev 90:544-84, and Goodchild J (1990) Bioconjugate Chem 1 : 165. Nucleic acid synthesis can also be performed using suitable recombinant methods that are well-known and conventional in the art, including cloning, processing, and / or expression of polynucleotides and gene products encoded by such polynucleotides. DNA shuffling by random fragmentation and PGR reassembly of gene fragments and synthetic polynucleotides are examples of known techniques that can be used to design and engineer polynucleotide sequences. Site-directed mutagenesis can be used to alter nucleic acids and the encoded proteins, for example, to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and the like. Suitable methods for transcription, translation and expression of nucleic acid sequences are known and conventional in the art.

[0199] The presence and / or quantity of one or more modified nucleotides in a self-amplifying RNA molecule can be determined using any suitable method. For example, a self-amplifying RNA can be digested to monophosphates (e.g., using nuclease P1) and dephosphorylated (e.g., using asuitable phosphatase such as CIAP), and the resulting nucleosides analyzed by reversed phase HPLC.

[0200] Optionally, the self-amplifying RNA molecules of the invention may include one or more modified nucleotides so that the self-amplifying RNA molecule will have less immunomodulatory activity upon introduction or entry into a host cell (e.g., a human cell) in comparison to the corresponding self-amplifying RNA molecule that does not contain modified nucleotides.

[0201] If desired, the self-amplifying RNA molecules can be screened or analyzed to confirm their therapeutic and prophylactic properties using various in vitro or in vivo testing methods that are known to those of skill in the art. For example, vaccines comprising self-amplifying RNA molecule can be tested for their effect on induction of proliferation or effector function of the particular lymphocyte type of interest, e.g., B cells, T cells, T cell lines, and T cell clones. For example, spleen cells from immunized mice can be isolated and the capacity of cytotoxic T lymphocytes to lyse autologous target cells that contain a self-amplifying RNA molecule that encodes a polypeptide antigen. In addition, T helper cell differentiation can be analyzed by measuring proliferation or production of TH1 (IL-2 and IFN-y) and / or TH2 (IL-4 and IL-5) cytokines by ELISA or directly in CD4+ T cells by cytoplasmic cytokine staining and flow cytometry after antigen stimulation.

[0202] Self-amplifying RNA molecules that encode a polypeptide antigen can also be tested for ability to induce humoral immune responses, as evidenced, for example, by induction of B cell production of antibodies specific for an antigen of interest. These assays can be conducted using, for example, peripheral B lymphocytes from immunized individuals. Such assay methods are known to those of skill in the art. Other assays that can be used to characterize the self-amplifying RNA molecules of the invention can involve detecting expression of the encoded antigen by the target cells. For example, FACS can be used to detect antigen expression on the cell surface or within the cell. Another advantage of FACS selection is that one can sort for different levels of expression; sometimes lower expression may be desired. Other suitable methods for identifying cells which express a particular antigen involve panning using monoclonal antibodies on a plate or capture using magnetic beads coated with monoclonal antibodies.B. DNA Molecules

[0203] In embodiments where the bioactive agent is a DNA molecule, the DNA molecule may encode proteins of various types, including, without limitation, antigens, antibodies, toxins, growth factors, cytokines, and hormones. The DNA can include, without limitation, plasmid DNA, circular DNA, linear DNA, single-stranded DNA, modified DNA, antisense DNA, and aptamer DNA.C. Antigens

[0204] The bioactive agent described herein can be a nucleic acid molecule (e.g., DNA or RNA) that encodes an antigen, or a protein antigen or epitope. Suitable antigens include, but are not limited to, a bacterial antigen, a viral antigen, a fungal antigen, a protozoan antigen, a plant antigen, a cancer antigen, or a combination thereto. The antigen can be involved in, or derived from, for example, an allergy, cancer, infectious disease, or auto-immune disease.

[0205] An antigen may be any target epitope, molecule (including a biomolecule), molecular complex (including molecular complexes that contain biomolecules), subcellular assembly, cell or tissue against which elicitation or enhancement of immunoreactivity in a subject is desired. Frequently, the term antigen will refer to a polypeptide antigen of interest. In certain embodiments the antigen may be, or may be derived from, or may be immunologically cross-reactive with, an infectious pathogen and / or an epitope, biomolecule, cell, or tissue that is associated with infection, cancer, autoimmune disease, allergy, asthma, or any other condition where stimulation of an antigen-specific immune response would be desirable or beneficial.

[0206] Certain embodiments contemplate an antigen that is derived from at least one infectious pathogen such as a bacterium, a virus or a fungus, including an Actinobacterium such as M. tuberculosis or M. leprae or another mycobacterium; a bacterium such as a member of the genus Escherichia, Salmonella, Neisseria, Borrelia, Chlamydia, Clostridium or Bordetella; a virus such as a herpes simplex virus, a human immunodeficiency virus (HIV such as HIV-1 or HIV-2 ), an influenza virus, a parainfluenza virus, a measles virus, a mumps virus, a rubella virus, a coronavirus (such as SARS or MERS), a rotavirus, a norovirus, a picorna virus (such as a poliovirus, an enterovirus, or a coxsackie virus), a veterinary pathogen, for example, a feline immunodeficiency virus (FIV), cytomegalovirus, Varicella Zoster Virus, hepatitis virus, Epstein Barr Virus (EBV), a flavivirus virus (such as dengue virus, Japanese encephalitis virus, yellow fever virus, Zika virus, Powassan virus, or tick-borne encephalitis virus ), a henipah virus (such as hendra or nipah virus), a bunyavirus (such as Hantavirus or Rift Valley Fever virus), an arenavirus (such as lassa virus, junin virus, machupo virus, or guanarito virus), a filovirus (such as Ebola virus or Marburg virus), a lyssavirus (such as Rabies virus), respiratory syncytial virus, human papilloma virus (HPV) and a cytomegalovirus; ; a fungus such as Aspergillus, Blastomyces, Coccidioides, and Pneumocysti or a yeast, including Candida species such as C. albicans, C. glabrata, C. krusei, C. lusitaniae, C. tropicalis, and C. parapsilosis; a parasite such as a protozoan, for example, a Plasmodium species including P. falciparum, P. vivax, P. malariae, and P. ovale; or another parasite such as one or more of Acanthamoeba, Entamoeba histolytica, Angiostrongylus, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Cryptosporidium, Ancylostoma, Entamoeba histolytica, Entamoeba coll, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Wuchereria bancrofti, Giardia, Toxoplasma gondii,and Leishmania. In specific embodiments, the antigen may be from, or related to antigens involved in tuberculosis, influenza, amebiasis, HIV, hepatitis, or Leishmaniasis.

[0207] In some embodiments, the antigen is an influenza-related antigen. In some embodiments, the antigen is an influenza-causing antigen. In some embodiments, the antigen is from an influenza causing virus. In one embodiment, the antigen comprises hemagglutinin (HA) from H5N1. In one embodiment, the antigen comprises neuraminidase from H5N1.

[0208] For example, in certain embodiments, antigens are derived from Borreli a sp., the antigens may include nucleic acid, pathogen derived antigen or antigenic preparations, recombinantly produced protein or peptides, and chimeric fusion proteins. One such antigen is OspA. The OspA may be a full mature protein in a lipidated form by virtue of its biosynthesis in a host cell (Lipo-OspA) or may alternatively be a non-lipidated derivative. Such non-lipidated derivatives include the non- lipidated NS1 -OspA fusion protein which has the first 81 N-terminal amino acids of the non-structural protein (NS1) of the influenza virus, and the complete OspA protein, and another, MDP-OspA is a non-lipidated form of OspA carrying 3 additional N-terminal amino acids.

[0209] In certain embodiments the antigen is derived from a virus such as from a coronavirus (such as SARS or MERS), HIV-1 , (such as tat, nef, gp120 or gp160), human herpes viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSV1 or HSV2, cytomegalovirus ((esp. Human)(such as gB or derivatives thereof), Rotavirus (including live- attenuated viruses), Epstein Barr virus (such as gp350 or derivatives thereof), Varicella Zoster Virus (such as gpl, II and IE63), or from a hepatitis virus such as hepatitis B virus (for example Hepatitis B Surface antigen or a derivative thereof), hepatitis A virus, hepatitis C virus and hepatitis E virus, or from other viral pathogens, such as paramyxoviruses: Respiratory Syncytial virus (such as F and G proteins or derivatives thereof), parainfluenza virus, measles virus, mumps virus, human papilloma viruses (for example HPV6, 11 , 16, 18, etc.), flaviviruses (e.g., dengue virus, Japanese encephalitis virus, yellow fever virus, Zika virus, Powassan virus, tick-borne encephalitis virus) or Influenza virus (whole live or inactivated virus, split influenza virus, grown in eggs or MDCK cells, or whole flu virosomes (as described by Gluck, Vaccine, 1992, 10, 915-920) or purified or recombinant proteins thereof, such as HA, NP, NA, PB1 , PB2, PA, NS1 or M proteins, or combinations thereof).

[0210] In certain other embodiments, the antigen is derived from one or more bacterial pathogens such as Neisseria spp, including N. gonorrhea and N. meningitidis (for example capsular polysaccharides and conjugates thereof, transferrin-binding proteins, lactoferrin binding proteins, PilC, adhesins); S. pyogenes (for example M proteins or fragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S. mutans: H. ducreyi; Moraxella spp, including M. catarrhalis, also known as Branhamella catarrhalis (for example high and low molecular weight adhesins and invasins);Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium spp., including M. tuberculosis (for example ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coll (for example colonization factors, heat-labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof), enterohemorragic E. coll, enteropathogenic E. coll (for example shiga toxin-like toxin or derivatives thereof); Vibrio spp, including V. cholera (for example cholera toxin or derivatives thereof); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y. enterocolitica (for example a Yop protein), Y. pestis, Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (for example toxins, adhesins and invasins) and C. coll; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp., including L. monocytogenes; Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis; Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp., including C. tetani (for example tetanus toxin and derivative thereof), C. botulinum (for example botulinum toxin and derivative thereof), C. difficile (for example Clostridium toxins A or B and derivatives thereof); Bacillus spp., including B. anthracis (for example botulinum toxin and derivatives thereof); Corynebacterium spp., including C. diphtheriae (for example diphtheria toxin and derivatives thereof); Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp. including C. trachomatis (for example MOMP, heparin-binding proteins), C. pneumoniae (for example MOMP, heparin-binding proteins), C. psittaci; Leptospira spp., including L. interrogans; Treponema spp., including T. pallidum (for example the rare outer membrane proteins), T. denticola, T. hyodysenteriae; or other bacterial pathogens.

[0211] In certain other embodiments, the antigen is derived from one or more parasites (See, e.g., John, D.T. and Petri, W.A., Markell and Voge's Medical Parasitology-9th Ed., 2006, WB Saunders, Philadelphia; Bowman, D.D., Georgis' Parasitology for Veterinarians-8th Ed., 2002, WB Saunders, Philadelphia) such as Plasmodium spp., including P. falciparum; Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Tg34); Entamoeba spp., including E. histolytica; Babesia spp., including B. microti; Trypanosoma spp., including T. cruzi; Giardia spp., including G. lamblia; Leshmania spp., including L. major; Pneumocystis spp., including P. carinii; Trichomonas spp., including T. vaginalis; or from a helminth capable of infecting a mammal, such as: (I) nematodeinfections (including, but not limited to, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria, Necator americanus, Ancylostoma duodenale, Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, and Strongyloides stercoralis); (II) trematode infections (including, but not limited to, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongi, Opisthorchis sinensis, Paragonimus sp, Fasciola hepatica, Fasciola magna, Fasciola gigantica); and (ill) cestode infections (including, but not limited to, Taenia saginata and Taenia solium). In certain embodiments, the antigen is derived from Schisostoma spp., Schistosoma mansonii, Schistosoma haematobium, and / or Schistosoma japonicum, or derived from yeast such as Candida spp., including C. albicans; Cryptococcus spp., including C. neoformans.

[0212] Other specific antigens are derived from M. tuberculosis, for example Th Ra12, Tb H9, Tb Ra35, Tb38-1 , Erd 14, DPV, MTI, MSL, mTTC2 and hTCC1 (WO 99 / 51748). Proteins for M. tuberculosis also include fusion proteins and variants thereof where at least two, three, or four or more, polypeptides of M. tuberculosis are fused into a larger protein. Certain fusions include Ra12- TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL, Erd14DPV-MTI-MSL-mTCC2, Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, TbH9-DPV-MTI (WO 99151748). Other antigens that may be used include antigens, combination of antigens, and fusion proteins described in US 2010 / 0129391 and WO 2008 / 124647. In one exemplary embodiment, the fusion protein is ID93. In one exemplary embodiment, the fusion protein is ID91.

[0213] The fusion protein ID91 includes a fusion of four Mtb proteins: Rv3619 (a virulence factor, EsX family), Rv2389 (produced under hypoxic conditions, resuscitation factor D), Rv3478 (a member of the PE / PPE family) and Rv1886 (Ag85A, secreted / membrane protein; mycolyltransferase). Mtb antigens included in ID91 were prioritized based on lack of human sequence homology and human PBMC IFN-g secretion from PPD+ donors (and not PPDdonors) after stimulation with antigen to ensure immunogenicity in the human population (Bertholet et al., J Immunol. 181 (11)7948-57 (2008)). ID91 protein combined with synthetic Toll-like receptor 4 (TLR4) agonist glucopyranosyl lipid adjuvant in a stable emulsion (GLA-SE) demonstrates protection against Mtb H37Rv four weeks after one immunization in a preclinical mouse model (Orr et al., J Immunol. 193(6):2911-18 (2014)). With this subunit vaccine a robust TH1 response (IFN-g, TNF, and IL-2) to ID91 was observed. Id.

[0214] In some embodiments, ID91 may include restriction enzymes well known to those of skill in the art. Exemplary restriction enzymes, include but are not limited to, Ndel, Kpnl, BamHI, EcoRI, and / or Hindlll.

[0215] Other specific antigens are derived from Chlamydia and include for example the High Molecular Weight Protein (HWMP) (WO 99 / 17741), ORF3 (EP 366 412), and putative membraneproteins (Pmps). Other Chlamydia antigens can be selected from the group described in WO 99128475. Certain antigens may be derived from Streptococcus spp, including S. pneumoniae (for example capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline-binding proteins) and the protein antigen Pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25, 337-342), and mutant detoxified derivatives thereof (WO 90 / 06951; WO 99 / 03884). Other bacterial vaccines comprise antigens derived from Haemophilus spp., including H. influenzae type B (for example PRP and conjugates thereof), non-typeable H. influenzae, for example OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin derived peptides (U.S. Pat. No. 5,843,464) or multiple copy variants or fusion proteins thereof.

[0216] Other specific antigens are derived from Hepatitis B. Derivatives of Hepatitis B Surface antigen are well known in the art and include, inter alia, those PreS1 , PreS2, S antigens set forth described in European Patent applications EP-A414 374; EP-A-0304 578, and EP 198474.

[0217] In other embodiments, the antigen is derived from the Human Papilloma Virus (HPV) considered to be responsible for genital warts (HPV 6 or HPV 11 and others), and the HPV viruses responsible for cervical cancer (HPV16, HPV18 and others). Particular antigens include L1 particles or capsomers, and fusion proteins comprising one or more antigens selected from the HPV 6 and HPV 11 proteins E6, E7, L1 , and L2. Certain forms ef fusion protein include L2E7 as disclosed in WO 96 / 26277, and protein D(1 / 3)-E7 disclosed in GB 9717953.5 (PCT / EP98 / 05285). Additional possible antigens include HPV 16,18, 33, 58 antigens. For example, L1 or L2 antigen monomers, or L1 or L2 antigens presented together as a virus like particle (VLP) or the L1 alone protein presented alone in a VLP or capsomer structure. Such antigens, virus like particles and capsomer are per se known. See for example W094 / 00152, WO94 / 20137, WO94 / 05792, and WO93 / 02184.

[0218] In other embodiments, the antigen is a fusion protein. Fusion proteins may be included alone or as fusion proteins such as E7, E2 or F5 for example; particular embodiments include a VLP comprising L1 E7 fusion proteins (WO 96 / 11272). Particular HPV 16 antigens comprise the early proteins E6 or F7 in fusion with a protein D carrier to form Protein D-E6 or E7 fusions from HPV 16, or combinations thereof; or combinations of E6 or E7 with L2 (WO 96 / 26277). Alternatively, the HPV 16 or 18 early proteins E6 and E7, may be presented in a single molecule, for example a Protein D- E6 / E7 fusion. Compositions may optionally contain either or both E6 and E7 proteins front HPV 18, for example in the form of a Protein D-E6 or Protein D-E7 fusion protein or Protein D E6 / E7 fusion protein. Compositions may additionally comprise antigens from other HPV strains, for example from strains HPV 31 or 33.

[0219] Antigens may also be derived from parasites that cause Malaria. For example, antigens from Plasmodia falciparum include RTS,S and TRAP. RTS is a hybrid protein comprising substantiallyall the C-terminal portion of the circumsporozoite (CS) protein of P.falciparum linked via four amino acids of the preS2 portion of Hepatitis B surface antigen to the surface (S) antigen of hepatitis B virus. Its full structure is disclosed in the International Patent Application No. PCT / EP92 / 02591 , published as WO 93 / 10152 claiming priority from UK patent application No.9124390.7. When expressed in yeast RTS is produced as a lipoprotein particle, and when it is co-expressed with the S antigen from HBV it produces a mixed particle known as RTS,S.

[0220] TRAP antigens are described in the International Patent Application No. PCT / GB89 / 00895 published as WO 90 / 01496. An embodiment of the present invention is a Malaria vaccine wherein the antigenic preparation comprises a combination of the RTS, S and TRAP antigens. Other plasmodia antigens that are likely candidates to be components of a multistage Malaria vaccine are P. faciparum MSP1 , AMA1 , MSP3, EBA, GLURP, RAP1 , RAP2, Sequestrin, PfEMPI , Pf332, LSA1 , LSA3, STARP, SALSA, PfEXPI , Pfs25, Pfs28, PFS27125, Pfs16, Pfs48 / 45, Pfs230 and their analogues in Plasmodium spp.

[0221] In one embodiment, the antigen is derived from a cancer cell, as may be useful for the immunotherapeutic treatment of cancers. For example, the antigen may be a tumor rejection antigen such as those for prostate, breast, colorectal, lung, pancreatic, renal or melanoma cancers. Exemplary cancer or cancer cell-derived antigens include MAGE 1, 3 and MAGE 4 or other MAGE antigens such as those disclosed in WO99 / 40188, PRAME, BAGE, Lage (also known as NY Eos 1) SAGE and HAGE (WO 99 / 53061) or GAGE (Robbins and Kawakami, 1996 Current Opinions in Immunology 8, pp. 628-636; Van den Eynde et al., International Journal of Clinical & Laboratory Research (1997 &1998); Correale et al. (1997), Journal of the National Cancer Institute 89, p. 293. These non-limiting examples of cancer antigens are expressed in a wide range of tumor types such as melanoma, lung carcinoma, sarcoma and bladder carcinoma. See, e.g., U.S. Patent No. 6,544,518.

[0222] Other tumor-specific antigens include, but are not restricted to, tumor-specific or tumor- associated gangliosides such as GM2, and GM3 or conjugates thereof to carrier proteins; or a selfpeptide hormone such as whole length Gonadotrophin hormone releasing hormone (GnRH, WO 95 / 20600), a short 10 amino acid long peptide, useful in the treatment of many cancers. In another embodiment prostate antigens are used, such as Prostate specific antigen (PSA), PAP, PSCA (e.g., Proc. Nat. Acad. Sci. USA 95(4) 1735-1740 1998), PSMA or, in one embodiment an antigen known as Prostase. (e.g., Nelson, et al., Proc. Natl. Acad. Sci. USA (1999) 96: 3114-3119; Ferguson, et al. Proc. Natl. Acad. Sci. USA 1999. 96, 3114-3119; WO 98 / 12302; U.S. Pat. No. 5,955,306; WO 98 / 20117; U.S. Pat. Nos. 5,840,871 and 5,786, 148; WO 00 / 04149. Other prostate specific antigens are known from WO 98 / 137418, and WO / 004149. Another is STEAP (PNAS 96 14523 14528 7-121999).

[0223] Other tumor associated antigens useful in the context of the present invention include: Plu -1 (J Biol. Chem 274 (22) 15633-15645, 1999), HASH-1 , HasH-2, Cripto (Salomon et al Bioessays 199, 21 :61-70, U.S. Pat. No. 5,654, 140) and Criptin (U.S. Pat. No. 5,981 ,215). Additionally, antigens particularly relevant for vaccines in the therapy of cancer also comprise tyrosinase and survivin.

[0224] In other embodiments, the agents used in the compositions of the invention include antigens associated with respiratory diseases, such as those caused or exacerbated by bacterial infection (e.g. pneumococcal), for the prophylaxis and therapy of conditions such as chronic obstructive pulmonary disease (COPD). COPD is defined physiologically by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and / or emphysema (Am J Respir Grit Care Med. 1995 Nov;152(5 Pt 2):S77-121). Exacerbations of COPD are often caused by bacterial (e.g. pneumococcal) infection (Clin Microbiol Rev. 2001 Apr; 14(2):336- 63).D. Antibody-Encoding nucleic acid

[0225] The bioactive agents described herein (e.g., RNA) may encode an antibody and / or antigen-binding fragment of an antibody, optionally operably linked to one or more expression control elements, such that delivery to a subject results in the production of said antibody or antigen-binding fragment in the subject. In some embodiments, the bioactive agent may contain the coding sequence of the heavy chain and light chain in a single open reading frame. In other embodiments, an NLC of the present invention may comprise two bioactive agents wherein one of the bioactive agents encodes a heavy chain whereas the other encodes a light chain. In other embodiments, the bioactive agent may contain the coding sequence of the variable regions of the heavy and light chains linked by a short flexible polypeptide sequence such that the expressed biomolecule binds the antigen of interest. In some particular embodiments, the produced antibody is capable of eliciting an immune response in an individual.E. RNA interference

[0226] In some embodiments the bioactive polynucleotide associated with the NLC is a noncoding RNA such as an RNA interference (RNAi) polynucleotide. RNAi is a molecule capable of inducing RNA interference through interaction with the RNA interference pathway machinery of mammalian cells to degrade or inhibit translation of messenger RNA (mRNA) transcripts of a transgene in a sequence specific manner. Two primary RNAi polynucleotides are small (or short) interfering RNAs (siRNAs) and micro RNAs (miRNAs). RNAi polynucleotides may be selected from the group comprising: siRNA, microRNA, double-strand RNA (dsRNA), short hairpin RNA (shRNA), and expression cassettes encoding RNA capable of inducing RNA interference. siRNA comprises a double stranded structure typically containing 15-50 base pairs and preferably 21-25 base pairs andhaving a nucleotide sequence identical (perfectly complementary) or nearly identical (partially complementary) to a coding sequence in an expressed target gene or RNA within the cell. An siRNA may have dinucleotide 3' overhangs. An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure.

[0227] MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nucleotides long that direct destruction or translational repression of their mRNA targets. If the complementarity between the miRNA and the target mRNA is partial, translation of the target mRNA is repressed. If complementarity is extensive, the target mRNA is cleaved. For miRNAs, the complex binds to target sites usually located in the 3' UTR of mRNAs that typically share only partial homology with the miRNA. A "seed region”— a stretch of about seven (7) consecutive nucleotides on the 5' end of the miRNA that forms perfect base pairing with its target— plays a key role in miRNA specificity. Binding of the RISC / miRNA complex to the mRNA can lead to either the repression of protein translation or cleavage and degradation of the mRNA.F. CRISPR RNAs

[0228] In some embodiments the NLC formulation comprises a synthetic short guide RNA (sgRNA) of the CRISPR / Cas9 genome editing thereby targeting a gene of interest. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages. The CRISPR system provides a form of acquired immunity. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a memory of past exposures. CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms. Cas9, an essential protein component in the Type II CRISPR / Cas9 system, forms an active endonuclease when complexed with two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), thereby slicing foreign genetic elements in invading phages or plasmids to protect the host cells.

[0229] The RNA-guided endonuclease based on CRISPR / Cas9 system been employed for eukaryotic genome editing. In certain embodiments of the present invention, the bioactive agent is RNA that encodes sgRNAs and / or Cas9 endonucleases. In some embodiments, the RNA comprises one or more polynucleotides encoding Cas9 and two guide RNAs, the first guide RNA comprising a spacer sequence that is complementary to a segment of the 5' double-stranded break (DSB) locus, and the second guide RNA comprising a spacer sequence that is complementary to a segment of the 3' DSB locus. Both guide RNAs may be provided as single-molecule guide RNAs (comprisingtracrRNA and crRNA), or either or both may be provided as double-molecule guide RNAs comprising a crRNA and a tracrRNA that are not joined to each other but rather are separate molecules.G. Polypeptides

[0230] In some embodiments the one or more bioactive agents is a polypeptide. The polypeptide can be a full-length protein or a fragment thereof. In some embodiments the polypeptide is a peptide. In some embodiments, the polypeptide is a fusion protein. In some particular embodiments, the fusion protein is capable of eliciting an immune response upon administration to an individual. In some embodiments, the polypeptide is an antigen, as further described above. Polypeptides may be made by any suitable method known to one of skill in the art, including, for example, recombinant expression.H. Small Molecules

[0231] In certain embodiments, the present disclosure generally relates to a NLC composition where the one or more bioactive agents is a small molecule or therapeutic agent for drug delivery. A close association of drug molecule and the NLC may be influenced by drug physicochemical properties, surfactant type and concentration, lipid type, and production method. In certain embodiments, the small molecule drug is encapsulated by the NLC, which is enabled by the liquid lipid phase component of the oil core that provides high drug solubility (Beloqui, A., et al. Nanomedicine 2016; 12(1): 143-161).

[0232] The NLC compositions provided herein may be suitable for drug delivery through various routes of administration, including, without limitation, dermal, transdermal, oral, intranasal, pulmonary, or ophthalmological routes of administration.I. Hormones

[0233] In some embodiments the one or more bioactive agents associated with the NLC is a polynucleotide or polypeptide that encodes a hormone or analog of a hormone. In some embodiments, the NLC comprises a lipid that is conjugated to a hormone. The hormone may be selected from the group comprising human growth hormone, adrenocorticotropin, gonadotropin releasing hormone, oxytocin, leutinizing-hormone-releasing-hormone, follicle stimulating hormone, insulin, insulin-like growth factor, leptin, parathyroid hormone, thyroid stimulating hormone, or some combination thereof. In certain embodiments the NLC formulation comprises a hormone or analog of a hormone in combination with a small molecule therapeutic compound as described above.J. Adjuvants

[0234] The lipid-based nanoparticle compositions and / or formulations of the disclosure may be adjuvanted. There are many known adjuvants that have been combined with vaccine formulations. However, it was unknown and difficult to predict how a specific adjuvant would interact with a formulation that includes saRNA. In some embodiments, the lipid-based nanoparticle composition isfor vaccine delivery and one or more of the bioactive agents is an adjuvant or alternatively, the lipid- based nanoparticle compositions provided herein may be co-administered with an adjuvant. As used herein, the term adjuvant refers to a substance that enhances or potentiates an immune response. The immune response can be, for example, an antigen-specific immune response e.g., to an exogenous antigen.

[0235] In some embodiments, the adjuvant is a saponin. The saponin may be extracted from a natural source or synthetically produced. The saponin may be a Quillaja saponaria (QS) saponin such as QS-7, QS-17, QS-18, or QS-21. The saponin may be an extract from Aesculus hippocastanum such as Asecin or escin. The saponin may be an extract of Digitalis purpurea such as digitonin. The saponin may be an extract of a Gypsophila species. The saponin may be an extract of Chenopodium quinoa. The saponin may be a saponin analogue. Saponin analogues include synthetic and semisynthetic analogues. Saponins are discussed in Wang, P. Natural and Synthetic Saponins as Vaccine Adjuvants. Vaccines 2021 , 9, 222 and Marie-Aleth Lacaille-Dubois, Updated insights into the mechanism of action and clinical profile of the immunoadjuvant QS-21 : A review, Phytomedicine, Volume 60, 2019.

[0236] The molecular adjuvant QS-21 is a triterpenoid that belongs to a class of amphiphilic molecules called saponins. It is found in the bark of the Chilean soap bark tree Quillaja saponaria. QS-21 is an acylated triterpene glycoside with a molecular formula of C92O46H148 and a molecular weight of 1 ,990 Da. In recent years, QS-21 has attracted much attention as a vaccine adjuvant component due to its ability to elicit both humoral and cellular immune responses toward a wide range of vaccine antigens. Although its specific mechanisms of action are not yet fully elucidated, it has been shown that QS-21 facilitates vaccine uptake and processing in antigen presenting cells, provides a costimulatory signal to T cells, activates the NLRP3 inflammasome, and synergizes with other adjuvants (see M.-A. Lacaille-Dubois, Updated insights into the mechanism of action and clinical profile of the immunoadjuvant QS-21 : A review. Phytomedicine Int. J. Phytother. Phytopharm. 60, 152905 (2019). The success of QS-21 as a vaccine adjuvant component is demonstrated by three commercial vaccines, namely Shingrix (a blockbuster shingles vaccine approved by the US Food and Drug Administration (FDA) in 2017 for older adults), Mosquirix (the world's first malaria vaccine, approved by the European Medicines Agency (EMA) in 2015 and prequalified by the World Health Organization (WHO) in 2022 for infants and young children in endemic regions), and Nuvaxovid / Covovax (a SARS-CoV-2 spike protein antigen vaccine authorized by the FDA in 2022 for teenagers and adults). The adjuvant formulation in the Shingrix and Mosquirix vaccines is a liposomal system containing the adjuvant components QS-21 and monophosphoryl lipid A (MPL). The adjuvant formulation in the Nuvaxovid / Covovax vaccine contains a mixture of saponins (including QS-21) andlipid excipients. In addition, numerous other vaccine candidates covering a wide range of diseases in the research and clinical pipelines contain QS-21 as a key adjuvant component, including a promising tuberculosis vaccine candidate which demonstrated 50% protection in a Phase 2b clinical trial (see D. R. Tait, et al., Final analysis of a trial of M72 / AS01 E vaccine to prevent tuberculosis. N. Engl. J. Med. 381 , 2429-2439 (2019). Thus, QS-21 has a long and reliable history as a safe and immunogenic vaccine adjuvant component in human vaccines. Furthermore, it has been shown that the addition of saponins like QS-21 is useful in SARS-CoV-2 protein vaccines, but this concept has not been proposed or tested with RNA formulations (see R. Sharma, et al., Exploring the possible use of saponin adjuvants in COVID-19 vaccine. Hum. Vaccines Immunother. 16, 2944-2953 (2020).

[0237] In some embodiments, the lipid-based nanoparticle composition comprises from 0.1% to about 10% saponin (e.g, about 0.1 , 0.2, 0.3, 0.4, 0.5, O.6., 0.7, 0.8, 0.9. 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10% saponin). In some embodiments, the lipid-based nanoparticle composition comprises about 0.1 mg / ml to about 10 mg / ml saponin (e.g,, about 0.1, 0.2, 0.3, 0.4, 0.5, O.6., 0.7, 0.8, 0.9. 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10 mg / ml saponin). In some embodiments, the lipid-based nanoparticle composition comprises from 0.1 % to about 10% QS-21 (e.g., about 0.1 , 0.2, 0.3, 0.4, 0.5, O.6., 0.7, 0.8, 0.9. 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10% QS-21). In some embodiments, the lipid-based nanoparticle composition comprises about 0.1 mg / ml to about 10 mg / ml QS-21 (e.g,, about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10 mg / ml QS-21). In some embodiments, the lipid-based nanoparticle composition comprises an NLC of the disclosure and comprises from 0.1% to about 10% QS-21 (e.g, about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10% QS-21). In some embodiments, the lipid-based nanoparticle composition comprises an NLC particle of the disclosure and comprises about 0.1 mg / ml to about 10 mg / ml QS-21 (e.g,, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10 mg / ml QS-21).

[0238] In some embodiments, the bioactive agent is a nucleic acid and the saponin is present at about 0.4 to about 10 pg / 1 pg of nucleic acid (e.g, about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10 pg / 1 pg of nucleic acid). In some embodiments, the saponin is present at about 2 pg / 1 pg of nucleic acid. In some embodiments, the composition is an NLC, the bioactive agent is a nucleic acid and the saponin is present at about 0.4 to about 10 pg / 1 pg of nucleic acid (e.g, about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10 pg / 1 pg of nucleic acid). In some embodiments, the composition is an NLC, the bioactive agent is a nucleic acid and the saponin is present at about 2 pg / 1 pg of nucleic acid. In some embodiments, the composition is an NLC, the bioactive agent is a saRNA and the saponin is present at about 0.4 to about 10 pg / 1 pg of nucleic acid (e.g, about 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1 .0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10 pg / 1pg of nucleic acid). In some embodiments, the composition is an NLC, the bioactive agent is a saRNA and the saponin is present at about 2 pg / 1 pg of nucleic acid.

[0239] In some embodiments, the composition is an NLC and comprises from about 0.2% to about 40% w / v liquid phase lipid, from about 0.1% to about 10% w / v solid phase lipid, from about 0.1 % to about 10% sterol, from about 0.2% to about 10% w / v cationic lipid, from about 0.25% to about 5% w / v hydrophobic surfactant, from about 0.5% to about 10% w / v hydrophilic surfactant, and from 0.1 % to about 10% saponin. In some embodiments, the composition is an NLC and comprises about 37.3 mg / ml liquid phase lipid, about 2.4 mg / ml solid phase lipid, about 1 mg / ml sterol, about 30 mg / ml cationic lipid, about 37 mg / ml hydrophobic surfactant, about 37.2 mg / ml hydrophilic surfactant, and about 2 mg / ml saponin.

[0240] Certain exemplary compositions employ adjuvant systems designed to induce an immune response predominantly of the Th1 type. High levels of Th 1-type cytokines (e.g., IFN-y, TNF- a, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a compositions as provided herein, a patient may support an immune response that includes Thl and Th2-type responses. Within an exemplary embodiment, in which a response is predominantly Thltype, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mossman & Coffman, Ann. Rev. Immunol. 7:145-173 (1989).

[0241] Certain adjuvants for use in eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, for example 3-de-O-acylated monophosphoryl lipid A (3D-MPLTM), together with an aluminum salt (U.S. Pat. Nos. 4,436,727; 4,877,611 ; 4,866,034; and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96 / 02555, WO 99 / 33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352 (1996).

[0242] VI. Methods of Making Exemplary Compositions Comprising Bioactive agents and Lipid-based Nanoparticles

[0243] As provided herein, one method of making the NLCs described herein comprises (a) mixing the solid phase lipid, the liquid phase lipid, the optional sterol, the cationic lipid, and the hydrophobic surfactant (e.g., sorbitan ester) to form an oil phase mixture; (b) mixing the hydrophilicsurfactant, the saponin, and water to form an aqueous phase; and (c) mixing the oil phase mixture with the aqueous phase mixture to form the NLC. In some embodiments, a further step comprises combining the bioactive agent with the NLC such that the bioactive agent associates with the surface of the NLC particle by non-covalent interactions or by reversible covalent interactions. Such embodiments are preferred where the bioactive agent is negatively charged, such as an RNA molecule or a DNA molecule. The negative charges on the bioactive agent interact with the cationic lipid in the NLC, thereby associating the negatively charged bioactive agent with the NLC. In other embodiments, where the bioactive agent is hydrophobic, it is combined with the components in step (a) to form part of the oil phase mixture. In some embodiments, the bioactive agent may be attached to a component of the surface of the NLC via covalent interactions.

[0244] Mixing the solid phase lipid, the liquid phase lipid, the cationic lipid, and the hydrophobic surfactant (e.g., sorbitan ester) to form an oil phase mixture may be achieved, for example, by heating and sonication. Mixing the oil phase mixture with the aqueous phase mixture may be achieved, for example, by various emulsification methods, including, without limitation, high shear emulsification and microfluidization.

[0245] Preparation of LNPs is accomplished by dissolving the lipids in an organic solvent such as ethanol and mixing an aqueous solution of the saRNA using a microfluidic device, rapid injection, or hand mixing by pipette or syringe. The adjuvant may be added following LNP manufacture or as a component of the ethanol or aqueous solutions.

[0246] VII. Compositions Comprising the Lipid-based Nanoparticles

[0247] Provided herein are formulations, compositions, and pharmaceutical compositions comprising the lipid-based nanoparticles compositions described herein.

[0248] The compositions comprising the lipid-based nanoparticles and bioactive agent can optionally further comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0249] The compositions described herein can be administered to a subject for any vaccination, therapeutic or diagnostic purposes.

[0250] Provided here are pharmaceutical compositions comprising the presently disclosed compositions further in combination with a pharmaceutically acceptable carrier, excipient, or diluent.

[0251] In some embodiments provided herein, the pharmaceutical compositions provided herein are capable of being filtered through a 0.45 micron filter. In some embodiments, the pharmaceutical composition is capable of being filtered through a 0.20 micron filter. In some embodiments, the pharmaceutical composition is capable of being filtered through a 0.22 micron filter.

[0252] In one embodiment, the present invention is drawn to a pharmaceutical composition comprising a lipid-based nanoparticle of the disclosure and an associated bioactive agent. Such acomposition may be administered to a subject to stimulate an immune response, e.g., a non-specific immune response or an antigen-specific immune response, for the purpose of diagnosis, treating or preventing a disease or other condition, such as an infection by an organism.

[0253] In some other embodiments, the pharmaceutical composition is a vaccine composition that comprises the compositions described herein in combination with a pharmaceutically acceptable carrier, excipient or diluent. Illustrative carriers are usually nontoxic to recipients at the dosages and concentrations employed.

[0254] In some aspects, the pharmaceutical compositions provided herein are administered to a subject to generate a response in the subject, for example, for generating an immune response in the subject. Typically, a therapeutically effective amount is administered to the subject.

[0255] The term "effective amount” or "therapeutically effective amount” refers to an amount that is sufficient to achieve or at least partially achieve the desired effect, e.g., sufficient to generate the desired immune response. An effective amount of a pharmaceutical composition is administered in an "effective regime”. The term "effective regime” refers to a combination of amount of the composition being administered and dosage frequency adequate to accomplish the desired effect.

[0256] Actual dosage levels may be varied so as to obtain an amount that is effective to achieve a desired response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors in combination with the particular compositions employed, the age, sex, weight, condition, general health, and prior medical history of the subject being treated, and like factors well-known in the medical arts.

[0257] In exemplary therapeutic embodiments provided herein, a dosage of about 1 pig / kg to about 10 mg / kg of a therapeutic pharmaceutical composition is administered. It will be evident to those skilled in the art that the number and frequency of administrations will be dependent upon the response of the subject.

[0258] In exemplary vaccine-based embodiments provided herein, about 1 pig-100 pig of the antigen or 0.1 pig-10 mg of the nucleic acid encoding the antigen will be administered per administration. Exemplary formulations of the present disclosure permit a human dose of from about 0.1 pig, about 1 pig, about 5 pig or about 10 pig to about 500 pig of replicon RNA Exemplary formulations of the present permit a human dose of about 5 pig to about 20 pig replicon RNA.

[0259] It will be evident to those skilled in the art that the number and frequency of administrations will be dependent upon the response of the subject. Exemplary formulations allow for therapeutic efficacy after as little as one immunization.

[0260] "Pharmaceutically acceptable carriers” for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at physiological pH may be used. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents may be used. Id.

[0261] The pharmaceutical compositions may be in any form which allows for the composition to be administered to a patient. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, intranasal, intramucosal, pulmonary or subcutaneous. The term parenteral as used herein includes iontophoretic, sonophoretic, thermal, transdermal administration and also subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernous, intrathecal, intrameatal, intraurethral injection or infusion techniques. In some embodiments, a composition as described herein (including vaccine and pharmaceutical compositions) is administered intradermally by a technique selected from iontophoresis, microcavitation, sonophoresis, jet injection, or microneedles. In one preferred embodiment, a composition as described herein is administered intradermally using the microneedle device manufactured by NanoPass Technologies Ltd., Nes Ziona, Israel, e.g., MicronJet600 (see, e.g., US Patent No. 6,533,949 and 7,998, 119 and Yotam, et al., Human vaccines & immunotherapeutics 11 (4): 991-997 (2015), each of which is incorporated herein by reference in its entirety.

[0262] The pharmaceutical composition can be formulated so as to allow the active ingredients contained therein to be bioavai lable upon administration of the composition to a subject. Compositions that will be administered to a subject take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units.

[0263] For oral administration, an excipient and / or binder may be present. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethyl cellulose. Coloring and / or flavoring agents may be present. A coating shell may be employed.

[0264] The composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, compositions can contain one or more of a sweetening agent, preservatives, dye / colorant, and flavor enhancer. In a composition intended to be administered by injection by needle and syringe or needle free jet injection, one or more of a surfactant, preservative,wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonic agent may be included.

[0265] A liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, may include one or more of the following carriers or excipients: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as squalene, squalane, mineral oil, a mannide monooleate, cholesterol, and / or synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0266] In another embodiment, a composition of the present disclosure is formulated in a manner which can be aerosolized.

[0267] It may also be desirable to include other components in a pharmaceutical composition, such as delivery vehicles including but not limited to aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Examples of additional immunostimulatory substances (co-adjuvants) for use in such vehicles are also described above and may include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL- 12, GM-CSF, gamma interferon and IL-12.

[0268] While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of the present disclosure, the type of carrier will vary depending on the mode of administration and whether a sustained release is desired. For parenteral administration, such as subcutaneous injection, the carrier can comprise water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075, 109. In this regard, it is preferable that the microsphere be larger than approximately 25 microns.

[0269] Pharmaceutical compositions may also contain diluents such as buffers, antioxidants such as ascorbic acid, polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents.For example, a product may be formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents.

[0270] The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of the antigen (e.g., GLA-antigen vaccine composition) or GLA (e.g., immunological adjuvant composition; GLA is available from Avanti Polar Lipids, Inc., Alabaster, AL; e.g., product number 699800) of from about 0.1 to about 10% w / v (weight per unit volume).

[0271] The composition may be intended for rectal administration, in the form, e.g., of a suppository which can melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol. In the methods of the invention, the pharmaceutical compositions / adjuvants may be administered through use of insert(s), bead(s), timed-release formulation(s), patch(es) or fast-release formulation(s).

[0272] Optionally, to control tonicity, the NLC may comprise a physiological salt, such as a sodium salt. Sodium chloride (NaCI), for example, may be used at about 0.9% (w / v) (physiological saline). Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, magnesium chloride, calcium chloride, etc. Non-ionic tonicifying agents can also be used to control tonicity. Monosaccharides classified as aldoses such as glucose, mannose, arabinose, and ribose, as well as those classified as ketoses such as fructose, sorbose, and xylulose can be used as non-ionic tonicifying agents in the presently disclosed compositions. Disaccharides such a sucrose, maltose, trehalose, and lactose can also be used. In addition, alditols (acyclic polyhydroxy alcohols, also referred to as sugar alcohols) such as glycerol, mannitol, xylitol, and sorbitol are non-ionic tonicifying agents useful in the presently disclosed compositions. Non-ionic tonicity modifying agents can be present at a concentration of from about 0.1% to about 10% or about 1% to about 10%, depending upon the agent that is used. If NLCs are formulated for parenteral administration, it is preferable to make the osmolarity of the NLC composition the same as normal physiological fluids, preventing post-administration consequences, such as post-administration swelling or rapid absorption of the composition.

[0273] Optionally, lipid-based nanoparticles may be formulated with cryoprotectants comprising trehalose, sucrose, mannitol, sorbitol, Avicel PH102 (microcrystalline cellulose), Avicel RC591 (mixture of microcrystalline cellulose and sodium carboxymethyl cellulose), Mircrocelac® (mixture of lactose and Avicel), or a combination thereof. Optionally, NLCs may be formulated with a preservative agent such as, for example, Hydrolite 5.

[0274] VIII. Stable Emulsions

[0275] In some embodiments, stable emulsions are provided, wherein the stable emulsions comprise at least one adjuvant. Nonlimiting exemplary adjuvants that may be formulated with a stable emulsion include TLR3 agonists and Rig-I agonists. Nonlimiting exemplary such adjuvants include double-stranded RNA, RIBOXXOL, poly(l:C), and Hiltonol®.

[0276] In some embodiments, a stable emulsion (SE) is an oil-in-water emulsion. In some such embodiments, the oil-in-water emulsion is a squalene in water emulsion. WO 99 / 12565 discusses an improvement to these squalene emulsions with the addition of a sterol into the oil phase.

[0277] WO08 / 153541 discusses oil-in-water emulsions as conventionally having amounts of the components present in the range of from 2 to 10% oil, such as squalene;; and from 0.3 to 3% of a surfactant, such as polyoxyethylene sorbitan monooleate or Poloxamer 188 (copolymer of polyoxyethylene and polyoxypropylene). The ratio of oiksurfactant may be equal or less than 1 to improve stability of the emulsion. Span 85 may also be present at a level of about 1 %. In some cases it may be advantageous that the vaccines further contain a stabiliser. In one embodiment, the stabilizer may be a triglyceride, such as tricaprylin (C 27 H 50 O 6) (see, e.g., WO 98 / 56414). In some embodiments, an oil-in-water emulsion comprises 0.5% to 5%, or 0.5% to 5%, or 0.5% to 3%, or 1% to 3% glycerol. In some embodiments, an oil-in-water emulsion comprises 0.5% to 5%, or 0.5% to 5%, or 0.5% to 3%, or 1 % to 3% 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). A nonlimiting exemplary oil-in-water emulsion is discussed in the examples herein.

[0278] The size of the oil droplets found within the stable oil in water emulsion are preferably less than 1 micron, may be in the range of substantially 30-600 nm, preferably substantially around 30-500 nm in diameter, and most preferably substantially 150-500 nm in diameter, and in particular about 150 nm in diameter as measured by photon correlation spectroscopy. In this regard, 80% of the oil droplets by number should be within the preferred ranges, more preferably more than 90% and most preferably more than 95% of the oil droplets by number are within the defined size ranges The amounts of the components present in the oil emulsions of the present invention are conventionally in the range of from 2 to 10% oil, such as squalene;; and from 0.3 to 3% surfactant, such as polyoxyethylene sorbitan monooleate. Span 85 may also be present at a level of about 1 %. In somecases it may be advantageous that the vaccines of the present invention will further contain a stabiliser.

[0279] Methods of producing oil in water emulsions are well known to the person skilled in the art. Commonly, the method comprises the mixing the oil phase with a surfactant such as a PBS / TWEEN80® solution, followed by homogenization using a homogenizer. For instance, a method that comprises passing the mixture once, twice or more times through a syringe needle would be suitable for homogenizing small volumes of liquid. Equally, the emulsification process in a microfluidiser (M110S microfluidics machine, maximum of 50 passes, for a period of 2 minutes at maximum pressure input of 6 bar (output pressure of about 850 bar)) could be adapted to produce smaller or larger volumes of emulsion. This adaptation could be achieved by routine experimentation comprising the measurement of the resultant emulsion until a preparation was achieved with oil droplets of the required diameter.

[0280] IX. Methods of Using the Compositions of the Present Disclosure

[0281] A. Therapeutics

[0282] In some embodiments the compositions of the disclosure are useful for therapeutic purposes. Thus, in some embodiments, the compositions described comprise a lipid-based nanoparticle of the disclosure, and further comprise a bioactive agent for the treatment of a disease, condition, or disorder. In some embodiments, the compositions comprise an NLC provided herein, and further comprise a bioactive agent for the treatment of a disease, condition, or disorder.

[0283] In some embodiments the agent is useful for the treatment or prevention of allergy, cancer, infectious disease, autoimmunity, or addiction. In some embodiments the agent is useful for stimulating, enhancing and / or modulating an immune response.

[0284] In some aspects of the disclosed embodiments, the compositions comprise cancer antigens or nucleic acids encoding a cancer antigen. In some embodiments, a vaccine composition comprises a cancer antigen will be useful against any cancer characterized by tumor associated antigen expression, such as HER-2 / neu expression or other cancer-specific or cancer-associated antigens.

[0285] Compositions and methods according to certain embodiments of the present disclosure may also be used for the prophylaxis or therapy of autoimmune diseases, which include diseases, conditions or disorders wherein a host's or subject's immune system detrimentally mediates an immune response that is directed against "self' tissues, cells, biomolecules (e.g., peptides, polypeptides, proteins, glycoproteins, lipoproteins, proteolipids, lipids, glycolipids, nucleic acids such as RNA and DNA, oligosaccharides, polysaccharides, proteoglycans, glycosaminoglycans, or the like, and other molecular components of the subjects cells and tissues) or epitopes (e.g., specificimmunologically defined recognition structures such as those recognized by an antibody variable region complementarity determining region (CDR) or by a T cell receptor CDR.

[0286] Autoimmune diseases are thus characterized by an abnormal immune response involving either cells or antibodies that are in either case directed against normal autologous tissues. Autoimmune diseases in mammals can generally be classified in one of two different categories: cell- mediated disease (i.e., T-cell) or antibody-mediated disorders. Non-limiting examples of cell-mediated autoimmune diseases include multiple sclerosis, rheumatoid arthritis, Hashimoto thyroiditis, type I diabetes mellitus (Juvenile onset diabetes) and autoimmune uvoretinitis. Antibody-mediated autoimmune disorders include, but are not limited to, myasthenia gravis, systemic lupus erythematosus (or SLE), Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune asthma, cryoglobulinemia, thrombic thrombocytopenic purpura, primary biliary sclerosis and pernicious anemia. The antigen(s) associated with: systemic lupus erythematosus is small nuclear ribonucleic acid proteins (snRNP); Graves' disease is the thyrotropin receptor, thyroglobulin and other components of thyroid epithelial cells; pemphigus is cadherin-like pemphigus antigens such as desmoglein 3 and other adhesion molecules; and thrombic thrombocytopenic purpura is antigens of platelets.

[0287] The compositions provided herein may be used for inducing protective immunity, for example against tuberculosis include the use of polypeptides that contain at least one immunogenic portion of one or more Mycobacterium proteins and DNA and RNA molecules encoding such polypeptides. In addition, such compounds may be formulated into vaccines and / or pharmaceutical compositions for immunization against Mycobacterium infection.

[0288] In other embodiments, the compositions of the present disclosure include antigens associated with respiratory diseases, such as those caused or exacerbated by bacterial infection (e.g., pneumococcal), for the prophylaxis and therapy of conditions such as chronic obstructive pulmonary disease (COPD).

[0289] In addition to direct in vivo procedures, ex vivo procedures may be used in which cells are removed from a host, modified, and placed into the same or another host animal. It will be evident that one can utilize any of the compositions noted above for introduction of antigen-encoding nucleic acid molecules into tissue cells in an ex vivo context. Protocols for viral, physical and chemical methods of uptake are well known in the art.

[0290] In some embodiments, the compositions of the present disclosure are used to boost or enhance an immune response in a subject. In some such embodiments, the bioactive agent is an adjuvant. Nonlimiting exemplary adjuvants include TLR agonists (including TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 agonists), Rig-1 agonists, saponins, carbohydrates, carbohydrate polymers,conjugated carbohydrates, whole viral particles, virus-like particles, viral fragments, and cellular fragments. Examples of such adjuvants include, but are not limited to, double-stranded RNA, RIBOXXOL, poly(l:C), and Hiltonol®. In some embodiments, the composition comprises a stable emulsion and / or a nanostructured lipid carrier. In some embodiments, the composition comprises a stable emulsion and / or a nanostructured lipid carrier that comprises squalene. The present inventors have found that squalene-based formulations unexpectedly potentiate, for example, TLR3 agonists.

[0291] In some embodiments, the compositions of the present disclosure are useful for enhancing or eliciting, in a host, a patient or in cell culture, an immune response. As used herein, the term "subject” refers to any mammal. A patient may be afflicted with an infectious disease, cancer, such as breast cancer, or an autoimmune disease, or may be normal (i.e., free of detectable disease and / or infection). A "cell culture” is any preparation containing immunocompetent cells or isolated cells of the immune system (including, but not limited to, T cells, macrophages, monocytes, B cells and dendritic cells). Such cells may be isolated by any of a variety of techniques well known to those of ordinary skill in the art (e.g., Ficoll-hypaque density centrifugation). The cells may (but need not) have been isolated from a patient afflicted with cancer and may be reintroduced into a patient after treatment.

[0292] In some embodiments, the therapeutic compositions of the present disclosure are useful for enhancing or eliciting an immune response in a subject or patient afflicted with an infectious disease. In some embodiments, the infectious disease is associated with a bacterial, viral or fungal infection. In some embodiments, the subject is infected with at least one infectious pathogen such as a bacterium, a virus or a fungus, including an Actinobacterium such as M. tuberculosis or M. leprae or another mycobacterium; a bacterium such as a member of the genus Escherichia, Salmonella, Neisseria, Borrelia, Chlamydia, Clostridium or Bordetella; a virus such as a herpes simplex virus, a human immunodeficiency virus (HIV such as HIV-1 or HIV-2 ), an influenza virus, a parainfluenza virus, a measles virus, a mumps virus, a rubella virus, a coronavirus (such as SARS or MERS), a rotavirus, a norovirus, a picorna virus (such as a poliovirus, an enterovirus, or a coxsacchie virus), a veterinary pathogen, for example, a feline immunodeficiency virus (FIV), cytomegalovirus, Varicella Zoster Virus, hepatitis virus, Epstein Barr Virus (EBV), a flavivirus virus (such as dengue virus, Japanese encephalitis virus, yellow fever virus, Zika virus, Powassan virus or tick-borne encephalitis virus ), a henipah virus (such as hendra or nipah virus), a bunyavirus (such as Hantavirus or Rift Valley Fever virus), an arenavirus (such as lassa virus, junin virus, machupo virus, or guanarito virus), a filovirus (such as Ebola virus or Marburg virus), a lyssavirus (such as Rabies virus), respiratory syncytial virus, human papilloma virus (HPV) and a cytomegalovirus; ; a fungus such as Aspergillus, Blastomyces, Coccidioides and Pneumocysti or a yeast, including Candida species such as C.albicans, C. glabrata, C. krusei, C. lusitaniae, C. tropicalis and C. parapsilosis; a parasite such as a protozoan, for example, a Plasmodium species including P. falciparum, P. vivax, P. malariae and P. ovale; or another parasite such as one or more of Acanthamoeba, Entamoeba histolytica, Angiostrongylus, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Cryptosporidium, Ancylostoma, Entamoeba histolytica, Entamoeba coll, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Wuchereria bancrofti, Giardia, Toxoplasma gondii, and Leishmania. In specific embodiments, the antigen may be from, or related to antigens involved in tuberculosis, influenza, amebiasis, HIV, hepatitis, or Leishmaniasis.

[0293] In some embodiments, the therapeutic compositions of the present disclosure are useful for treating an allergic condition in a subject in need of treatment.

[0294] B. Vaccine

[0295] The present disclosure thus provides compositions for altering (i.e., increasing or decreasing in a statistically significant manner, for example, relative to an appropriate control as will be familiar to persons skilled in the art) immune responses in a host capable of mounting an immune response. As will be known to persons having ordinary skill in the art, an immune response may be any active alteration of the immune status of a host, which may include any alteration in the structure or function of one or more tissues, organs, cells or molecules that participate in maintenance and / or regulation of host immune status. Typically, immune responses may be detected by any of a variety of well-known parameters, including but not limited to in vivo or in vitro determination of: soluble immunoglobulins or antibodies; soluble mediators such as cytokines, lymphokines, chemokines, hormones, growth factors and the like as well as other soluble small peptide, carbohydrate, nucleotide and / or lipid mediators; cellular activation state changes as determined by altered functional or structural properties of cells of the immune system, for example cell proliferation, altered motility, induction of specialized activities such as specific gene expression or cytolytic behavior; cellular differentiation by cells of the immune system, including altered surface antigen expression profiles or the onset of apoptosis (programmed cell death); or any other criterion by which the presence of an immune response may be detected.

[0296] Determination of the induction of an immune response by the compositions of the present disclosure may be established by any of a number of well-known immunological assays with which those having ordinary skill in the art will be readily familiar. Such assays include, but need not be limited to, to in vivo or in vitro determination of: soluble antibodies; soluble mediators such as cytokines, lymphokines, chemokines, hormones, growth factors and the like as well as other soluble small peptide, carbohydrate, nucleotide and / or lipid mediators; cellular activation state changes as determined by altered functional or structural properties of cells of the immune system, for examplecell proliferation, altered motility, induction of specialized activities such as specific gene expression or cytolytic behavior; cellular differentiation by cells of the immune system, including altered surface antigen expression profiles or the onset of apoptosis (programmed cell death). Procedures for performing these and similar assays are widely known and may be found, for example in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998; see also Current Protocols in Immunology; see also, e.g., Weir, Handbook of Experimental Immunology, 1986 Blackwell Scientific, Boston, MA; Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, 1979 Freeman Publishing, San Francisco, CA; Green and Reed, 1998 Science 281 :1309 and references cited therein.).

[0297] Detection of the proliferation of antigen-reactive T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring the rate of DNA synthesis, and antigen specificity can be determined by controlling the stimuli (such as, for example, a specific desired antigenor a control antigen-pulsed antigen presenting cells) to which candidate antigen-reactive T cells are exposed. T cells which have been stimulated to proliferate exhibit an increased rate of DNA synthesis. A typical way to measure the rate of DNA synthesis is, for example, by pulse-labeling cultures of T cells with tritiated thymidine, a nucleoside precursor which is incorporated into newly synthesized DNA. The amount of tritiated thymidine incorporated can be determined using a liquid scintillation spectrophotometer. Other ways to detect T cell proliferation include measuring increases in interleukin-2 (IL-2) production, Ca2+ flux, or dye uptake, such as 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Alternatively, synthesis of lymphokines (such as interferon-gamma) can be measured or the relative number of T cells that can respond to a particular antigen may be quantified.

[0298] Detection of antigen-specific antibody production may be achieved, for example, by assaying a sample (e.g., an immunoglobulin containing sample such as serum, plasma or blood) from a host treated with a vaccine according to the present disclosure using in vitro methodologies such as radioimmunoassay (RIA), enzyme linked immunosorbent assays (ELISA), equilibrium dialysis or solid phase immunoblotting including Western blotting. In embodiments ELISA assays may further include antigen-capture immobilization of the target antigen with a solid phase monoclonal antibody specific for the antigen, for example, to enhance the sensitivity of the assay. Elaboration of soluble mediators (e.g., cytokines, chemokines, lymphokines, prostaglandins, etc.) may also be readily determined by enzyme-linked immunosorbent assay (ELISA), for example, using methods, apparatus and reagents that are readily available from commercial sources (e.g., Sigma, St. Louis, MO; see also R & D Systems 2006 Catalog, R & D Systems, Minneapolis, MN).

[0299] Any number of other immunological parameters may be monitored using routine assays that are well known in the art. These may include, for example, antibody dependent cell-mediated cytotoxicity (ADCC) assays, secondary in vitro antibody responses, flow immunocytofluorimetric analysis of various peripheral blood or lymphoid mononuclear cell subpopulations using well established marker antigen systems, immunohistochemistry or other relevant assays. These and other assays may be found, for example, in Rose et al. (Eds.), Manual of Clinical Laboratory Immunolog, 5th Ed., 1997 American Society of Microbiology, Washington, DC.

[0300] Accordingly, it is contemplated that the compositions provided herein will be capable of eliciting or enhancing in a host at least one immune response that is selected from a Th1-type T lymphocyte response, a TH2-type T lymphocyte response, a cytotoxic T lymphocyte (CTL) response, an antibody response, a cytokine response, a lymphokine response, a chemokine response, and an inflammatory response. In certain embodiments the immune response may comprise at least one of production of one or a plurality of cytokines wherein the cytokine is selected from interferon-gamma (IFN-y), tumor necrosis factor-alpha (TNF-a), production of one or a plurality of interleukins wherein the interleukin is selected from IL-1 , IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18 and IL- 23, production one or a plurality of chemokines wherein the chemokine is selected from MIP-1a, MIP- 1p, RANTES, CCL2,CCL4, CCL5, CXCL1 , and CXCL5,and a lymphocyte response that is selected from a memory T cell response, a memory B cell response, an effector T cell response, a cytotoxic T cell response and an effector B cell response.

[0301] In some embodiments, the compositions provided herein can be used to treat an allergic condition, i.e., as a tolerizing vaccine.

[0302] C. Diagnostic Agents

[0303] In some embodiments the bioactive agent is a diagnostic agent. Thus, in these embodiments, the compositions described comprise the NLC provided herein, and further comprise a diagnostic agent and are useful for the diagnosis of any disease, condition, or disorder.

[0304] In some embodiments, the diagnostic agents are useful for the detection cancer. Compositions and methods are known in the art for identifying subjects having or suspected of being at risk for developing cancer are described herein. Diagnosis of cancer in a subject having or suspected of being at risk for having cancer may be accomplished by any of a wide range of art- accepted methodologies, which may vary depending on a variety of factors including clinical presentation, degree of progression of the cancer, the type of cancer, and other factors. Examples of cancer diagnostics include histopathological, histocytochemical, immunohistocytochemical, and immunohistopathological examination of patient samples (e.g., blood, skin biopsy, other tissue biopsy, surgical specimens, etc.), PCR tests for defined genetic (e.g., nucleic acid) markers, serological testsfor circulating cancer-associated antigens or cells bearing such antigens, or for antibodies of defined specificity, or other methodologies with which those skilled in the art will be familiar.

[0305] In some embodiments, the diagnostic agents are useful for the detection of an autoimmune disease. Detection of an autoantibody thus permits early discovery or recognition of presence or risk for developing an autoimmune disease. Based on these findings, a variety of autoantibodies against autoantigens have been discovered and the autoantibodies against autoantigens have been measured in clinical tests.

[0306] In one embodiment, the diagnostic agents are useful for the detection of infectious diseases. Compositions and methods are known in the art for identifying subjects having, or suspected of being at risk for having, an infection with an infectious pathogen as described herein.

[0307] For example, the bacterium Mycobacterium tuberculosis cases tuberculosis (TB). Thus, in some embodiments, the compositions comprising any of the NLCs described herein further comprise an agent for diagnosing tuberculosis.

[0308] In some embodiments, the compositions comprising any of the NLCs described herein further comprise an agent for diagnosing malaria.

[0309] Polynucleotides have been described in the art that encode species-specific P. vivax malarial peptide antigens which are proteins or fragments of proteins secreted into the plasma of a susceptible mammalian host after infection, as have monoclonal or polyclonal antibodies directed against these antigens. The peptide antigens, monoclonal antibodies, and / or polyclonal antibodies are utilized in assays used to diagnose malaria, as well as to determine whether Plasmodium vivax is the species responsible for the infection. Species-specific P. vivax malarial peptide antigens have also been reported which are proteins or fragments of proteins secreted into the plasma of a susceptible mammalian host after infection, as have monoclonal or polyclonal antibodies directed against these antigens. The peptide antigens, monoclonal antibodies, and / or polyclonal antibodies are utilized in assays used to diagnose malaria, as well as to determine whether Plasmodium vivax is the species responsible for the infection.

[0310] A recombinant Plasmodium falciparum (3D7) AMA-1 ectodomain has also been expressed by a method that produces a highly purified protein which retains folding and disulfide bridging of the native molecule. The recombinant AMA-1 is useful as a diagnostic reagent, for use in antibody production, and as a vaccine. Similarly known are the expression and purification of a recombinant Plasmodium falciparum (3D7) MSP-142, which retains folding and disulfide bridging of the native molecule. The recombinant MSP-142 is useful as a diagnostic reagent, for use in antibody production, and as a vaccine.

[0311] In some embodiments, the compositions described herein further comprise an agent useful for diagnosing Leishmaniasis.

[0312] In some embodiments, the compositions described herein further comprise an agent useful for diagnosing HIV.

[0313] In some embodiments, the compositions described herein further comprise an agent useful for diagnosing a corona virus infection. In some embodiments, the compositions comprising any of the NLCs described herein further comprise an agent useful for diagnosing a SARS-CoV-2 corona virus infection.

[0314] X. Methods of Generating an Immune Response

[0315] Provided herein are methods of generating an immune response in a subject, including the step of administering to a subject in need thereof a therapeutically effective amount of a composition described herein, where the bioactive agent is a protein antigen or a nucleic acid molecule encoding a protein antigen. In exemplary embodiments, the bioactive agent is a RNA (e.g., mRNA) or a DNA molecule encoding a protein antigen. In some embodiments, methods of boosting or enhancing an immune response are provided, wherein the bioactive agent is an adjuvant.

[0316] Typical routes of administration of the therapeutically effective amount of the composition include, without limitation, oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, intranasal, intramucosal, or subcutaneous. In some exemplary embodiments, administration of the composition is intramuscular, ocular, parenteral, or pulmonary.

[0317] In exemplary embodiments, the compositions disclosed herein are vaccine compositions and are used as vaccines. The compositions described herein can be used for generating an immune response in the subject (including a non-specific response and an antigen-specific response). In some embodiments, the immune response comprises a systemic immune response. In some embodiments, the immune response comprises a mucosal immune response. Generation of an immune response includes stimulating an immune response, boosting an immune response, or enhancing an immune response. In some embodiments, the compositions described herein can also be used as tolerizing vaccines to allergens.

[0318] The compositions described herein may be used to enhance protective immunity against a virus. Such viruses and viral antigens include, for example, HIV-1 , (such as tat, nef, gp120 or gp160), human herpes viruses (such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSV1 or HSV2), cytomegalovirus ((esp. Human, such as gB or derivatives thereof), Rotavirus (including live-attenuated viruses), Epstein Barr virus (such as gp350 or derivatives thereof), Varicella Zoster Virus (such as gpl, II and IE63), or from a hepatitis virus such as hepatitis B virus (for example Hepatitis B Surface antigen or a derivative thereof), hepatitis A virus, hepatitis C virus and hepatitis Evirus, or from other viral pathogens, such as paramyxoviruses: Respiratory Syncytial virus (such as F and G proteins or derivatives thereof), parainfluenza virus, measles virus, mumps virus, human papilloma viruses (for example HPV6, 11 , 16, 18, etc.), flaviviruses (e.g., dengue virus, Japanese encephalitis virus, yellow fever virus, Zika virus, Poswanan virus, tick-borne encephalitis virus )or Influenza virus (whole live or inactivated virus, split influenza virus, grown in eggs or MDCK cells, or whole flu virosomes (as described by Gluck, Vaccine, 1992, 10, 915-920) or purified or recombinant proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof).

[0319] The compositions described herein may be used to enhance protective immunity against one or more bacterial pathogens such as Neisseria spp, including N. gonorrhea and N. meningitidis (for example capsular polysaccharides and conjugates thereof, transferrin-binding proteins, lactoferrin binding proteins, PilC, adhesins); S. pyogenes (for example M proteins or fragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S. mutans: H. ducreyi; Moraxella spp, including M. catarrhalis, also known as Branhamella catarrhalis (for example high and low molecular weight adhesins and invasins); Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium spp., including M. tuberculosis (for example ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coll (for example colonization factors, heat- labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof), enterohemorragic E. coll, enteropathogenic E. coll (for example shiga toxin-like toxin or derivatives thereof); Vibrio spp, including V. cholera (for example cholera toxin or derivatives thereof); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y. enterocolitica (for example a Yop protein), Y. pestis, Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (for example toxins, adhesins and invasins) and C. coll; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp., including L. monocytogenes; Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis; Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp., including C. tetani (for example tetanus toxin and derivative thereof), C. botulinum (for example botulinum toxin and derivative thereof), C. difficile (for example Clostridium toxins A or B and derivatives thereof); Bacillus spp., including B. anthracis (for example botulinum toxin and derivatives thereof); Corynebacterium spp., including C. diphtheriae (for example diphtheria toxin and derivatives thereof); Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia spp., including E.equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp. including C. trachomatis (for example MOMP, heparin-binding proteins), C. pneumoniae (for example MOMP, heparin-binding proteins), C. psittaci; Leptospira spp., including L. interrogans; Treponema spp., including T. pallidum (for example the rare outer membrane proteins), T. denticola, T. hyodysenteriae; or other bacterial pathogens.

[0320] The compositions described herein may be used to enhance protective immunity against one or more parasites (See, e.g., John, D.T. and Petri, W.A., Markell and Voge's Medical Parasitology- 9th Ed., 2006, WB Saunders, Philadelphia; Bowman, D.D., Georgis' Parasitology for Veterinarians- 8th Ed., 2002, WB Saunders, Philadelphia) such as Plasmodium spp., including P. falciparum; Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Tg34); Entamoeba spp., including E. histolytica; Babesia spp., including B. microti; Trypanosoma spp., including T. cruzi; Giardia spp., including G. lamblia; Leshmania spp., including L. major; Pneumocystis spp., including P. carinii; Trichomonas spp., including T. vaginalis; or from a helminth capable of infecting a mammal, such as: (I) nematode infections (including, but not limited to, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria, Necator americanus, Ancylostoma duodenale, Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, and Strongyloides stercoralis); (ii) trematode infections (including, but not limited to, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongi, Opisthorchis sinensis, Paragonimus sp, Fasciola hepatica, Fasciola magna, Fasciola gigantica); and (ill) cestode infections (including, but not limited to, Taenia saginata and Taenia solium). In certain embodiments, the antigen is derived from Schisostoma spp., Schistosoma mansonii, Schistosoma haematobium, and / or Schistosoma japonicum, or derived from yeast such as Candida spp., including C. albicans; Cryptococcus spp., including C. neoformans. infectious pathogen such as a bacterium, a virus or a fungus, including an Actinobacterium such as M. tuberculosis or M. leprae or another mycobacterium; a bacterium such as a member of the genus Salmonella, Neisseria, Borrelia, Chlamydia or Bordetella; a virus such as a herpes simplex virus, a human immunodeficiency virus (HIV), a feline immunodeficiency virus (FIV), cytomegalovirus, Varicella Zoster Virus, hepatitis virus, Epstein Barr Virus (EBV), Zika virus (ZIKV) respiratory syncytial virus, human papilloma virus (HPV) and a cytomegalovirus; HIV such as HIV-1 or HIV-2; a fungus such as Aspergillus, Blastomyces, Coccidioides and Pneumocysti or a yeast, including Candida species such as C. albicans, C. glabrata, C. krusei, C. lusitaniae, C. tropicalis and C. parapsilosis; a parasite such as a protozoan, for example, a Plasmodium species including P. falciparum, P. vivax, P. malariae and P. ovale; or another parasite such as one or more of Acanthamoeba, Entamoeba histolytica, Angiostrongylus, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Cryptosporidium, Ancylostoma, Entamoeba histolytica,Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Wuchereria bancrofti, Giardia, and Leishmania.

[0321] Methods for determining whether a composition of the present inventions is capable of effectively delivering the bioactive agent and / or having the desired effect in a subject are known in the art and not described herein in detail. In one aspect, immune responses against an antigen can be determined by monitoring the level antigen-specific antibody before and after administration (e.g., systemic IgM, IgG (lgG1, lgG2a, et al.) or IgA) in blood samples or from mucosal sites. Cellular immune responses also can be monitored after administration by assessing T and B cell function after antigen stimulation.

[0322] Another way of assessing the immunogenicity of the compositions or vaccines disclosed herein where the nucleic acid molecule (e.g., the RNA) encodes a protein antigen is to express the recombinant protein antigen for screening patient sera or mucosal secretions by immunoblot and / or microarrays. A positive reaction between the protein and the patient sample indicates that the patient has mounted an immune response to the protein in question. This method may also be used to identify immunodominant antigens and / or epitopes within protein antigens.

[0323] The efficacy of the compositions can also be determined in vivo by challenging appropriate animal models of the pathogen of interest infection.

[0324] In the embodiments provided herein, the subject is a mammal (e.g., an animal including farm animals (cows, pigs, goats, horses, etc.), pets (cats, dogs, etc.), and rodents (rats, mice, etc.), or a human). In one embodiment, the subject is a human. In another embodiment, the subject is a non-human mammal. In another embodiment, the non-human mammal is a dog, cow, or horse.

[0325] XI. Methods of Delivering a Bioactive Agent to a Cell

[0326] Provided herein are methods of delivering a bioactive agent to a cell, including the step of contacting the cell with a composition described herein. In some embodiments, the bioactive agent is a nucleic acid. In some embodiments, contacting the cell with the composition includes a step of administering the composition to a subject where the cell is in the subject. Such methods are useful in the delivery of antigen or antigen-encoding nucleic acids for generation of an immune response. Such methods are also useful for the delivery of antibody-encoding nucleic acids, protein or small molecule drugs, hormones, non-coding RNA molecules, and other bioactive agents for treatment of disease and health conditions.

[0327] The methods described herein for delivering a bioactive agent to a cell may find use in the treatment of diseases and health conditions including, without limitation, cancer, such as meningiomas, hepatic cell carcinoma, pancreatic tumors; allergy; infectious diseases including fungal,bacterial, or parasitic diseases; inflammatory diseases including psoriasis and arthritis and atrial- ventricular malformations; autoimmune diseases; and neurological diseases.

[0328] In embodiments of methods of delivering a composition to a cell including the step of administering the composition to a subject where the cell is in the subject, typical routes of administration of the therapeutically effective amount of the composition include, without limitation, oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, intranasal, intramucosal, or subcutaneous. In preferred embodiments, administration of the composition is intramuscular, parenteral, or intradermal. In such embodiments, the subject is a mammal (e.g., an animal including farm animals (cows, pigs, goats, horses, etc.), pets (cats, dogs, etc.), and rodents (rats, mice, etc.), or a human). In one embodiment, the subject is a human. In another embodiment, the subject is a non-human mammal. In another embodiment, the non-human mammal is a dog, cow, or horse.

[0329] In some embodiments, multiple modes of delivery may be used to obtain greater immune response. For example, the composition can be administered 1 , 2, 3, or 4 times. In some embodiment, the one or more administrations may occur as part of a so-called "prime-boost” protocol. In some embodiments the "prime-boost” approach comprises administration in several stages that present the same antigen through different vectors or multiple doses. In some embodiments, administration may occur more than twice, e.g., three times, four times, etc., so that the first priming administration is followed by more than one boosting administration. When multiple vectors or doses are administered, they can be separated from one another by, for example, one week, two weeks, three weeks, one month, six weeks, two months, three months, six months, one year, or longer. In some embodiments, a prime-boost approach comprises a RNA stage and a protein stage. The RNA stage may include, for example, administration of RNA carrying a gene coding for the antigenic protein, translation of the RNA into the antigen, and production of the corresponding antibodies in the subject. The protein stage may include, for example, administration of the antigen directly in the form of a protein. In some embodiments, the subject is administered (e.g., primed with) an oncolytic virus (which may be formulated with an NLC or without an NLC) that encodes a neoantigen, and then subsequently administered (e.g., boosted with) an NLC comprising an RNA construct that encodes the neoantigen.

[0330] XII. Methods for Optimizing Delivery of RNA to a Cell

[0331] Provided herein are methods for optimizing delivery of RNA to a cell comprising a step of selecting a molar ratio of nitrogen (N) to phosphate (P) that optimizes antibody titers produced in a subject comprising the cell. In an exemplary embodiment, actual N to P ratio is used to provide an interpretation of the RNA-NLC binding and corresponding in vitro expression of the RNA-encoded protein. Exemplary molar ratio of N to P may be from 1 to 200, from 1 to 100, preferably from 1 to 50,more preferably from about 5 to about 50 or from about 5 to about 40. In some exemplary embodiments, the molar ratio of N to P may be from 1 to 15 or from 1 to 7.

[0332] XIII. Methods for Treating a Disease or Condition

[0333] Provided herein are methods for treating a disease, condition, or disorder in a subject. In some embodiments, the subject is infected with a pathogen. In some embodiments the pathogen is a bacterium, virus, fungus or parasite. In some embodiments, the disease is associated with a bacterial, viral, fungal or parasitic infection. In some embodiments, the bacterium is a mycobacterium. In some embodiments, the pathogen is cryptosporidium. In some embodiments, the virus is a coronavirus (e.g., SARS-CoV-2), a Zika virus, an influenza virus (e.g., H1 N1, H5 andH7 subtypes), a yellow fever virus, HIV, human papillomavirus, chikungunya virus, VZV, RSV, or EBV.

[0334] In some embodiments, the compositions of the disclosure can be used to treat an allergic condition. For example, in a particular embodiment, the compositions are used in allergy desensitization therapy. Such therapy involves the stimulation of the immune system with gradually increasing doses of the substances to which a person is allergic, wherein the substances are formulated in compositions of the disclosure. In some embodiments, the compositions are used in the treatment of allergies to food products, pollen (tree, grass), mites, cats, stinging insects (e.g., bees, hornets, yellow jackets, wasps, velvet ants, fire ants), or Japanese Red Cedar antigens (CryJ 1 / CryJ2). Formulations comprising a saponin have been demonstrated to preferentially induce a Th1 immune response, associated with a protective immune response, and to down regulate a Th2 response, the type associated with an allergic response (e.g., high IL-2 and IFN-g, low IL-5 and IL-10). Administration of allergen with a composition of the disclosure will be expected to down regulate an existing allergic (Th2) response. In another embodiment, compositions of the disclosure can be administered to an allergic individual without co-administration of allergen. In such a case, endogenous allergen is present in an allergic individual with symptoms, and compositions of the disclosure interact with endogenous allergen to switch an allergic response to a protective response. Thus, in one embodiment the disclosure provides a method comprising selecting an allergic individual with symptoms and administering a composition of the disclosure to the individual in an amount effective to reduce or eliminate the symptoms.

[0335] In some embodiments, the methods comprise administering to a subject in need thereof a therapeutically effective amount of a composition of the disclosure, wherein the bioactive agent is a protein antigen or a nucleic acid molecule encoding a protein antigen or epitope.

[0336] XIV. Kits and Articles of Manufacture

[0337] Also contemplated in certain embodiments are kits comprising the herein described nanostructured lipid carriers (NLCs) and compositions, which may be provided in one or morecontainers. In one embodiment all components of the compositions are present together in a single container. In other embodiments, components of the compositions may be in two or more containers. In a preferred embodiment, the NLC is provided in one container, and the bioactive agent is provided in another container.

[0338] In some embodiments, one vial of the kit comprises an NLC provided herein, and a second vial of the kit contains an RNA molecule. In some embodiments, the kit comprises a third vial containing an optional component.

[0339] The kits of the invention may further comprise instructions for use as herein described or instructions for mixing the materials contained in the vials. In some embodiments, the material in the vial is dry or lyophilized. In some embodiments, the material in the vial is liquid.

[0340] A container according to such kit embodiments may be any suitable container, vessel, vial, ampule, tube, cup, box, bottle, flask, jar, dish, well of a single-well or multi-well apparatus, reservoir, tank, or the like, or other device in which the herein disclosed compositions may be placed, stored and / or transported, and accessed to remove the contents. Typically, such a container may be made of a material that is compatible with the intended use and from which recovery of the contained contents can be readily achieved. Non-limiting examples of such containers include glass and / or plastic sealed or re-sealable tubes and ampules, including those having a rubber septum or other sealing means that is compatible with withdrawal of the contents using a needle and syringe. Such containers may, for instance, by made of glass or a chemically compatible plastic or resin, which may be made of, or may be coated with, a material that permits efficient recovery of material from the container and / or protects the material from, e.g., degradative conditions such as ultraviolet light or temperature extremes, or from the introduction of unwanted contaminants including microbial contaminants. The containers are preferably sterile or sterilizeable, and made of materials that will be compatible with any carrier, excipient, solvent, vehicle or the like, such as may be used to suspend or dissolve the herein described vaccine compositions and / or immunological adjuvant compositions and / or antigens and / or recombinant expression constructs, etc.

[0341] The following Examples are offered by way of illustration and not by way of limitation.EXAMPLESDevelopment of NLC formulations

[0342] Nanostructured Lipid Carrier (NLC) compositions were prepared using procedures described previously in WO2018 / 232257 and J. H. Erasmus et al., A Nanostructured Lipid Carrier for Delivery of a Replicating Viral RNA Provides Single, Low-Dose Protection against Zika. Mol Ther 26, 2507-2522 (2018) with modifications to add cholesterol and QS-21.

[0343] The oil phase was composed of squalene - the liquid-phase of the oil core - a non-ionic sorbitan ester surfactant sorbitan monostearate (Span® 60), the sterol cholesterol, the cationic lipid DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), and the solid lipid (glyceryl trimyristate Dynasan® 114). The aqueous phase was a 10 mM sodium citrate trihydrate buffer containing the non-ionic PEGylated surfactant Tween® 80, and the saponin QS-21. Briefly, trimyristin (Sigma-Aldrich, St. Louis, MO), squalene (SAFC Supply Solutions, St. Louis, MO), cholesterol, sorbitan monostearate (Spectrum Chemical Mfg. Corp., New Brunswick, NJ), and the cationic lipid DOTAP (Lipoid, Ludwigshafen, Germany) were mixed and heated at 60°C in a bath sonicator to create the oil phase. Polysorbate 80 (MilliporeSigma, Burlington, MA) and QS-21 were separately diluted in 10mM sodium citrate trihydrate and heated to 60°C in a bath sonicator to create the aqueous phase. After dispersion of components in each phase, a high-shear mixer (Silverson Machines, East Longmeadow, MA) was used at -5,000 rpm to mix the oil and aqueous phases. Particle size of the mixture was then further decreased by high-pressure homogenization by processing at 30,000 psi for ten discrete passes using an M110P Microfluidizer (Microfluidics, Westwood, MA). The NLC product was then filtered through a 0.22 m PES filter and stored at 2°C- 8°C until use.

[0344] The make-up of the formulations depicted in the examples shown include 37.3 mg / ml squalene, 2.4 mg / ml dynasan 114, 30 mg / ml DOTAP, Tween 80, and 10 mM sodium citrate. QH933: The normal NLC contains the components listed above. QH932: The QS-21 NLC formulation also includes 1 mg / ml cholesterol and either 0.4, 2.0, or 10.0 pg of QS-21. The cholesterol NLC contains 1 mg / ml cholesterol without QS-21. QH931 : The solanesol NLC uses solanesol instead of squalene but is otherwise the same as the standard NLC. QH934: The GLC NLC includes 0.25 mg / mL GLA without QS-21 or cholesterol. QH849: The 3M-052 NLC contains 0.12 mg / mL 3M-052 without QS-21 or cholesterol.

[0345] To synthesize NLC formulations, the oil phase was first prepared by mixing the liquid phase lipid, solid phase lipid, cholesterol, positively charged lipid, and hydrophobic surfactant in a Blend Vessel, which was placed in a sonicating water bath (70 ± 5°C) to facilitate solubilization. Preparation of the aqueous phase involved dilution of a hydrophilic surfactant, preferably Tween 80, and the saponin, preferable QS-21, in ultrapure water for injection (WFI) or an aqueous buffer, such as 10 mM sodium citrate trihydrate, followed by stirring for complete dissolution. The aqueous composition was heated to 60-70°C in, for example, a bath sonicator, before blending with the oil phase. In some instances, the two phases were both heated separately to 60°C in a bath sonicator. A high shear mixer was used to combine the oil and aqueous phases by high shearing of the composite mixture. The blending speed was gradually increased to 5,000 RPM, or a maximum of10,000 RPM, in a high-speed laboratory emulsifier (Silverson Machines, Inc.), and mixing then occurred for a period of ten minutes to one hour to produce a crude mixture containing micron-sized oil droplets. The positioning of the Silverson mixing probe was adjusted as necessary for uniform dispersal of oil and complete emulsification. Further particle size reduction was achieved by high- shear homogenization in a M-110P microfluidizer (Microfluidics, Corp.). NLC particles were obtained from the crude emulsion using a M-110P Microfluidizer Materials Processor (Microfluidics, Corp.). Each emulsion was processed for approximately 5 passes on the microfluidizer at 45°C at 30,000 psi. The final pH was between 6.5-6.8. The resulting NLC particle suspension was strained using a 0.2pm sterile filter (e.g., 0.2 pm polyethersulfone membrane syringe filter) to collect the final NLC formulation and store at 2-8°C, which was subsequently assessed for particle size.Vaccine complexing and characterization

[0346] Vaccine complexes for immediate in vivo injection were created by mixing aqueous RNA 1 : 1 by volume with NLC diluted in a buffer containing 10mM sodium citrate and 20% w / v sucrose. All vaccines were prepared at a nitrogen:phosphate (N:P) ratio of 15, representing the ratio of amine groups on the NLC DOTAP to phosphate groups on the RNA backbone. This produced a vaccine solution containing the intended dose of complexed saRNA / NLC in an isotonic 10% w / v sucrose, 5mM sodium citrate solution (with small (<4 mM) amounts of Tris buffer present from the bulk RNA material). Vaccine was incubated on ice for 30 minutes after mixing to ensure complete complexing.Determining the N / P ratio

[0347] The Nitrogen to Phosphate (N / P) ratio is a theoretical representation of the molar stoichiometry of cationic nitrogens (positive charge) and anionic phosphate groups (negative charge) available to form the RNA-NLC complex. The cationic lipid DOTAP chloride (N-[1-(2,3- Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) used in NLCs contains a quaternary trimethylammonium head group and carries a positive charge that is independent of pH. Since each DOTAP molecule contains one trimethylammonium head group, nitrogen concentration (or the amount of positive charge) is essentially equal to DOTAP concentration. On the other hand, each ribonucleotide monophosphate in a RNA copy consists of a single 1 approximately proportional to the RNA concentration normalized to the average molecular weight of ribonucleotide monophosphates (339.5 g / mol). Thus, N / P where [DOTAP] and [RNA] are molar concentrations ofDOTAP and RNA, respectively.

[0348] Furthermore, RNA binding to NLC as a function of the theoretical N / P is characterized using a gel retardation assay (GRA). AAHI-SC2 saRNA was complexed with the various NLCs at an N:P ratio of 15, for a 1 pig dose in 100 pil with a 10% sucrose, 5 mM sodium citrate excipientbackground. The RNA-NLC mixtures were allowed to complex for 30 minutes on ice and then electrophoresed at 120 V for about an hour in a 1 % agarose gel. Optical densitometry analysis of RNA bands was performed to determine the relative amount of RNA bound to NLC formulation as a function of N / P. saRNA expression plasmid design, cloning, and production

[0349] A plasmid encoding an SP6 promoter followed by the 5' and 3' untranslated regions (UTRs) and nonstructural genes of Venezuelan equine encephalitis virus (VEEV) strain TC-83 as well as enhanced GFP under the control of the VEEV subgenomic promoter, termed pSP6-VEE-Rep-GFP, was kindly provided by Dr. Scott Weaver. A control reporter rvRNA, encoding secreted human embryonic alkaline phosphatase (SEAP) was also designed.

[0350] Three saRNA plasmids each with a unique candidate SARS-CoV-2 spike open reading frame were created, along with a fourth saRNA plasmid expressing secreted embryonic alkaline phosphatase (SEAP) instead of a vaccine antigen as an appropriate vector control. The SARS-CoV- 2 spike open reading frame sequence from GenBank MT246667.1 incorporating the additional D614G mutation was used as the "baseline” sequence, with additional modifications to create two additional vaccine candidates: D614G-2P represents the baseline sequence with a substitution of PP for KV at amino acid positions 987-988 and an addition of nine N-terminal codons from the reference genome encoding amino acid sequence MFLLTTKRT; D614G-2P-3Q refers to the baseline sequence with the diproline substitution and an additional substitution of QQAQ for RRAR at the furin cleavage site at amino acid positions 683-686. These sequences were then codon-optimized for mammalian (human) expression by Codex DNA (San Diego, CA) using a proprietary algorithm, synthesized by BioXp (Codex DNA), and inserted into AAHI's backbone saRNA expression vector by Gibson cloning. The SEAP-expressing plasmid was created by a similar process to insert the SEAP expression sequence in place of the vaccine antigen. Plasmid sequences were all confirmed by Sanger sequencing. Template plasmids were amplified in E. coli and extracted using Qiagen (Germantown, MD) maxior gigaprep kits, followed by Nofl linearization (New England Biolabs [NEB], Ipswich, MA). Linearized DNA was purified by Qiagen DNA purification kits.RNA manufacture

[0351] Vaccine saRNA was generated by T7 polymerase-mediated in vitro transcription (IVT) using Nofl-linearized DNA plasmids as templates. An in-house optimized IVT protocol was used with commercially available rNTP mix (NEB) and commercially available T7 polymerase, RNase inhibitor, and pyrophosphatase enzymes (Aldevron, Fargo, ND). DNA plasmids were digested away (DNase I, Aldevron), and Cap 0 structures were added to the transcripts by treatment with guanylyltransferase (Aldevron), GTP, and S-adenosylmethionine (NEB). RNA was chromatographically purified usingCapto Core 700 resin (GE Healthcare, Chicago, IL) followed by diafiltration and concentration through tangential flow filtration using a 750 kDa molecular weight cut-off (MWCO) modified polyethersulfone (mPES) membrane (Repligen, Waltham, MA). The final bulk RNA contained 10 mM Tris-HCI pH 8. Terminal filtration of the saRNA material was done using a 0.22 pm PES filter, and the saRNA materials were stored at -80°C until use / complexation. Agarose gel electrophoresis was used to characterize saRNA size and integrity. All gels that derive from the same experimental timepoint were processed in parallel. RNA concentration was quantified by UV absorbance (NanoDrop 1000) and RiboGreen assay (Thermo Fisher Scientific, Waltham, MA).

[0352] RNA for versions of saRNA with modified nucleosides was made with n1- methylpseudouridine-5'-triphosphate (TriLink BioTechnologies, San Diego, CA) substituted for DTP. The other three NTPs were used as normal, and production proceeded normally as described above.Evaluation of NLC Formulations as SARS-CoV-2 Vaccine Candidates

[0353] The NLC formulations were combined with self-amplifying viral RNA (saRNA) derived from a strain of Venezuelan equine encephalitis virus (VEEV, strain TC83) where the VEEV structural genes were replaced with a SARS-Cov-2 spike protein cassette. RNA replicon systems were developed to facilitate heterologous prime-boost strategies. The formulated saRNA was administered via the intramuscular route using a conventional needle.Materials and Methods

[0354] RNA production

[0355] Following transformation and amplification in Top10 cells (Invitrogen) and isolation using maxi-prep kits (Qiagen), plasmids were linearized by restriction digest with Notl enzyme (New England Biolabs) and purified using phenol-chloroform. RNA was then transcribed in vitro using T7 megascript kit (Invitrogen), followed by lithium chloride precipitation and capping with a Vaccinia capping kit (New England Biolabs). Capped transcripts were then precipitated in lithium chloride and resuspended in nuclease-free water to a final concentration of 1 pg / pl and analyzed by agarose-gel electrophoresis. All RNA was aliquoted and stored at -80°C.

[0356] Human Peripheral Blood Mononuclear Cell Assay

[0357] Studies involving human donors were approved by the Western Institutional Review Board. Heparinized whole blood was attained from 6 normal donors upon informed consent and peripheral blood mononuclear cells (PBMCs) were isolated as previously described. See Seubert et al., J Immunol. 180 (8): 5402- 12 (2008). One million cells per 150 pl volume of serum-free RPMI were plated into U-bottom TC grade 96-well plates and formulation / rvRNA complexes were added to the cells in a 50 pl volume and incubated at 37°C. Twenty-four hours later, plates were centrifuged for 10min at 1.8K rpm, and supernatants were harvested and stored at -20 °C. Mip-1 p ELISA was performed as previously described. See Seubert et al., J Immunol. 180 (8): 5402- 12 (2008).

[0358] Study 1

[0359] C57BL / 6 mice obtained from The Jackson Laboratory (Harbor, ME), 6-8 weeks old at study onset were used, with n=8 mice / group. Mice were non-specifically and blindly distributed into their respective groups. No exclusion criteria were established prior to beginning the studies. A total of 100 pl of vaccine was administered intramuscularly to the rear quadriceps muscles (50 pl / each rear leg) of each mouse. Mice received a single vaccine dose of 1 pg of the AAHI-SC2 saRNA, complexed with either normal NLC or cholesterol-containing NLC complexed with the various doses of QS-21. The vector control was the SEAP-expressing saRNA complexed with the cholesterol NLCs with a 10 pg QS-21 dose / mouse. Serum were taken from the mice three weeks after the single vaccine dose.Serum IgG, titers by ELISA (FIG. 2A)

[0360] SARS CoV-2 spike protein-binding IgG antibodies in mouse serum were measured by ELISA. Plates (384-well high-binding plates, Corning, Corning, NY) were coated with 1 pg / mL of Recombinant SARS-CoV-2 Spike His Protein, Carrier Free, (R&D Systems, Minneapolis, MN; #10549-CV) in phosphate-buffered saline (PBS) and incubated overnight at 4°C. The coating solution was removed, and blocking buffer (2% dry milk, 0.05% Tween 20, and 1% goat serum) was applied for at least 1 hour. On a separate, low-binding plate, each sample was diluted 1 :40 and then serially 1 :2 to create a 14-point dilution curve for each sample. Naive mouse serum, used as the negative control, was diluted identically to the samples. A SARS-CoV-2 neutralizing monoclonal antibody (mAb; GenScript, Piscataway, NJ; #A02057), was used as a positive control at a known starting concentration of 3.2 ng / pL followed by serial 1 :2 dilutions similarly to each sample and negative control. SARS-CoV-2 spike protein-coated and blocked assay plates were washed, and serially diluted samples were then transferred onto the coated plates followed by a 1 -hour incubation. Plates were then washed, and spike protein-bound antibodies were detected using an Anti-Mouse IgG (Fc Specific)-Alkaline Phosphatase antibody (Sigma-Aldrich, #A2429) at a 1 :4000 dilution in blocking buffer. Plates were washed and then developed using phosphatase substrate (Sigma-Aldrich, #S0942) tablets dissolved in diethanolamine substrate buffer (Fisher Scientific, Waltham, MA; #PI34064) at a final concentration of 1 mg / mL. After a 30-minute development, plates were read spectrophotometrically at 405 nm. A 4-point logistic curve (4PL) was used to fit the antibody standard titration curve. Sample concentrations were interpolated off the linear region of each sample dilution curve using the standard curve for absolute quantification of antibody titers.

[0361] For lgG1 and lgG2a isotype-specific ELISAs, the identical plate coating and blocking procedures were conducted. For the lgG1 assay, the standard curve was run using an lgG1 SARS- CoV-2 neutralizing antibody (GenScript #A02055) or an lgG2a SARS-CoV-2 neutralizing antibody (GenScript #BS-M0220) for full quantification. Sample dilution and incubation were identical to the total IgG curve, and plates were probed with IgGland lgG2a-specific secondary alkaline phosphatase-conjugated detection antibodies prior to development, reading, and quantification as described above.Pseudovirus neutralization assay (FIGS. 2B and 2C)

[0362] The SARS-CoV-2 pseudovirus neutralizing antibody titers of immunized mouse sera were measured by pseudovirus neutralization assays using procedures adapted from Crawford, K. H. D. et al. Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 spike protein for neutralization assays. Viruses 12, 513, doi: 10.3390 / v 12050513 (2020). Lentiviral SARS-CoV-2 spike protein pseudotyped particles were prepared by following the Bioland Scientific (Paramount, CA) BioT plasmid transfection protocol. Briefly, HEK-293 cells (American Type Culture Collection, Manassas, VA; #CRL-3216) were plated at 4 x 105 cells / mL in 6-well plates 18-24 hours before the assay to achieve 50-70% confluency at assay start. Cellular growth medium was then replaced with serum- free Gibco Dulbecco's Modified Eagle Medium (DMEM) with GlutaMAX immediately prior to transfection. The HEK-293 cells were co-transfected with several plasmids: a plasmid containing a lentiviral backbone expressing luciferase and ZsGreen (BEI Resources, Manassas, VA; #NR-52516), plasmids containing lentiviral helper genes (BEI Resources; #NR-52517, NR-52518, and NR-52519), and a plasmid expressing a delta19 cytoplasmic tail-truncated SARS-CoV-2 spike (Wuhan strain, B.1.1.7, and B.1.351 plasmids; and B.1.617.2 plasmid). BioT transfection reagent (Bioland Scientific) was used to mediate the co-transfection. The assay plates were incubated for 24 hours at standard cell culture conditions (37°C and 5% CO2). Then the serum-free media was aspirated off, and fresh growth medium (Gibco DMEM with GlutaMAX and 10% fetal bovine serum [FBS]) was added to the plates before returning them to the incubator for an additional 48 hours. Pseudovirus stocks were collected by harvesting the supernatants from the plates, filtering them through a 0.2 pm PES filter (Thermo Scientific), and freezing at -80°C until titration and use.

[0363] Mouse serum samples were diluted 1 :10 in medium (Gibco DMEM with GlutaMAX and 10% FBS) and then serially diluted 1 :2 for 11 total dilutions in 96-well V-bottom plates. Polybrene (Sigma-Aldrich) was then added at a concentration of 5 pig / mL to every well on the plate, and pseudovirus, diluted to a titer of 1 x 108 total integrated intensity units / mL as per titers determined independently for each pseudovirus batch, was added 1 :1 to the diluted serum samples. The plates were incubated for 1 hour at 37°C and 5% CO2. Serum-virus mix was then added in duplicate toHuman Angiotensin-Converting Enzyme 2 (hACE2)-expressing HEK-293 cells (BEI Resources, #NR- 52511) seeded at 4 x 105 cells / mL on a 96-well flat-bottom plate and incubated at 37°C and 5% CO2 for 72 hours. To determine 50% inhibitory concentration (IC50) values, plates were scanned on a high-content fluorescent imager (ImageXpress Pico Automated Cell Imaging System, Molecular Devices, San Jose, CA) for ZsGreen expression. Total integrated intensity per well was used to calculate the percent of pseudovirus inhibition in each well. Neutralization data for each sample were fit with a four-parameter sigmoidal curve that was used to interpolate IC50 values.

[0364] A Wuhan-strain pseudoneutralization test using the World Health Organization (WHO) standard for neutralization assays with an official IC50 of 1000 resulted in an IC50 of 7800 using our assay (FIG. 2C), suggesting that our test is more sensitive than the WHO assay but may overreport IC50 values— by less than one Iog10— an effect likely most pronounced for strongly neutralizing samples.

[0365] Fig. 4A shows neutralizing titers of serum neutralizing antibodies for five different NLC formulations and a vector control (SEAP). Various NLCs were complexed with RNA encoding AAHI- SC2 saRNA and neutralizing antibodies and assayed 4 months after a single 1 pig IM injection. Four months after vaccination, when antibody responses are in a waning mode, the QS-21 adjuvanted saRNA / NLC vaccinated mice display significantly higher serum neutralizing antibody titers relative to mice vaccinated with the standard, unadjuvanted saRNA / NLC vaccine. Only the NLC formulation adjuvanted with QS-21 achieved significantly higher antibody titers than the standard saRNA / NLC vaccine. It was unexpected that adjuvanting with QS-21 would produce an enhancement of antibody titers relative to the unadjuvanted control particularly because other adjuvants— GLA, solanesol, and 3M-052— fail to give such enhancement. This indicates that the inclusion of QS-21 uniquely enhances serum neutralizing antibody titers induced by saRNA vaccination, while inclusion of other standard vaccine adjuvants fails to do so. FIG. 4B shows titers of serum neutralizing antibodies in the same mice, using serum collected 6 weeks and 4 months after a single 1 pig IM injection. Very little waning of serum neutralizing antibody titers is seen with the QS-21 adjuvanted saRNA / NLC vaccinated mice over a four month period of time after a single low-dose vaccination. Data was analyzed by an ordinary two-way ANOVA with test followed by Dunnett's multiple comparisons test comparing all groups to the standard saRNA / NLC vaccine responses, on log-normalized antibody titer data.Splenocyte harvest, intracellular cytokine staining, and flow cytometry (FIGS. 3 and 5)

[0366] Spleens were dissociated in 4 mL of RPMI medium by manual maceration through a cell strainer using the end of a syringe plunger. Homogenized samples were briefly centrifuged at 400 x g (15-20 sec) to pellet fat cells. Samples were then carefully resuspended and fat clumps removed by pipette. The supernatants containing lymphocytes were transferred to 5-mL mesh-cap tubes to strainout any remaining tissue debris or were lysed with ammonium-chloride-potassium (ACK) buffer and washed. Cell counts for each sample were obtained on a Guava easyCyte (Luminex, Austin, TX). Each spleen sample was seeded in 96-well round-bottom plates at 1-2 x 106cells per well in RPMI medium containing 10% FBS, 50pM beta-mercaptoethanol, CD28 costimulatory antibody (BD Biosciences #553294), and brefeldin A. Cells were stimulated with one of three stimulation treatments: 0.0475% dimethyl sulphoxide (DMSO) as a negative stimulation control, 0.2 pg / well (1 pg / mL) per peptide of spike peptide pool (JPT Peptide Technologies, Berlin, Germany; #PM-WCPV-S-1) in an equivalent amount of DMSO, or 10 pg / well of phorbol myristate acetate (PMA) / ionomycin solution. After 6 hours of incubation at 37°C with 5% CO2, plates were centrifuged at 400 x g for 3 minutes, the supernatants were removed by pipetting, and cells were resuspended in PBS. Plates were centrifuged, the supernatants were removed, and cells were stained for flow cytometry. Splenocytes were stained for viability with Zombie Green (BioLegend, San Diego, CA) in 50 pL of PBS, and then Fc receptors were blocked with CD16 / CD32 antibody (Invitrogen #14-0161-86). Cells were then surface stained with fluorochrome-labeled mAbs specific for mouse CD4, CD8, CD44, and CD107a in 50 pL of staining buffer (PBS with 0.5% bovine serum albumin and 0.1 % sodium azide). Cells were washed twice, permeabilized using the Fixation / Permeabilization Kit (BD Biosciences, Franklin Lakes, NJ), and stained with fluorochrome-labeled mAbs specific for mouse TNFo, IL-2, IFNy, IL-5, IL-10, and IL-17A. After two washes in staining buffer, cells were resuspended in 100 pL of staining buffer and analyzed on an LSRFortessa flow cytometer (BD Biosciences). After initial gating for live CD4+or CD8+lymphocytes, cells were gated for cytokine positivity. Quality of the response was determined by gating on cells that were double or triple positive for these markers. Cells triple positive for TNFo, IL-2, and IFNy were considered activated polyfunctional T cells (FIG. 3C, 5C). n = 6 mice for SEAP control group and n = 8 for dosing groups (FIGS 3A-3C, 5A-5C).Whole Blood Assay (FIG. 6)

[0367] To assess the ability of the NLC formulations to stimulate human whole blood cells to release chemokines, human whole blood assays were performed. Heparinized whole blood was obtained from two normal donors. The indicated formulations (stock at 5% oil) were added to whole blood (0.4% oil final). Whole blood was incubated at 37°C-CO2 for 24 hours. The plasma supernatant was aspirated and assayed for the beta-chemokine CCL4 / MIP-1 p (ELISA) and IFNo / p Type I interferon (HEK-Blue Type I IFN Reporter assay).

[0368] Human PMBCs were isolated from 2 blood donors and resuspended in serum-free DMEM at a concentration of 1 x 106cells / mL, 200 pil / well in a 96 well plate. The following solutions were serially 1 :2 diluted in serum-free DMEM:

[0369] Cholesterol-containing NLC (lot QJ932)

[0370] Cholesterol-containing NLC (lot QJ932) complexed with p505 AAHI-SC2 Covid-19 saRNA vaccine RNA.

[0371] Cholesterol-containing NLC (lot QJ932) complexed with QS-21_(0.2 mg / mL final in mix) and with p505 AAHI-SC2 Covid-19 saRNA vaccine RNA.

[0372] 2 x 105primary human peripheral blood mononuclear cells (PBMCs) from two different blood donors were stimulated with NLC alone, NLC + saRNA, and NLC + saRNA + QS-21 to give a final concentration of QS-21 at 0.5 pig / mL and 0.25 pig / mL in 250 piL total volume. After 18 hours, supernatants were harvested and assayed for the monocyte / macrophage recruiting chemokine Ml P- 1p (ELISA) and IFNo / p Type I interferon (HEK-Blue Type I IFN Reporter assay) (FIG. 6A-6D).

[0373] NLC alone stimulated negligible levels of type I IFN and low levels of the chemokine MIP- 1p. Addition of saRNA to the NLC clearly stimulated type I IFN responses in human PBMCs and stimulated a clear increase in MIP-1 p secretion relative to the NLC alone. The key result is that addition of QS-21 to the NLC-saRNA vaccines dramatically increased both type I IFN and MIP-1 p secretion, illustrating the complementary immune stimulation due to QS-21 relative to the immunostimulatory effects of the squalene and saRNA components of the standard vaccine. The enhancement of type I IFN responses by QS-21 is notable as type I interferons restrict saRNA replication; however, antigen expression remains sufficient in vivo for robust vaccine responses, and the strong stimulation of APC-recruiting chemokines such as MIP-1 p is hypothesized to be the mechanism behind the observed in vivo enhancement of neutralizing antibody and CD8+ T cell responses as demonstrated in FIGS. 4 and 5. These data indicate that the inclusion of QS-21 in the NLC formulation in the context of an RNA vaccine is indeed changing the human innate immune response to the vaccine. For an RNA vaccine, suppression of innate immunity is generally considered beneficial.

[0374] Previous data from the inventors indicates that liposomal QS-21 robustly stimulates pro- inflammatory cytokines IL-1 p, TNFo, and IL-6; neutrophil-recruiting cytokines IL-8 and CXCL5; and macrophage-recruiting cytokines MCP-1 and MIP-1 p without stimulating either IFNy or IP-10, further suggesting that the mode of action for QS-21 enhancement of saRNA vaccine responses involves recruitment of APCs and other immune cells to the site of vaccination.

[0375] Study 2

[0376] This example demonstrates that QS-21 -containing saRNA-qNLC Vaccines Drive Enhanced Neutralizing Antibody and T Cell Responses.

[0377] A single injection of SARS-CoV-2 spike saRNA complexed with QS-21 -adjuvanted NLCs (qNLCs) at different QS-21 doses was injected intramuscularly into C57BL / 6 mice at a single low 1- pig RNA dose. SARS-CoV-2 serum neutralizing antibody titers were measured 3 weeks post-primeby pseudovirus neutralization assay and found to be significantly and repeatably enhanced by a 2-pig dose of QS-21 relative to the unadjuvanted controls (cholesterol-contai ning and standard NLCs) (FIG. 7 A). Notably, CD8+ T cell splenocyte populations were markedly increased by this same addition of 2 pig QS-21 (pcO.001), with a more-than-doubled mean CD8+ T cell response in the adjuvanted saRNA-qNLC vaccine (FIG. 7B). The dose-response curve shows a clear benefit of 2 pig of added QS-21 but little to no benefit of a higher 10-pig dose - likely demonstrating the balance between optimally stimulating key immune responses, including APC-recruiting chemokine production, while not yet overwhelming saRNA replication and antigen expression with type I IFN responses.

[0378] Study 3

[0379] This example demonstrates that QS-21 -Adjuvanted saRNA-qNLC Vaccines Show Improved Immune Durability in Mice.

[0380] A follow-up study of immune durability improvement by the optimal 2-pig QS-21- adjuvanted saRNA-qNLC vaccine dose was conducted, again in C57BL / 6 mice. A single low 1-pig RNA vaccine dose was delivered to mice using standard NLC or qNLC, and mice were monitored for serum neutralizing antibody titers over a 4-month period post-vaccination, at which point the mice were euthanized and splenocytes isolated for long-term T cell analyses (FIG. 8). While the normal saRNA-NLC formulation induced strong neutralizing antibody titers, these clearly waned over a period of 4 months post-vaccination, with waning apparent starting just 3 weeks post-vaccination. In contrast, the QS-21 adjuvanted saRNA-qNLC vaccine showed no significant waning of serum antibody titers over the 4-month post-vaccination period (FIG. 8A). CD8+ T cell responses 4 months after a single 1-pig saRNA dose were also significantly higher in mice dosed with the adjuvanted vaccine relative to the standard vaccine (FIG. 8B), at levels surprisingly similar to those seen 3 weeks post-vaccination as observed in FIG. 7B. The fact that these whole-percentage levels of polyfunctional (IFNy+, IL-2+, TNFa+) CD8+ T cells were still present a full 4 months after a single vaccination suggests that the addition of QS-21 to the standard NLCs is substantially increasing stimulation and establishment of key vaccine antigen-directed cytotoxic T cell populations.

[0381] Study 4

[0382] This example shows that QS-21 -Adjuvanted saRNA-qNLC Vaccines Show Efficacy in Preventing Viral Disease.

[0383] To demonstrate the effective potential of a QS-21-adjuvanted saRNA-qNLC vaccine, we vaccinated C57BL / 6 mice with a single 1-pig qNLC-formulated saRNA dose. This saRNA-qNLC vaccine is designed to express the Zika virus prM and E proteins using the same VEEV replicon backbone as the SARS-CoV-2 replicon proposed in this contract. Three weeks post-vaccination, vaccinated and vector control-dosed (1 pig qNLC-formulated saRNA expressing the nonimmunogenicreporter SEAP protein) mice were transiently immunocompromised with an IFNAR-blocking monoclonal antibody (mAb) the day before, day after, and 4 days post-challenge and challenged with 1 x 106PFU of mouse-adapted Zika Dakar virus via the rear footpads.

[0384] The mice in the QS-21-adjuvanted Zika saRNA-qNLC-vaccinated group were completely protected from viral-induced morbidity and mortality over the 2 weeks post-challenge, while vector control-vaccinated mice demonstrated 80% mortality and uniform significant weight loss over the 6- 12 days after challenge (FIGS. 9A and 9B).

[0385] Study 5

[0386] This example shows adding a NLC containing alpha-tocopherol in place of some of the squalene as an additional adjuvant. Alpha-tocopherol is a vitamin E type molecule used as part of various successful vaccine adjuvant systems (such as AS03, which uses a combination of it and squalene as the main immune stimulatory compounds). The mice used, Covid vaccine RNA construct, and dosing are the same as above. The first three groups listed in FIGS. 10A and 10B show that saRNA can be successfully delivered using LNP and CNE platforms. The fourth group shows the superior results provided by the NLC platform of this disclosure adjuvated with cholesterol and QS-21 as described above. The fifth group shows results for the NLC platform of this disclosure without an adjuvant. Interestingly, adjuvanting with alpha-tocopherol, the fifth group, resulted in a greatly decreased antibody response. This shows that some known adjuvants will have a deleterious effect when combined with the NLC platform and saRNA. The seventh group is a control that uses SEAP RNA instead of the Covid spike protein RNA.EXEMPLARY EMBODIMENTS

[0387] The disclosure provides the following exemplary embodiments.1 . A composition comprising a lipid-based nanoparticle for delivery of a bioactive agent to a cell, wherein the lipid-based nanoparticle comprises a saponin.2. The composition of embodiment 1 , wherein the lipid-based nanoparticle is selected from a nanostructured lipid carrier (NLC), liposome, lipid nanoparticle (LNP), solid lipid nanoparticle (SLN), oil-in-water emulsion, cationic lipid-nucleic acid complex, cationic nanoemulsion (CNE), charge-altering releasable transporter (CARTs), or polymeric nanoparticle.3. The composition of embodiment 2, wherein the NLC particle comprises:(a) an oil core comprising a mixture of a liquid phase lipid and a solid phase lipid,(b) a cationic lipid;(c) a hydrophobic surfactant;(d) a hydrophilic surfactant; and(e) a saponin.4. The composition of embodiment 3, wherein the liquid phase lipid is a naturally occurring or synthetic terpenoid.5. The composition of any one of embodiments 3 or 4, wherein the liquid phase lipid is squalene, synthetic squalene, sunflower oil, soybean oil, olive oil, grapeseed oil, squalane, capric / caprylic triglyceride, lauroyl polyoxylglyceride, monoacylglycerol, soy lecithin, or a combination thereof.6. The composition of any one of embodiments 3 to 5, wherein the liquid phase lipid is squalene.7. The composition of any one of embodiments 3 to 6, wherein the solid phase lipid is a glycerolipid.8. The composition of any one of embodiments 3 to 7, wherein the solid phase lipid is a microcrystalline triglyceride.9. The composition of embodiment 8, wherein the microcrystalline triglyceride is trimyristin (Dynasan®114), tristearin (Dynasan®118), or tripalmitin (Dynasan®116).10. The composition of embodiment 9, wherein the microcrystalline triglyceride is trimyristin.11 . The composition of any one of embodiments 3 to 10, wherein the liquid phase lipid is squalene and the solid phase lipid is trimyristin.12. The composition of any of embodiments 1 to 11, further comprising a sterol.13. The composition of embodiment 12, wherein the sterol is cholesterol, synthetic cholesterol, semi-synthetic cholesterol, 3p-[N— (N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol (DC Cholesterol), phytosterol, or a cholesterol analogue.14. The composition of embodiment 13, wherein the sterol is cholesterol.15. The composition of any one of embodiments 1 to 14, wherein the cationic lipid is 1,2- dioleoyloxy-3-(trimethylammonio)propane (DOTAP), dimethyldioctadecylammonium (DDA), 1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP), dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N- dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), or 1 ,2-dilinoleyloxy-3- dimethylaminopropane (DLinDMA).16. The composition of embodiment 15, wherein the cationic lipid is 1 ,2-dioleoyloxy-3- (trimethylammonio)propane (DOTAP).17. The composition of any one of embodiments 1 to 16, wherein the hydrophobic surfactant is a sorbitan ester.18. The composition of embodiment 17, wherein the sorbitan ester has a hydrophilic-lipophilic balance (HLB) value from 1 to 5.19. The composition of embodiment 18, wherein the sorbitan ester having a HLB value from 4 to 5.20. The composition of any one of embodiments 1 to 19, wherein the sorbitan ester is a sorbitan monoester.21 . The composition of embodiment 20, wherein the sorbitan monoester is sorbitan trioleate (Span®85)22. The composition of embodiment 20, wherein the sorbitan monoester is sorbitan monooleate (Span®80).23. The composition of embodiment 20, wherein the sorbitan monoester is sorbitan monostearate (Span®60).24. The composition of any one of embodiments 1 to 23, wherein the hydrophilic surfactant is a hydrophilic non-ionic surfactant.25. The composition of embodiment 24, wherein the hydrophilic non-ionic surfactant is a polysorbate.26. The composition of embodiment 24, wherein the hydrophilic non-ionic surfactant is a polyoxyethylene sorbitan ester.27. The composition of any of embodiments 24 to 26, wherein the hydrophilic surfactant is polysorbate 80 (TWEEN® 80).28. The composition of any of embodiments 1 to 27, wherein the hydrophobic surfactant is sorbitan monostearate (Span®60) and the hydrophilic surfactant is polysorbate 80 (TWEEN® 80).29. The composition of any of embodiments 1 to 28, wherein the saponin is extracted from a natural source or synthetically produced.30. The composition of any of embodiments 1 to 28, wherein the saponin is a Quillaja saponaria (QS) saponin.31 . The composition of embodiment 29, wherein the QS saponin is QS-7, QS-17, QS-18, or QS-21.32. The composition of embodiment 31, wherein the QS saponin is QS-21.33. The composition of any of embodiments 1 to 28, wherein the saponin is an extract from Aesculus hippocastanum.34. The composition of embodiment 33, wherein the saponin is Asecin or escin.35. The composition of any of embodiments 1 to 28, wherein the saponin in an extract ofDigitalis purpurea.36. The composition of embodiment 35, wherein the saponin is digitonin.37. The composition of any of embodiments 1 to 28, wherein the saponin in an extract of aGypsophila species.38. The composition of any of embodiments 1 to 28, wherein the saponin in an extract of Chenopodium quinoa.39. The composition of any of embodiments 1 to 36, comprising 0.1 to 1000.0 pig of saponin.40. The composition of any one of embodiments 1 to 40, wherein the liquid phase lipid is squalene, the solid phase lipid is trimyristin, the sterol is cholesterol, the cationic lipid is DOTAP, the hydrophobic surfactant is sorbitan monostearate (Span®60), the hydrophilic surfactant is polysorbate 80 (TWEEN® 80), and the saponin is QS-21.41 . The composition of any of embodiments 1 to 40, further comprising:(f) the bioactive agent.42. The composition of embodiment 41, wherein the bioactive agent is not encapsulated by the NLC particles and is associated with the surface of the NLC particles.43. The composition of any one of embodiments 41 to 42, wherein the bioactive agent is a protein, RNA or DNA.44. The composition of embodiment 43, wherein the bioactive agent is mRNA, oncolytic viral RNA, non-coding RNA, or self-amplifying RNA.45. The composition of embodiment 44, wherein the bioactive agent is self-amplifying RNA.46. The composition of any one of embodiments 43 to 45, wherein the RNA is a replicon.47. The composition of any one of embodiments 43 to 46, wherein the RNA encodes an antigen.48. The composition of embodiment 47, wherein the antigen is an allergen.49. The composition of any one of embodiments 43 to 46, wherein the RNA encodes an antibody.50. The composition of any one of embodiments 43 to 49, wherein the bioactive agent encodes a protein or a protein antigen.51. The composition of embodiment 47 or 50, wherein the antigen is derived from, or immunologically cross-reactive with, an infectious pathogen and / or an epitope, biomolecule, cell, or tissue that is associated with infection, cancer, autoimmune disease, or allergy.52. The composition of embodiment 47, wherein the infectious pathogen is a bacterium, a virus, or a parasite.53. The composition of embodiment 48, wherein the bacterium is a mycobacterium.54. The composition of embodiment 48, wherein the virus is a coronavirus, a Zika virus, an influenza virus, a yellow fever virus, HIV, human papillomavirus, chikungunya virus, VZV, RSV, or EBV.55. The composition of embodiment 48, wherein the parasite is cryptosporidium.56. The composition of embodiment 51, wherein the infectious pathogen is SARS-CoV-2 and / or the epitope is the SARS-CoV-2 spike protein.57. The composition of any one of embodiments 43 to 56, wherein the bioactive agent is complexed with the NLC particles at a N:P ratio of about 10 to about 20, or about 15.58. The composition of any one of embodiments 1 to 56, having an oil to surfactant molar ratio of about 0.5 to about 12, about 0.5 to about 9, or about 0.5 to about 1.59. The composition of any one of embodiments 1 to 56, having a hydrophilic surfactant to cationic component ratio of about 0.2 to about 1 .5 or about 0.2 to about 1 .60. The composition of any one of embodiments 1 to 59, having a loading capacity for RNA of at least about 1 pg / 100 pl RNA.61 . The composition of any one of embodiments 1 to 60, wherein the bioactive agent is a nucleic acid and the saponin is present at about 0.4 to about 10 pg / 1 pg of nucleic acid.62. The composition of embodiment 61, wherein the saponin is present at about 2 pg / 1 pg of nucleic acid.63. The composition of any one of embodiments 1 to 60, comprising from about 0.2% to about 40% w / v liquid phase lipid, from about 0.1% to about 10% w / v solid phase lipid, from about 0.1% to about 10% sterol, from about 0.2% to about 10% w / v cationic lipid, from about 0.25% to about 5% w / v hydrophobic surfactant, from about 0.5% to about 10% w / v hydrophilic surfactant, and from 0.1% to about 10% saponin.64. The composition of any one of embodiments 1 to 60, comprising about 37.3 mg / ml liquid phase lipid, about 2.4 mg / ml solid phase lipid, about 1 mg / ml sterol, about 30 mg / ml cationic lipid, about 37 mg / ml hydrophobic surfactant, about 37.2 mg / ml hydrophilic surfactant, and about 2 mg / ml saponin.65. The composition of embodiment 68, wherein the virus is a coronavirus, a Zika virus, an influenza virus, a yellow fever virus, HIV, human papillomavirus, chikungunya virus, VZV, RSV, or EBV.66. The composition of embodiment 73, wherein the parasite is cryptosporidium.67. A method of generating an immune response in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of embodiments 1 to 64, wherein the bioactive agent is a nucleic acid molecule encoding a protein antigen.68. The method of embodiment 65, wherein the bioactive agent is RNA.69. The method of embodiment 65 or 66, wherein administration of the composition is intramuscular, parenteral, or intradermal.70. The method of any one of embodiments 65 to 67, wherein the immune response is a humoral immune response.71. The method of any one of embodiments 65 to 67, wherein the immune response is a CD8 T cell response.72. The method of any one of embodiments 65 to 69, wherein the immune response remains elevated about 4 months or about 6 months after administration.73. The method of any one of embodiments 65 to 70, wherein the immune response does not stimulate an innate immune response.74. The method of embodiment 65, wherein the protein antigen is an allergen, and the immune response is a predominantly Th1 immune response combined with a down-regulated Th2 immune response.75. The method of any one of embodiments 65 to 71 , wherein the immune response is greater than the immune response when the subject is administered the NLC particles without the sterol and the saponin.76. A method of delivering a bioactive agent to a cell, comprising contacting the cell with the composition of any one of embodiments 41 to 56.77. The method of embodiment 74, wherein the bioactive agent is a nucleic acid.78. A method of making the composition of any one of embodiments 1 to 64, comprising:(a) mixing the solid phase lipid, the liquid phase lipid, the sterol, the cationic lipid, and the hydrophobic surfactant to form an oil phase mixture;(b) mixing the hydrophilic surfactant, the saponin, and water to form an aqueous phase mixture; and(c) mixing the oil phase mixture with the aqueous phase mixture to form the NLC particles.79. The method of embodiment 76, further comprising:(d) combining the bioactive agent with the NLC particles such that the bioactive agent associates with the surface of the NLC particles by non-covalent interactions or by reversiblecovalent interactions.80. A method of treating a subject infected with a pathogen, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of embodiments 1 to 64, wherein the bioactive agent is a protein antigen or a nucleic acid molecule encoding a protein antigen.81 . The method of embodiment 84, wherein the pathogen is a bacterium, virus, fungus, or parasite.82. The method of embodiment 85, wherein the bacterium is a mycobacterium.83. The composition of embodiment 68, wherein the virus is a coronavirus, a Zika virus, an influenza virus, a yellow fever virus, HIV, human papillomavirus, chikungunya virus, VZV, RSV, or EBV.84. The composition of embodiment 73, wherein the parasite is cryptosporidium.CONCLUSION

[0388] In the application, unless specified otherwise, the terms "comprising”, "comprise”, and grammatical variants thereof, intended to represent "open” or "inclusive” language such that they include recited elements but also permit inclusion of additional, non-explicitly recited elements.

[0389] Throughout this disclosure, certain embodiments may be disclosed in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0390] Numerous embodiments of the invention are possible. The previous exemplary embodiments are intended to merely illustrate, and not limit, the breadth and depth of embodiments that can fall within the scope of the appended claims and future claims, which define the invention.

[0391] It will be apparent that various other modifications and adaptations of the application will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the application and it is intended that all such modifications and adaptations come within the scope of the appended claims.

Claims

CLAIMS A composition comprising a lipid-based nanoparticle for delivery of a bioactive agent to a cell, wherein the lipid-based nanoparticle comprises a saponin. The composition of claim 1 , wherein the lipid-based nanoparticle is selected from a nanostructured lipid carrier (NLC), liposome, lipid nanoparticle (LNP), solid lipid nanoparticle (SLN), oil-in-water emulsion, cationic lipid-nucleic acid complex, cationic nanoemulsion (CNE), charge-altering releasable transporter (CARTs), or polymeric nanoparticle. The composition of claim 2, wherein the lipid-based nanoparticle is a NLC and the NLC particle comprises:(g) an oil core comprising a mixture of a liquid phase lipid and a solid phase lipid,(h) a cationic lipid;(I) a hydrophobic surfactant;(j) a hydrophilic surfactant; and(k) the saponin. The composition of claim 3, wherein the liquid phase lipid is a naturally occurring or synthetic terpenoid. The composition of claim 4, wherein the liquid phase lipid is squalene, synthetic squalene, sunflower oil, soybean oil, olive oil, grapeseed oil, squalane, capric / caprylic triglyceride, lauroyl polyoxylglyceride, monoacylglycerol, soy lecithin, or a combination thereof. The composition of claim 5, wherein the liquid phase lipid is squalene. The composition of claim 3, wherein the solid phase lipid is a glycerolipid. The composition of claim 3, wherein the solid phase lipid is a microcrystalline triglyceride.The composition of claim 8, wherein the microcrystalline triglyceride is trimyristin (Dynasan®114), tristearin (Dynasan®118), or tripalmitin (Dynasan®116). The composition of claim 9, wherein the microcrystalline triglyceride is trimyristin. The composition of any of one of claims 3 to 10, wherein the liquid phase lipid is squalene and the solid phase lipid is trimyristin. The composition of any one of claims 3 to 10, further comprising a sterol. The composition of claim 12, wherein the sterol is cholesterol, synthetic cholesterol, semisynthetic cholesterol, 3p-[N— (N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol (DC Cholesterol), phytosterol, or a cholesterol analogue. The composition of claim 13, wherein the sterol is cholesterol. The composition of claim 3, wherein the cationic lipid is 1 ,2-dioleoyloxy-3- (trimethylammonio)propane (DOTAP), dimethyldioctadecylammonium (DDA), 1,2- Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP), dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N- dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), or 1 ,2-dilinoleyloxy-3- dimethylaminopropane (DLinDMA). The composition of claim 15, wherein the cationic lipid is 1 ,2-dioleoyloxy-3- (trimethylammonio)propane (DOTAP). The composition of claim 3, wherein the hydrophobic surfactant is a sorbitan ester. The composition of claim 17, wherein the sorbitan ester has a hydrophilic-lipophilic balance (HLB) value from 1 to 5. The composition of claim 18, wherein the sorbitan ester has a HLB value from 4 to 5.The composition of any one of claims 17 to 19, wherein the sorbitan ester is a sorbitan monoester. The composition of claim 20, wherein the sorbitan monoester is sorbitan trioleate (Span®85) The composition of claim 20, wherein the sorbitan monoester is sorbitan monooleate (Span®80). The composition of claim 20, wherein the sorbitan monoester is sorbitan monostearate (Span®60). The composition of claim 3, wherein the hydrophilic surfactant is a hydrophilic non-ionic surfactant. The composition of claim 24, wherein the hydrophilic non-ionic surfactant is a polysorbate. The composition of claim 24, wherein the hydrophilic non-ionic surfactant is a polyoxyethylene sorbitan ester. The composition of any of claims 24 to 26, wherein the hydrophilic surfactant is polysorbate 80 (TWEEN® 80). The composition of claim 3, wherein the hydrophobic surfactant is sorbitan monostearate (Span®60) and the hydrophilic surfactant is polysorbate 80 (TWEEN® 80). The composition of claim 3, wherein the saponin is extracted from a natural source or synthetically produced. The composition of any one of claims 1 to 3, wherein the saponin is a Quillaja saponaria (QS) saponin. The composition of claim 30, wherein the QS saponin is QS-7, QS-17, QS-18, or QS-21.The composition of claim 31, wherein the QS saponin is QS-21. The composition of any one of claims 1 to 3, wherein the saponin is an extract from Aesculus hippocastanum. The composition of claim 33, wherein the saponin is Asecin or escin. The composition of any one of claims 1 to 3, wherein the saponin in an extract of Digitalis purpurea. The composition of claim 35, wherein the saponin is digitonin. The composition of any one of claims 1 to 3, wherein the saponin in an extract of a Gypsophila species. The composition of any one of claims 1 to 3, wherein the saponin in an extract of Chenopodium quinoa. The composition of any of claims 1 to 3, comprising 0.1 to 1000.0 pig of the saponin. The composition of claim 12, wherein the liquid phase lipid is squalene; the solid phase lipid is trimyristin; the sterol is cholesterol; the cationic lipid is DOTAP; the hydrophobic surfactant is sorbitan monostearate (Span®60); the hydrophilic surfactant is polysorbate 80 (TWEEN® 80); and the saponin is QS-21. The composition of any of claims 1 to 3, further comprising:(I) the bioactive agent. The composition of claim 41, wherein the bioactive agent is not encapsulated by the NLC particles and is associated with the surface of the NLC particles. The composition of any one of claims 41 to 42, wherein the bioactive agent is a protein, RNA or DNA.The composition of claim 43, wherein the bioactive agent is mRNA, oncolytic viral RNA, non-coding RNA, or self-amplifying RNA. The composition of claim 44, wherein the bioactive agent is self-amplifying RNA. The composition of claim 43, wherein the RNA is a replicon. The composition of any one of claims 44 to 46, wherein the RNA encodes an antigen. The composition of claim 47, wherein the antigen is an allergen. The composition of any one of claims 44 to 46, wherein the RNA encodes an antibody. The composition of claim 41, wherein the bioactive agent encodes a protein or a protein antigen. The composition of 50, wherein the antigen is derived from, or immunologically cross- reactive with, an infectious pathogen and / or an epitope, biomolecule, cell, or tissue that is associated with infection, cancer, autoimmune disease, or allergy. The composition of claim 51, wherein the infectious pathogen is a bacterium, a virus, or a parasite. The composition of claim 52, wherein the bacterium is a mycobacterium. The composition of claim 52, wherein the virus is a coronavirus, a Zika virus, an influenza virus, a yellow fever virus, HIV, human papillomavirus, chikungunya virus, VZV, RSV, or EBV. The composition of claim 52, wherein the parasite is cryptosporidium. The composition of claim 52, wherein the infectious pathogen is SARS-CoV-2 and / or the epitope is the SARS-CoV-2 spike protein.The composition of claim 44, wherein the bioactive agent is complexed with the NLC particles at a N:P ratio of about 10 to about 20, or about 15. The composition of claim 3, having an oil to surfactant molar ratio of about 0.5 to about 12, about 0.5 to about 9, or about 0.5 to about 1 . The composition of claim 3, having a hydrophilic surfactant to cationic component ratio of about 0.2 to about 1 .5 or about 0.2 to about 1 . The composition of any one of claims 1 to 59, having a loading capacity for RNA of at least about 1 pg / 100 pl RNA. The composition of any one of claims 1 to 60, wherein the bioactive agent is a nucleic acid and the saponin is present at about 0.4 to about 10 pg / 1 pg of nucleic acid. The composition of claim 61 , wherein the saponin is present at about 2 pg / 1 pg of nucleic acid. The composition of any one of claims 3 to 60, comprising from about 0.2% to about 40% w / v liquid phase lipid, from about 0.1 % to about 10% w / v solid phase lipid, from about 0.1 % to about 10% sterol, from about 0.2% to about 10% w / v cationic lipid, from about 0.25% to about 5% w / v hydrophobic surfactant, from about 0.5% to about 10% w / v hydrophilic surfactant, and from 0.1 % to about 10% saponin. The composition of any one of claims 3 to 60, comprising about 37.3 mg / ml liquid phase lipid, about 2.4 mg / ml solid phase lipid, about 1 mg / ml sterol, about 30 mg / ml cationic lipid, about 37 mg / ml hydrophobic surfactant, about 37.2 mg / ml hydrophilic surfactant, and about 2 mg / ml saponin. A method of generating an immune response in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of claims 1 to 64, wherein the bioactive agent is a nucleic acid molecule encoding a protein antigen. The method of claim 65 wherein the bioactive agent is RNA.The method of claim 65 or 66, wherein administration of the composition is intramuscular, parenteral, or intradermal. The method of any one of claims 65 to 67, wherein the immune response is a humoral immune response. The method of any one of claims 65 to 67, wherein the immune response is a CD8 T cell response. The method of any one of claims 65 to 69, wherein the immune response remains elevated about 4 months or about 6 months after administration. The method of any one of claims 65 to 70, wherein the immune response does not stimulate an innate immune response. The method of any one of claims 65 to 70, wherein the protein antigen is an allergen, and the immune response is a predominantly Th1 immune response combined with a down- regulated Th2 immune response. The method of any one of claims 65 to 72, wherein the immune response is greater than the immune response when the subject is administered the NLC particles without the sterol and the saponin. A method of delivering a bioactive agent to a cell, comprising contacting the cell with the composition of any one of claims 41 to 57. The method of claim 74, wherein the bioactive agent is a nucleic acid. A method of making the composition of any one of claims 1 to 64, comprising:(a) mixing the solid phase lipid, the liquid phase lipid, the sterol, the cationic lipid, and the hydrophobic surfactant to form an oil phase mixture;(b) mixing the hydrophilic surfactant, the saponin, and water to form an aqueous phase mixture; and(c) mixing the oil phase mixture with the aqueous phase mixture to form the NLCparticles. The method of claim 76, further comprising:(d) combining the bioactive agent with the NLC particles such that the bioactive agent associates with the surface of the NLC particles by non-covalent interactions or by reversible covalent interactions. A method of treating a subject infected with a pathogen, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of claims 1 to 64, wherein the bioactive agent is a protein antigen or a nucleic acid molecule encoding a protein antigen. The method of claim 78, wherein the pathogen is a bacterium, virus, fungus, or parasite. The method of claim 79, wherein the bacterium is a mycobacterium. The composition of claim 79, wherein the virus is a coronavirus, a Zika virus, an influenza virus, a yellow fever virus, HIV, human papillomavirus, chikungunya virus, VZV, RSV, or EBV. The composition of claim 79, wherein the parasite is cryptosporidium.

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