EP4583841A1 - Composition de vaccin immunogène incorporant une saponine - Google Patents

Composition de vaccin immunogène incorporant une saponine

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Publication number
EP4583841A1
EP4583841A1 EP23772597.3A EP23772597A EP4583841A1 EP 4583841 A1 EP4583841 A1 EP 4583841A1 EP 23772597 A EP23772597 A EP 23772597A EP 4583841 A1 EP4583841 A1 EP 4583841A1
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EP
European Patent Office
Prior art keywords
composition
lipid
nlc
rna
virus
Prior art date
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Pending
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EP23772597.3A
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German (de)
English (en)
Inventor
Christopher Bradford FOX
Emily VOIGT
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Access to Advanced Health Institute
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Access to Advanced Health Institute
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Publication of EP4583841A1 publication Critical patent/EP4583841A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure relates generally to the fields of pharmaceutical and vaccine formulations.
  • 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.
  • 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).
  • RNA-based platforms 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.
  • 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).
  • the formulations comprise nanostructured lipid carrier (NLC)- based formulations.
  • NLC nanostructured lipid carrier
  • 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.
  • FIG. 1 shows an exemplary QS-21 adjuvanted saRNA vaccine delivery formulation of the disclosure.
  • 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. 1 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
  • 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).
  • SEAP vector control
  • 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.
  • 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.
  • FIGS. 8A and 8B demonstrate cholesterol/QS-21-formulated NLC saRNA vaccine immunogenicity enhancement also improves immune durability.
  • 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.
  • mice were transiently immunocompromised by injection with IFNAR- blocking monoclonal antibody and challenged with 10 6 PFU of mouse-adapted Zika strain Dakar.
  • 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.
  • adjuvanting with QS-21 + cholesterol resulted in SARS-CoV- 2 spike binding IgG titers similar to other formulations and the highest neutralizing antibody titers.
  • 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.
  • LNPs solid lipid nanoparticles
  • 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 yields NLC cores with structural integrity and stability.
  • 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).
  • a mixture of surfactants typically a sorbitan ester and a hydrophilic surfactant
  • a cationic component typically a cationic lipid or phospholipid.
  • bioactive agents such as small molecule drugs
  • 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).
  • nucleic acids e.g., self-amplifying RNA (saRNA)
  • saRNA self-amplifying RNA
  • 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.
  • nucleic acid e.g., synthetic saRNA expressing a protective antigen
  • 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.
  • micromolecule refers to large molecules exemplified by, but not limited to, peptides, proteins, oligonucleotides and polynucleotides of biological or synthetic origin.
  • 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.
  • polypeptide As 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.
  • polynucleotides or polypeptides containing one or more analogs of a nucleotide or an amino acid including, for example, unnatural amino acids, etc.
  • amino acid including, for example, unnatural amino acids, etc.
  • isolated means the molecule has been removed from its natural environment.
  • liquid phase lipid refers to a lipid that, prior to mixing with any other component, is liquid at ambient temperature.
  • Additional suitable cationic lipids include 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).
  • DOTMA N-[1 -(2,3-dioleyloxy)propyl]-N,N, N-trimethylammonium chloride
  • DODAC N,N-dioleoyl-N,N- dimethylammonium chloride
  • DOEPC 2-diol eoy
  • the NLCs may comprise one or any combination of two or more of the cationic lipids described herein.
  • 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.
  • DOTAP 1,2- dioleoyloxy-3-(trimethylammonio)propane
  • DC Cholesterol dimethyldioctadecylam
  • 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 ly, 1
  • 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).
  • 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.
  • the cationic lipid is DC Cholesterol.
  • the NLC may comprise DC Cholesterol at from about 0.1 mg/ml to about 5 mg/ml DC Cholesterol.
  • the cationic lipid is DDA.
  • the NLC may comprise, for example, from about 0.5 mg/ml to about 50 mg/ml DODAC.
  • the cationic lipid is DODAP.
  • the NLC may comprise, for example, from about 0.5 mg/ml to about 50 mg/ml DODAP.
  • 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), from about 1% to about 5%
  • a cationic lipid that is soluble in the oil core it may be desirable to use a cationic lipid that is soluble in the oil core.
  • DOTAP DOEPC, DODAC, and DOTMA are soluble in squalene or squalane.
  • 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.
  • 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).
  • lipids having one or two unsaturated fatty acid chains are soluble in squalene or squalane; whereas lipids having saturated fatty acid chains (e.g., stearoyl tails) are not soluble in squalene.
  • 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).
  • 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.
  • DLS Dynamic Light Scattering
  • PCS Photon Correlation Spectroscopy
  • 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.
  • the immune response to encoded proteins in the bioactive nucleic acid can be modulated by selection of sorbitan ester used in the NLC.
  • use of a sorbitan monoester was particularly effective at enhancing the effectiveness of the NLC.
  • the acyl chain of the sorbitan monoester is saturated.
  • the sorbitan ester 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).
  • the solid lipid e.g., microcrystalline triglycerides
  • 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.
  • 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).
  • 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.
  • exemplary sorbitan monoesters may be salt forms (e.g., pharmaceutically acceptable salts) of Formulas I, la
  • 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)).
  • sorbitan monostearate is represented by Formula II or Ila or a salt form thereof
  • exemplary sorbitan monooleate is represented by Formula III or Illa or a salt form thereof.
  • 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 surfactants including sorbitan-based non-ionic surfactants) and a hydrophilic surfactant.
  • 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.
  • the acyl chains of the sorbitan diester or triester will be saturated.
  • sorbitan esters may have chiral centers and may occur, for example, as racemates, racemic mixtures, and as individual enantiomers and diastereomers.
  • 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.
  • 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.
  • nonionic polymer e.g., a poly(alkyl glycol) such as polyethylene glycol, polypropylene glycol, or polybutlyene glycol
  • nonionic surfactant e.g., a nonionic surfactant, a nonionic surfactant, or nonionic surfactant.
  • NLC compositions can be described by the molar ratios of various components.
  • 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.
  • potential toxicity of the formulations can be reduced.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the hydrophilic surfactant can be present at 0.2% to about 10 % or from about 0.2% to about 5 % w/v.
  • 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.
  • 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.
  • 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.
  • 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.
  • any of the NLC compositions/formulations described herein can be diluted or concentrated for use in the present invention.
  • the formulation when mixed 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.
  • dilution occurs when mixing the formulation with a bioactive agent (e.g., RNA or DNA) for delivery.
  • a bioactive agent e.g., RNA or DNA
  • 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.
  • 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.
  • 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.
  • the sorbitan ester is a sorbitan monoester, diester, or triester;
  • 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;
  • the liquid phase lipid is squalene;
  • the solid phase lipid is a glycerolipid;
  • the solid phase lipid is microcrystalline triglyceride;
  • the solid phase lipid is trimyristin;
  • the cationic lipid is DOTAP;
  • the hydrophilic surfactant is polysorbate 80 (also referred to
  • 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 [00154], 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.
  • 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 .
  • the hydrophilic SurfactanbCationic component e.g., cationic lipid
  • 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 .
  • 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
  • a hydrophilic SurfactanbCationic component e.g., cationic lipid
  • 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.
  • 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
  • a hydrophilic SurfactanbCationic component e.g., cationic lipid
  • 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.
  • 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
  • a hydrophilic SurfactanbCationic component e.g., cationic lipid
  • 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.
  • 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.
  • the size of the NLC refers to the Z-average diameter.
  • 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
  • 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, is about 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.
  • bioactive agents may be associated with the formulations of the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • TLR toll-like receptors
  • RIG-I non TLR pathways
  • 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.
  • 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).
  • IRES internal ribosome entry site
  • 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.
  • 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.
  • Sindbis virus Sindbis virus
  • Semliki Forest virus Semliki Forest virus
  • VEE Venezuelan equine encephalitis virus
  • C capsid
  • E envelope glycoproteins
  • 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.
  • 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.
  • 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.
  • SFV Semliki Forest virus
  • Sindbis virus Sindbis virus
  • VEE Venezuelan equine encephalitis virus
  • RRV Ross-River virus
  • chikungunya virus or other viruses belonging to the alphavirus genus.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • a replicon useful with the invention can, for example, have two open reading frames.
  • the first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes an antigen.
  • the RNA may have additional (e.g. downstream) open reading frames e.g. to encode additional antigens or to encode accessory polypeptides.
  • 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.
  • AAUAAA poly-A polymerase recognition sequence
  • Replicons can have various lengths, but they are typically 5000-25000 nucleotides long e.g. 8000-15000 nucleotides, or 9000-12000 nucleotides.
  • Suitable oncolytic viruses are known in the art and are available from sequence depositories, such as the American Type Culture Collection, Rockville, Md.
  • 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.
  • the oncolytic virus is genetically engineered for tumour selectivity.
  • the oncolytic virus is naturally occurring.
  • Naturally occurring oncolytic viruses include, but are not limited to, reovirus and senecavirus.
  • 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.
  • the selfamplifying RNA molecules of the invention contain at least about 3 kb.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the term antigen will refer to a polypeptide antigen of interest.
  • 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.
  • 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.
  • the antigen may be from, or related to antigens involved in tuberculosis, influenza, amebiasis, HIV, hepatitis, or Leishmaniasis.
  • 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.
  • HA hemagglutinin
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • Neisseria spp including N. gonorrhea and N. meningitidis
  • 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.
  • B. pertussis for example pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 Veterinarian
  • T. cruzi Trypanosoma spp., including T. cruzi
  • Giardia spp. including G. lamblia
  • Leshmania spp. including L. major
  • Pneumocystis spp. including P. carinii
  • Trichomonas spp. including T.
  • 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
  • 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
  • cestode infections including, but not limited
  • 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.
  • the fusion protein is ID93.
  • the fusion protein is ID91.
  • the adjuvant formulation in the Nuvaxovid/Covovax vaccine contains a mixture of saponins (including QS-21) and lipid excipients.
  • 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).
  • QS-21 has a long and reliable history as a safe and immunogenic vaccine adjuvant component in human vaccines.
  • 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).
  • 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.
  • 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).
  • 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).
  • 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).
  • the saponin is present at about 2 pg /1 pg of nucleic acid.
  • 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).
  • 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.
  • the composition is an NLC
  • the bioactive agent is a saRNA
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • Th2-type cytokines e.g., IL-4, IL-5, IL-6 and IL-10
  • a patient may support an immune response that includes Thl and Th2-type responses.
  • 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).
  • 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 hydrophilic surfactant, 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.
  • 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.
  • 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.
  • the bioactive agent is hydrophobic, it is combined with the components in step (a) to form part of the oil phase mixture.
  • the bioactive agent may be attached to a component of the surface of the NLC via covalent interactions.
  • 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.
  • 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.
  • compositions Comprising the Lipid-based Nanoparticles
  • formulations, compositions, and pharmaceutical compositions comprising the lipid-based nanoparticles compositions described herein.
  • compositions comprising the lipid-based nanoparticles and bioactive agent can optionally further comprise a pharmaceutically acceptable carrier, excipient, or diluent.
  • stable emulsions are provided, wherein the stable emulsions comprise at least one adjuvant.
  • 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®.
  • a stable emulsion is an oil-in-water emulsion.
  • 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.
  • 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).
  • 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.
  • the stabilizer may be a triglyceride, such as tricaprylin (C 27 H 50 O 6) (see, e.g., WO 98/56414).
  • 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.
  • 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).
  • DMPC diimyristoyl-sn-glycero-3-phosphocholine
  • 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.
  • 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 some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser.
  • the method comprises the mixing the oil phase with a surfactant such as a PBS/TWEEN80® solution, followed by homogenization using a homogenizer.
  • a surfactant such as a PBS/TWEEN80® solution
  • 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.
  • 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)
  • 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.
  • compositions of the disclosure are useful for therapeutic purposes.
  • 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.
  • the compositions comprise an NLC provided herein, and further comprise a bioactive agent for the treatment of a disease, condition, or disorder.
  • 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.
  • compositions comprise cancer antigens or nucleic acids encoding a cancer antigen.
  • 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.
  • 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., specific immunologically defined recognition structures such as those recognized by an antibody variable region complementarity determining region (CDR) or by a T cell receptor CDR.
  • autoimmune diseases include diseases, conditions or disorders wherein a host's or subject's immune system detrimentally mediates an immune response that is directed against "
  • 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.
  • 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.
  • snRNP small nuclear ribonucleic acid proteins
  • 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
  • thrombic thrombocytopenic purpura is antigens of platelets.
  • 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.
  • such compounds may be formulated into vaccines and/or pharmaceutical compositions for immunization against Mycobacterium infection.
  • 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).
  • respiratory diseases such as those caused or exacerbated by bacterial infection (e.g., pneumococcal)
  • COPD chronic obstructive pulmonary disease
  • 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.
  • the compositions of the present disclosure are used to boost or enhance an immune response in a subject.
  • the bioactive agent is an adjuvant.
  • 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.
  • TLR agonists including TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 agonists
  • Rig-1 agonists include 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®.
  • 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.
  • compositions of the present disclosure are useful for enhancing or eliciting, in a host, a patient or in cell culture, an immune response.
  • 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.
  • 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.
  • the infectious disease is associated with a bacterial, viral or fungal infection.
  • 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.
  • 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
  • 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.
  • the antigen may be from, or related to antigens involved in tuberculosis, influenza, amebiasis, HIV, hepatitis, or Leishmaniasis.
  • the therapeutic compositions of the present disclosure are useful for treating an allergic condition in a subject in need of treatment.
  • 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.
  • Neisseria spp including N. gonorrhea and N. meningitidis
  • 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.
  • B. pertussis for example pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae
  • 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.
  • 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 derivative
  • 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.
  • 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.
  • 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.
  • 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,
  • 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
  • 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.
  • 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.
  • 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, Entam
  • 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.
  • level antigen-specific antibody e.g., systemic IgM, IgG (lgG1, lgG2a, et al.) or IgA
  • Cellular immune responses also can be monitored after administration by assessing T and B cell function after antigen stimulation.
  • nucleic acid molecule e.g., the RNA
  • the nucleic acid molecule encodes a protein antigen
  • 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.
  • compositions can also be determined in vivo by challenging appropriate animal models of the pathogen of interest infection.
  • 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).
  • the subject is a human.
  • the subject is a non-human mammal.
  • the non-human mammal is a dog, cow, or horse.
  • a bioactive agent to a cell, including the step of contacting the cell with a composition described herein.
  • the bioactive agent is a nucleic acid.
  • 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.
  • 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.
  • cancer such as meningiomas, hepatic cell carcinoma, pancreatic tumors
  • infectious diseases including fungal, bacterial, or parasitic diseases
  • inflammatory diseases including psoriasis and arthritis and atrial- ventricular malformations
  • autoimmune diseases and neurological diseases.
  • 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.
  • administration of the composition is intramuscular, parenteral, or intradermal.
  • 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).
  • the subject is a human.
  • the subject is a non-human mammal.
  • the non-human mammal is a dog, cow, or horse.
  • the composition can be administered 1 , 2, 3, or 4 times.
  • the one or more administrations may occur as part of a so-called "prime-boost” protocol.
  • the "prime-boost” approach comprises administration in several stages that present the same antigen through different vectors or multiple doses.
  • 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.
  • 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.
  • 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.
  • an oncolytic virus which may be formulated with an NLC or without an NLC
  • an NLC comprising an RNA construct that encodes the neoantigen
  • RNA-NLC binding a molar ratio of nitrogen (N) to phosphate (P) that optimizes antibody titers produced in a subject comprising the cell.
  • N nitrogen
  • P phosphate
  • 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.
  • the molar ratio of N to P may be from 1 to 15 or from 1 to 7.
  • the subject is infected with a pathogen.
  • the pathogen is a bacterium, virus, fungus or parasite.
  • the disease is associated with a bacterial, viral, fungal or parasitic infection.
  • the bacterium is a mycobacterium.
  • the pathogen is cryptosporidium.
  • 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.
  • coronavirus e.g., SARS-CoV-2
  • Zika virus e.g., an influenza virus (e.g., H1 N1, H5 andH7 subtypes)
  • influenza virus e.g., H1 N1, H5 andH7 subtypes
  • a yellow fever virus e.g., HIV, human papillomavirus, chikungunya virus, VZV, RSV, or EBV.
  • 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.
  • compositions of the disclosure can be administered to an allergic individual without co-administration of allergen.
  • 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.
  • 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.
  • 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.
  • 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 of 10,000 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.
  • 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
  • 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.
  • 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).
  • [DOTAP] and [RNA] are molar concentrations of DOTAP and RNA, respectively.
  • RNA binding to NLC as a function of the theoretical N/P is characterized using a gel retardation assay (GRA).
  • GAAHI-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 excipient background.
  • 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.
  • a control reporter rvRNA, encoding secreted human embryonic alkaline phosphatase (SEAP) was also designed.
  • 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.
  • SEAP secreted embryonic alkaline phosphatase
  • 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.
  • Vaccine saRNA was generated by T7 polymerase-mediated in vitro transcription (IVT) using Nofl-linearized DNA plasmids as templates.
  • IVT in vitro transcription
  • 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 using Capto 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).
  • 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.
  • RNA replicon systems were developed to facilitate heterologous prime-boost strategies.
  • the formulated saRNA was administered via the intramuscular route using a conventional needle.
  • 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.
  • 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.
  • 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.
  • 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-prime by 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).
  • 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.
  • a composition comprising a lipid-based nanoparticle for delivery of a bioactive agent to a cell, wherein the lipid-based nanoparticle comprises a saponin.
  • 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.
  • NLC nanostructured lipid carrier
  • LNP lipid nanoparticle
  • SSN solid lipid nanoparticle
  • oil-in-water emulsion oil-in-water emulsion
  • CNE cationic nanoemulsion
  • CARTs charge-altering releasable transporter
  • 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.
  • composition of embodiment 8, wherein the microcrystalline triglyceride is trimyristin (Dynasan®114), tristearin (Dynasan®118), or tripalmitin (Dynasan®116).
  • composition of embodiment 9, wherein the microcrystalline triglyceride is trimyristin.
  • 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.
  • composition of embodiment 20, wherein the sorbitan monoester is sorbitan monooleate (Span®80).
  • composition of embodiment 24, wherein the hydrophilic non-ionic surfactant is a polyoxyethylene sorbitan ester.
  • composition of any of embodiments 24 to 26, wherein the hydrophilic surfactant is polysorbate 80 (TWEEN® 80).
  • the hydrophobic surfactant is sorbitan monostearate (Span®60) and the hydrophilic surfactant is polysorbate 80 (TWEEN® 80).
  • QS Quillaja saponaria
  • composition of embodiment 29, wherein the QS saponin is QS-7, QS-17, QS-18, or QS-21.
  • composition of embodiment 31, wherein the QS saponin is QS-21.
  • composition of any of embodiments 1 to 36 comprising 0.1 to 1000.0 pig of saponin.
  • 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.
  • composition of embodiment 43, wherein the bioactive agent is mRNA, oncolytic viral RNA, non-coding RNA, or self-amplifying RNA.
  • composition of embodiment 44, wherein the bioactive agent is self-amplifying RNA.
  • 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.
  • the infectious pathogen is a bacterium, a virus, or a parasite.
  • composition of embodiment 51, wherein the infectious pathogen is SARS-CoV-2 and/or the epitope is the SARS-CoV-2 spike protein.
  • 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.
  • 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.
  • composition is intramuscular, parenteral, or intradermal.
  • 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.
  • 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.
  • composition of embodiment 73, wherein the parasite is cryptosporidium.

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Abstract

L'invention concerne des compositions de nanoparticules à base de lipides, et des procédés de fabrication et d'utilisation de celles-ci. Les compositions comprennent des transporteurs lipidiques nanostructurés (NLC), des liposomes, des nanoparticules lipidiques (LNP), des nanoparticules lipidiques solides (SLN), des émulsions huile dans l'eau, des complexes lipide cationique-acide nucléique, des nanoémulsions cationiques (CNE), des transporteurs libérables modifiant la charge (CART), ou des nanoparticules polymères, et comprennent en outre un adjuvant de saponine, et éventuellement un stérol et/ou un agent bioactif. L'agent bioactif peut être un ARN auto-amplifiant. Les compositions sont capables de délivrer une biomolécule en direction d'une cellule pour la génération d'une réponse immunitaire, par exemple à des fins vaccinales, thérapeutiques, de désensibilisation aux allergies ou diagnostiques. La présente invention porte également sur des compositions et sur des procédés associés à la fabrication des compositions et à leur utilisation pour stimuler une réponse immunitaire.
EP23772597.3A 2022-09-09 2023-09-09 Composition de vaccin immunogène incorporant une saponine Pending EP4583841A1 (fr)

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