Nanoparticulate vaccines for small molecules

Abstract

A nanoparticle vaccine composition includes a shell including one or more amphiphilic molecules and one or more hapten molecules, where at least one amphiphilic molecule is conjugated to the hapten molecule, and a substantially hydrophobic core including an immunogenic protein, a vaccine adjuvant, one or more inert biocompatible materials, or a combination of two or more thereof, where the core is encapsulated in the shell to form a nanoparticle.

Description

NANOPARTICULATE VACCINES FOR SMALL MOLECULESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 685,100, which was filed August 20, 2024, and is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to a nanoparticle vaccine composition for small molecules, its method of preparation, and its use for inducing immune response and preventing and treating opioid and other drug abuse.BACKGROUND

[0003] This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be construed as admissions about what is or is not prior art.

[0004] Small molecules are not immunogenic, so the immune system does not ‘see’ them and cannot make antibodies that bind and remove them from circulation. However, there are several classes of small molecules, e.g., opioids and neurotoxins, for which it would be desirable to induce the formation of antibodies reliably. It is reported in the art that small molecules can be made into immunogenic “haptens” by their conjugation onto a protein or other carrier.

[0005] Vaccination against drugs of abuse, such as opioids, is being investigated as a method to reduce the biological effects of opioids and other drugs of abuse and reduce the incidence of overdose. Opioid abuse disorder is a major public health crisis in the United States. Opioid vaccines have the potential to induce opioid-specific antibodies that bind the drug and neutralize its biological effects by prohibiting it from entering the brain and / or preventing respiratory depression that leads to overdosing effects. However, inducing effective opioid-specific neutralizing antibody responses requires the conjugation of the opioid to a carrier system with specific properties, e.g., proteinaceous character to provide T cell help, high opioid surface density, and proper formulation to allow administration.

[0006] There is room for improvement in the current vaccines for opioids and other drugs of abuse. For example, many candidate vaccines for drugs of abuse are immunogenic, i.e., they induce drugspecific antibodies, but the anti-drug antibodies they induce do not effectively block the biological activity of the drugs in vivo. The immunogenicity of an opioid vaccine requires the opioid hapten to be conjugated with a foreign carrier molecule to assist in developing an immune response. Proteins have often been used as carrier molecules for opioids and other drugs of abuse (Jalah et al., Bioconjugate chemistry, 2015, 26, 6, 1041-1053). Traditional protein carriers for conjugate vaccines include tetanus toxoid (TT) and cross-reactive material 197 (CRM197), a nontoxic form of diphtheria toxin. Nanoparticles such as polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) are carriers that may be utilized with conjugate vaccines. Nanoparticles can express the hapten on the surface, while encapsulating protein antigens in the interior of the nanoparticle. Another nanoparticle platform evaluated for vaccines for addictive drugs is bacteriophage Qp virus-like particle (VLP), which is an immunogenic nanoparticle due to foreign proteins in the QP VLP. The carrier protein or immunogenic nanoparticle used in candidate opioid vaccines provide CD4+ T cell help that enhances the host antibody response to the opioid and may improve the ability of the anti-opioid antibody to block subsequently the biological activity of the opioid when used as a drug of abuse. However, pre-existing immunity to carrier proteins or nanoparticles such as QP VLP may reduce the immunogenicity of the opioid conjugate vaccine if repeated doses of the vaccine are required to induce and maintain high-titer, anti-opioid antibodies.

[0007] In view of the above, it is an object of the present disclosure to provide nanoparticle (NP) vaccines for small drug molecules, such as opioids or neurotoxins, which can offer advantages over currently available vaccines. This and other objects and advantages, as well as inventive features, will be apparent from the detailed description provided herein.SUMMARY

[0008] Provided is a nanoparticle vaccine composition comprising:(i) a shell comprising (a) one or more amphiphilic molecules and (b) one or more hapten molecules, wherein at least one amphiphilic molecule is conjugated to the hapten molecule; and(ii) a substantially hydrophobic core comprising (a) an immunogenic protein, (b) a vaccine adjuvant, (c) one or more inert biocompatible materials; or (d) a combination of two or more thereof, wherein the core is encapsulated in the shell as a nanoparticle.

[0009] The hapten molecule can be selected from dinitrophenol, trinitrophenol (e.g., 2,4,6- trinitrophenol), morphine, heroin, fentanyl, cocaine, malathion, sarin, methyl phosphonic acid, p- aminophenyl 1,2,2-trimethylpropyl diester, dimethyl methylphosphonate, phosphocholine, diisopropyl methylphosphonate, diethyl methylthiophosphonate, diethyl phenylthiophosphonate, diethyl phenylphosphorothioate, phenyl diphenylphosphinothioate, diethyl 3- (dimethylamino)propylphosphonate, diethyl chlorophosphonate, diisopropyl fluorophosphonate, diethyl cyanophosphonate, isopropyldodecylflouro phosphonate, phenylthiophosphonic acid O- ethyl S-[2-(diisopropylamino)ethyl] ester (PhX), pinacolylmethylphosphonic acid, diethyl N,N- diethylphosphoramidate, dicyclohexyl methylphosphonate, ethyl N,N- diethylphosphoramidocyanidate, chloroethyl phenylsulfide, bis(2-chloroethyl)sulfide, p- nitrophenyl diphenylphosphate, tabun, soman GD, cyclosarin GF, VX, R-VX, parathion, parathion-methyl, paraoxon, diazinon, 4-nitrophenol, diethylchloro-thiophosphate, triazophos, fenthion, fenitrothion, chlorpyrifos, chlorpyrifos-methyl, atrazine, coumaphos, cyanophos, demeton, dichlorovos, dioxathion, glyphosate, fonofos, malaoxon, malathion, methamidophos, mevinphos, and oxydemeton-methyl.

[0010] In certain examples, the hapten molecule is morphine, heroin, fentanyl, cocaine, or chloropyrifos.

[0011] The amphiphilic molecule can be selected from a diblock polymer, an amphiphilic lipid, a surfactant, a polymer, a polymer-lipid, and a combination of two or more thereof. In some embodiments, the diblock polymer comprises (a) at least one hydrophobic block selected from polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), polystyrene (PS), and polylactic acid (PLA); (b) at least one hydrophilic block selected from polyethylene glycol (PEG), polyacrylic acid (PAA), polylysine, and poly[2 (dimethylamino)ethyl methacrylate] (DMAEMA); or (c) a combination of (a) and (b). In some embodiments, the diblock copolymer is poly(caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG).

[0012] In some embodiments, the amphiphilic molecule can be selected from tocopherol polyethylene glycol succinate, methoxypoly(ethylene glycol) ditetradecylacetamide (ALC-0159),l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), and a combination of two or more thereof.

[0013] The hapten molecule TNP can be conjugated to a hydrophilic section of the diblock polymer. The surface coverage of the hapten molecule on the shell is about 1% to about 100%.

[0014] In some embodiments, the vaccine adjuvant is a ligand that activates receptors of the innate immune system such as, but not limited to, toll-like receptor 4 ligands (monophosphoryl lipid A; MPLA) toll-like receptor 5 ligands (flagellin); toll-like receptor 9 ligands (CpG oligodeoxynucleotides); toll-like receptor 2 / 1 ligands (lipopeptide PAM3CSK4); toll-like receptor 7 / 8 ligands (imidazoquinoline); toll-like receptor 3 ligands (Polyinosine-polycytidylic acid); stimulator of interferon genes, (STING) receptor ligands (cyclic dinucleotide 2’3’-cGAMP); C- type lectin ligands (Trehalose-6,6-dibehenate or trehalos 6,6’ dimycolate); compounds that inhibit immune inhibitory pathways (small molecule NF-KB inhibitors), or hydrophobic salts thereof. In examples, the vaccine adjuvant is alum, mastoparan 7, mastoparan 17, or monophosphoryl Lipid A (MPL).

[0015] In some embodiments, the immunogenic protein can be a protein that induces an immune response such as, but not limited to, CRM197 (a genetically detoxified diphtheria toxin), genetically detoxified tenaus toxin, tetanus toxin heavy chain, influenza hemagglutinin, respiratory syncytial virus recombinant proteins (preF A and preF B recombinant proteins) and other proteins used in vaccines approved for human use, or hydrophobic salts thereof. In examples, the immunogenic protein can be selected from ovalbumin (OVA), CRM-197, cholera toxin, keyhole limpet hemocyanin, and a salt of any of the foregoing. In one example, the immunogenic protein is ovalbumin or a hydrophobic salt thereof

[0016] In some embombodiments, the inert biocompatible materials herein can be selected from among PCL, PLGA, Vitamin E, Vitamin E acetate (VitEAc), and PLA. In some embodiments, a vaccine adjuvant may be co-administered with vaccines with an inery biocompatible material core.

[0017] In some embodiments, the vaccine core is Vitamin E acetate (VitEAc). The non- immunogenic core can be selected from Vitamin E acetate. In some embodiments, a vaccine adjuvant may be co-administered with vaccines with an inert biocompatible material core. The vaccine adjuvant can be selected from OVA, CRM- 197, cholera toxin, keyhole limpet hemocyanin, and a salt of any of the foregoing.

[0018] Provided is a pharmaceutical composition comprising the above-described nanoparticle vaccine composition and a pharmaceutically acceptable carrier, excipient, or diluent.

[0019] Further provided is a method of inducing an immune response to a hapten molecule, which method comprises administering to a patient in need thereof a therapeutically effective amount of the nanoparticle vaccine composition or the pharmaceutical composition. The nanoparticle vaccine composition can be administered intramuscularly, intravenously, subcutaneously, intraperitoneally, intraocularly, intrathecally, intranasally, or intratracheally. The nanoparticle vaccine composition is effective in eliciting an antibody response to a hapten molecule.

[0020] Further provided is a method of preventing or treating an opioid or other drug use disorder by administering to a patient in need thereof a prophylactically or therapeutically effective amount of the nanoparticle vaccine composition or the pharmaceutical composition. The nanoparticle vaccine composition can induce opioid-specific or other drug-specific antibodies.

[0021] Still further provided is a method of preparing a nanoparticle vaccine composition, which method comprises:(i) dissolving one or more components selected from (a) one or more amphiphilic molecules and (b) one or more hapten molecules, wherein at least one amphiphilic molecule is conjugated to the one or more hapten molecules, in an organic solvent to obtain an organic feed solution;(ii) mixing homogeneously the organic feed solution of step (i) rapidly with an aqueous feed solution, which may contain (a) an immunogenic protein, (b) a vaccine adjuvant, (c) one or more inert biocompatible materials; or (d) a combination of two or more thereof, which leads to the rapid precipitation of the components from the organic feed solution; and(iii) allowing particles to assemble instantly by Flash NanoPrecipitation, whereupon the nanoparticle vaccine composition is formed.BRIEF DESCRIPTION OF DRAWINGS

[0022] The above and other objects, features, and advantages of the present invention will become more apparent when taken in conjunction with the following description and drawings whereinidentical reference numerals have been used, where possible, to designate identical features that are common to the figures, and wherein:

[0023] Fig. 1 is a schematic representation of a nanoparticle vaccine made by Flash NanoPrecipitation (FNP), which induces the formation of hapten- specific antibodies in vivo.

[0024] Fig. 2 is a 1H NMR spectra of PCL-PEG-TNP. The peak of the TNP functional group at 9.02 ppm and the linker group at 1.24, 2.20, and 3.45 confirms the successful conjugation between PCL-PEG-NH2 and TNP.

[0025] Fig. 3 illustrates the size distribution of PCL-PEG-TNP nanocarriers encapsulating VitEAc with varying percentages of TNP surface coverage (0%, 10%, and 100%).

[0026] Fig. 4A illustrates the nanoparticle size and poly dispersity index (PDI) of nanoparticle vaccine compositions, for examples, formulations F 1 to F5 with different surface coverage of TNP. Fig. 4B illustrates the size and PDI of PCL-PEG-TNP nanocarriers encapsulating OVA with 0% TNP surface coverage over 7 days. Fig. 4C is an image of a cryo transmission electron microscopy (TEM) of nanocarriers encapsulating OVA with 100% TNP surface coverage, illustrating the presence of mostly spherical NPs approximately 100 nm in diameter, supporting other size measurements.

[0027] Fig. 5 illustrates day 28 anti-trinitrophenol-bovine serum albumin (anti-TNP-BSA) serum immunoglobulin (IgG) geometric mean titers (GMT) following dosing with the different formulations of the nanoparticle vaccine composition.

[0028] Fig. 6 illustrates that ovalbumin-tri nitrophenol nanoparticle (OVA- TNP NP) vaccines are immunogenic after two intramuscular (IM) immunizations. OVA- TNP NP vaccines induce potent OVA- and TNP-specific antibody responses. Female BALB / c mice received two IM immunizations with monophosphoryl lipid (MPL) adjuvanted, OVA- TNP protein-conjugated vaccines or MPL-adjuvanted, OVA-encapsulated nanoparticles prepared by FNP with various densities of TNP surface coverage. The serum collected on day 35 was measured for OVA, OVA- TNP, and OVA-specific IgG by ELISA.

[0029] Fig. 7 illustrates that the TNP-specific antibody response depends on TNP concentration, i.e., TNP surface coverage percentage on the nanoparticles. Female BALB / c mice were immunized as described in Fig. 6. The data represent the OVA- TNP, OVA, and TNP-specific serum IgG responses of the individual mice within each vaccine group. The median antibody response is reported for each group, as indicated by the black horizontal line.DETAILED DESCRIPTION

[0030] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the claimed invention is thereby intended.

[0031] Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0032] The term “hapten” refers to a small molecule that stimulates the production of antibodies only when conjugated to a larger molecule, called a carrier molecule.

[0033] The terms “nanoparticle vaccine,” “nanoparticle vaccine composition,” and “nanoparticle” are used interchangeably.

[0034] Opioid abuse disorder is a major public health crisis in the United States. The present disclosure is predicated, at least in part, on the discovery that the vaccination against drugs of abuse, such as opioids, can reduce the biological effects of opioids and other drugs of abuse and reduce the incidence of overdose. Opioid vaccines have the potential to induce opioid-specific antibodies that bind the drug and neutralize its biological effects by prohibiting it from entering the brain and / or preventing respiratory depression that leads to overdosing effects. However, inducing effective opioid-specific neutralizing antibody responses requires the conjugation of the opioid to a carrier system with specific properties: (1) proteinaceous character to provide T cell help; (2) high opioid surface density; and (3) proper formulation to allow administration. Although vaccines for other drugs of abuse are immunogenic (i.e., they induce drug-specific antibodies) but, the anti-drug antibodies they induce do not effectively block the biological activity of the drugs in vivo.

[0035] In view of the above, provided are nanoparticle (NP) vaccine compositions for small molecules, such as opioids and other drugs of abuse. The NP vaccine composition may induce drug-specific antibodies that can neutralize drug overdosing effects and may prevent pre-existing antibodies from interfering with vaccine immunogenicity. Advantages of NP vaccine compositions disclosure herein may include, for example, (a) the ability to encapsulate protein at high efficiency; (b) the ability to formulate NPs with a high and tunable surface density of haptens of smallmolecules; (c) the ability to scale up from bench production to industrial production; and (d) the ability to inhibit, and desirably prevent, pre-existing antibodies from interfering with vaccine immunogenicity.

[0036] In particular examples herein, an NP vaccine composition may comprise:(i) a shell comprising (a) one or more amphiphilic molecules and (b) one or more hapten molecules, wherein at least one amphiphilic molecule is conjugated with the hapten molecule; and(ii) a substantially hydrophobic core comprising (a) an immunogenic protein, (b) a vaccine adjuvant, (c) one or more inert biocompatible materials; or (d) a combination of two or more thereof, wherein the core is encapsulated in the shell as a nanoparticle.Fig. 1 provides a cartoon illustration of an NP vaccine composition, herein which according to the illustrated example, comprises: (i) a shell 100 comprising (a) one or more amphiphilic molecules 102 and (b) one or more hapten molecules 104, wherein at least one amphiphilic molecule is conjugated with the hapten molecule; and (ii) a substantially hydrophobic core 106 comprising (a) an immunogenic protein 108, (b) a vaccine adjuvant 110, wherein the core 106 is encapsulated in the shell 100 as a nanoparticle.

[0037] Small molecules contemplated in examples herein may be opioids or neurotoxins. Nanoparticle vaccines may be prepared by utilizing Flash NanoPrecipitation (FNP) technology or other sufficiently rapid mixing techniques. The NP vaccine composition may include a core and a shell. The shell of the NP may include amphiphilic molecules and hapten molecules, wherein at least one amphiphilic molecule may be conjugated to a hapten molecule. The NP may have a high and tunable hapten surface density. In some embodiments, one or more amphiphilic molecules may be the same or different. In some embodiments, one or more hapten molecules may be the same or different.

[0038] In certain examples, the hapten molecule may be selected from a group consisting of dinitrophenol, trinitrophenol (e.g., 2,4,6-trinitrophenol), morphine, heroin, fentanyl, cocaine, malathion, sarin, methyl phosphonic acid, p-aminophenyl 1,2,2-trimethylpropyl diester, dimethyl methylphosphonate, phosphocholine, diisopropyl methylphosphonate, diethyl methylthiophosphonate, diethyl phenylthiophosphonate, diethyl phenylphosphorothioate, phenyl diphenylphosphinothioate, diethyl 3- (dimethylamino)propylphosphonate, diethyl chlorophosphonate, diisopropyl fluorophosphonate, diethyl cyanophosphonate,isopropyldodecylflouro phosphonate, phenylthiophosphonic acid O-ethyl S-[2- (diisopropylamino)ethyl] ester (PhX), pinacolylmethylphosphonic acid, diethyl N,N- diethylphosphoramidate, dicyclohexyl methylphosphonate, ethyl N,N- diethylphosphoramidocyanidate, chloroethyl phenyl sulfide, bis(2-chloroethyl)sulfide, p- nitrophenyl diphenylphosphate, tabun, soman GD, cyclosarin GF, VX, R-VX; organophosphate- based pesticides selected from parathion, parathion-methyl, paraoxon, diazinon, 4- nitrophenol,diethylchloro-thiophosphate, triazophos, fenthion, fenitrothion, chlorpyrifos, chlorpyrifos-methyl, atrazine, coumaphos, cyanophos, demeton, dichlorovos, dioxathion, glyphosate, fonofos, malaoxon, malathion, methamidophos, mevinphos, and oxydemeton-methyl. In one example, the hapten molecule is 2,4,6-trinitrophenol (TNP). In certain examples, the hapten molecule is morphine, heroin, fentanyl, cocaine, or chloropyrifos.

[0039] Amphiphilic molecules are chemical compounds that have both polar and nonpolar regions, giving them both hydrophilic (water-loving) and lipophilic (fat-loving) properties. As contemplated herein, tamphiphilic molecules herein are is capable of stabilizing the NP surface. Any suitable amphiphilic molecule, as well-known in the art, may be used. The amphiphilic molecules may be selected, for example, from a diblock polymer, an amphiphilic lipid, a surfactant, a polymer, a polymer-lipid, and a combination of two or more thereof. In some embodiments, the amphiphilic molecule is a diblock polymer. The diblock polymer may comprise (a) at least one hydrophobic block selected, for example, from polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), polystyrene (PS), and polylactic acid (PLA); (b) at least one hydrophilic block selected, for example, from polyethylene glycol (PEG), polyacrylic acid (PAA), polylysine, and poly[2-(dimethylamino)ethyl-methacrylate] (DMAEMA); or (c) a combination of (a) and (b). In some embodiments, the block copolymer is poly(caprolactone)-b-poly(ethylene glycol) (PCL-b- PEG).

[0040] In certain examples, the amphiphilic molecule may be tocopherol polyethylene glycol succinate, methoxypoly (ethylene glycol) ditetradecyl acetamide (ALC-0159), 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol (DMG-PEG), or a combination of two or more thereof.

[0041] In some embodiments, the vaccine adjuvant is a ligand that activates receptors of the innate immune system such as, but not limited to, toll-like receptor 4 ligands (monophosphoryl lipid A; MPLA) toll-like receptor 5 ligands (flagellin); toll-like receptor 9 ligands (CpG oligodeoxynucleotides); toll-like receptor 2 / 1 ligands (lipopeptide PAM3CSK4); toll-like receptor7 / 8 ligands (imidazoquinoline); toll-like receptors 3 ligands (Polyinosine-polycytidylic acid); stimulator of interferon genes, (STING) receptor ligands (cyclic dinucleotide 2’3’-cGAMP); C- type lectin ligands (Trehalose-6,6-dibehenate or trehalos 6,6’ dimycolate); compounds that inhibit immune inhibitory pathways (small molecule NF-KB inhibitors), or hydrophobic salts thereof.

[0042] In some embodiments, the immunogenic protein can be a protein that induces an immune response such as, but not limited to, CRM197 (a genetically detoxified diphtheria toxin), genetically detoxified tenaus toxin, tetanus toxin heavy chain, influenza hemagglutinin, respiratory syncytial virus recombinant proteins (preF A and preF B recombinant proteins) and other proteins used in vaccines approved for human use, or hydrophobic salts thereof.

[0043] The core may further comprise one or more inert biocompatible materials, which can be selected from among PCL, PLGA, Vitamin E, Vitamin E acetate (VitEAc), and PLA. In some embodiments, a vaccine adjuvant may be co-administered with vaccines with an inery biocompatible material core.

[0044] The hapten molecule, such as TNP, can be conjugated to the terminal end of the hydrophilic part of the diblock copolymer. For example, a hydrophobic block of PCL conjugated to a hydrophilic block of PEG can be conjugated covalently with TNP to form PCL-b-PEG-TNP, where m may between 30 and 100 units and n is between 40 and 200 units:PCL-PEG-TNPIn examples, m may between 30 and 90 units, 35 and 80 units, 40 and 70 units, 40 and 60 units, 40 and 60 units, or 40 and 50. In one particular example, m may be ~45 units. In the same or other examples, n may be between 50 and 190 units, 60 and 180 units, 70 and 170 units, 80 and 160 units, 90 and 150 units, 100 and 140 units, 100 and 130 units, 100 and 120 units, or 105 and 115 units. In one particular example n may be -110 units. In another example, m is -45 units and n is -110 units.

[0045] In some embodiments, TNP can be conjugated to PCL-b-PEG-NH2 by suspending about 1 gram (g) of PCL-PEG-NH2 HC1 polymer and about 480 milligrams (mg), (about 1.3 millimoles (mmol)) in about 20 milliliters (mL) of methylene chloride, dichloromethane (DCM) and reacting with about 300 microliters (%muL) (about 1.7 mmol) and letting stir around 16 hours at room temperature, between 20 and 22 degrees Celsius or 68 - 72 degrees Fahrenheit. The solution can then be concentrated to dryness under reduced pressure. The conjugate can then be purified through flash column chromatography using about 12 g of Residep Rf, SiC>2 and eluting with a gradient of 100% DCM to 5% methanol (MeOH) in DCM. After additional concentration to dryness under reduced pressures, the final product can be described as a glassy solid and can have a mass of about 120 milligrams (mg). As shown in Fig. 2, the final product was confirmed using1HNMR, specifically the presence of the peak of TNP functional group at 9.02 ppm and the linker group at 1.23, 2.20, and 2.34 ppm.PCL-PEG-NH2HC1Synthesis scheme for the conjugation of PCL-B-PEG-NH2 with TNP to create PCL-b-PEG- TNP.

[0046] In some embodiments, the surface density / coverage of TNP on NP shell may be about 1% to about 100%. In examples, the surface density / coverage of TNP on NP shell maybe about 10% to about 100%, 20% to about 100%, 30% to about 100%, 40% to about 100%, 50% to about 100%, 60% to about 100%, 70% to about 100%, 80% to about 100%, 80% to about 95%, or 85% to about 90%, 90% to about 100%, or 95% to about 100%. Such percentages indicate the number of PCL- b-PEG chains on the surface of the nanoparticle that are conjugated with TNP. If all the polymers on the NP surface are simply PCL-b-PEG (no conjugation with TNP), that means the surface coverage of TNP is 0%. If all the polymers on the NP surface are PCL-b-PEG- TNP, that means the surface coverage of TNP is 100%.Synthesis scheme for the conjugation of Opioids to PCL-PEG Diblock Copolymer.

[0047] Fentanyl hapten conjugation reaction:

[0048] Morphine hapten conjugation reaction:

[0049] Cocaine hapten conjugation reaction:

[0050] Chlorpyrifos hapten conjugation reaction:

[0051] The core of NP comprise an immunogenic protein capable of boosting the hapten immunogenicity. Any suitable immunogenic protein may be encapsulated in the core. For example, the immunogenic protein may be selected from ovalbumin, CRM-197, cholera toxin, keyhole limpet hemocyanin, and a salt of any of the foregoing. The immunogenic protein of certain examples may be ovalbumin or a hydrophobic salt thereof. In one example, the Ovalbumin may be an immunogenic CD4+ T cell helper protein.

[0052] The core may also optionally further comprise at least one vaccine adjuvant. Any suitable vaccine adjuvants, as well-known in the art, can be used. The vaccine adjuvant may further boost the hapten immunogenicity. In some embodiments, the vaccine adjuvant is selected from alum, mastoparan 7, mastoparan 17, and monophosphoryl Lipid A.

[0053] In examples, the NP vaccines may be prepared using the Flash NanoPrecipitation process. The process may be facilitated through a confined impinging jet mixer or multi-inlet vortex mixer. The process allows for precise control over the formulation process, resulting in efficient loading of the immunogenic protein into the core of the nanoparticulate vaccine while the amphiphilic shell with the hapten can be efficiently installed onto the NP surface. The rapid mixing of the two feed solutions leads to rapid precipitation of the components from the organic solution due to a sudden change in the solvent quality.

[0054] Provided herein are methods of preparing a nanoparticle vaccine composition. In examples, the method comprises:(i) dissolving one or more components selected from (a) one or more amphiphilic molecules and (b) one or more hapten molecules, wherein at least one amphiphilic molecule is conjugated to the hapten molecule, in an organic solvent to obtain an organic feed solution;(ii) mixing homogeneously the organic feed solution of step (i) rapidly with an aqueous feed solution, which may contain (a) an immunogenic protein, (b) a vaccine adjuvant, (c) one or more inert biocompatible materials; or (d) a combination of two or more thereof, which, when performed, leads to the rapid precipitation of the components from the organic feed solution; and(iii) allowing particles to assemble instantly by flash nano-precipitation, whereupon the nanoparticle vaccine composition is formed.

[0055] In some embodiments, the method may further comprise subjecting the formed NPs to a quench bath, which, when performed, directly affects NP size due to minimizing unimer exchange, and dialysis against water to remove excess solvent, which then eliminates further size change of the NPs. In some embodiments, the organic solvent that may be used for FNP consistent with the disclosure is a water-miscible organic solvent such as tetrahydrofuran.

[0056] NP vaccine compositions herein prepared by FNP may encapsulate an immunogenic protein in the core and co-encapsulate a vaccine adjuvant in the NP core. The protein improves theimmunogenicity of the nanoparticle, and co-localizing a vaccine adjuvant with the antigen further improves the performance of the NP vaccine compositions. However, FNP NPs by themselves lack immunogenicity, which can be advantageous in reducing anti-carrier immunity that may decrease vaccine efficacy. In comparison, the QP virus-like particles are immunogenic due to being composed of bacteriophage proteins, and the immunogenicity of the QP VLP will result in the induction of anti-QP immune responses. This anti-QP immunity may reduce the immunogenicity of QP VLP hapten conjugates, due to the pre-existing immunity to QP VLP, and interfere with the induction of anti-hapten antibodies. In contrast, FNP NP encapsulates the protein antigen on the core of the NP, which can prohibit pre-existing antibodies from interfering with vaccine immunogenicity. Therefore, the FNP NP vaccine composition cannot induce immune responses against the FNP NP because the FNP NP are produced from lipids and polymers and do not express antigens / haptens other than the conjugated hapten on its surface.

[0057] Provided are pharmaceutical compositions comprising a nanoparticle vaccine composition as described herein and a pharmaceutically acceptable carrier, excipient, or diluent. The carrier, excipient, or diluent can vary based on the particular route of administration (see, e.g., Remington’s The Science and Practice of Pharmacy, 23rded. (2020)). In examples, the pharmaceutical composition includes a nanoparticle vaccine composition comprising:(i) a shell comprising (a) one or more amphiphilic molecules and (b) one or more hapten molecules, wherein at least one amphiphilic molecule is conjugated to the hapten molecule; and(ii) a substantially hydrophobic core comprising (a) an immunogenic protein, (b) a vaccine adjuvant, (c) one or more inert biocompatible materials; or (d) a combination of two or more thereof, wherein the core is encapsulated in the shell as a nanoparticle; and and a pharmaceutically acceptable carrier, excipient, or diluent.

[0058] Provided is a method of inducing an immune response to small molecules in a patient in need thereof. The method comprises administering to the patient a therapeutically effective amount of the NP vaccine composition or a pharmaceutical composition comprising the NP vaccine composition and a pharmaceutically acceptable carrier, excipient, or diluent. In examples, the NP vaccine composition comprises:(i) a shell comprising (a) one or more amphiphilic molecules and (b) one or more hapten molecules, wherein at least one amphiphilic molecule is conjugated to the hapten molecule; and(ii) a substantially hydrophobic core comprising (a) an immunogenic protein, (b) a vaccine adjuvant, (c) one or more inert biocompatible materials; or (d) a combination of two or more thereof, wherein the core is encapsulated in the shell as a nanoparticle.In some embodiments, the hapten molecules can treat opioid or other drug abuse disorders. The NP can induce the formation of antibodies specific to haptens.

[0059] Provided is a method of preventing or treating an opioid or other drug use disorder. The method comprises administering to a patient in need thereof a prophylactically or therapeutically effective amount of the NP vaccine composition or a pharmaceutical composition comprising the nanoparticle vaccine composition and a pharmaceutically acceptable carrier, excipient, or diluent. In examples, the NP vaccine composition comprises:(i) a shell comprising (a) one or more amphiphilic molecules and (b) one or more hapten molecules, wherein at least one amphiphilic molecule is conjugated to the hapten molecule; and(ii) a substantially hydrophobic core comprising (a) an immunogenic protein, (b) a vaccine adjuvant, (c) one or more inert biocompatible materials; or (d) a combination of two or more thereof, wherein the core is encapsulated in the shell as a nanoparticle.The NP vaccine is capable of inducing opioid-specific or other drug-specific antibodies and, thus, induce an immune response to the opioid and other drugs. In some embodiments, the drug of abuse is an opioid drug.

[0060] The nanoparticle vaccine composition consistent with the disclosure may be administered by any suitable route, examples of which include intramuscularly, intravenously, subcutaneously, intraperitoneally, intraocularly, intrathecally, intranasally, and intratracheally.

[0061] An NP vaccine composition for opioids is also provided. The composition comprises:(i) a shell comprising (a) one or more amphiphilic molecules and (b) one or more opioid molecules, wherein at least one amphiphilic molecule is conjugated to the opioid molecule; and(ii) a substantially hydrophobic core comprising (a) an immunogenic protein, (b) a vaccine adjuvant, (c) 1 or more inert biocompatible materials, or (d) a combination of (a) and / or (b), and / or (c) wherein the core is encapsulated in the shell to form a nanoparticle.

[0062] The NP vaccine for opioid (opioid vaccine) may utilizes a precisely controlled opioid hapten density on the outer surface of the NP. The opioid hapten may first be conjugated to an amphiphilic molecule, e.g., block copolymers PCL-b-PEG that serves as the surface stabilizer of the NP during the FNP process, and opioid hapten may be presented on the surface of the NP. The flow of the PCL-PEG-opioid may be modulated during the mixing process to control reproducibly and precisely the density of an opioid on the surface of the NP. The NP vaccine may be used for any suitable opioid. In some embodiments, the opioid is morphine, heroin, fentanyl, dinitrophenol, trinitrophenol (e.g., 2,4,6-trinitrophenol), or cocaine. The opioid vaccine composition may be prepared using the FNP process.

[0063] In some embodiments, the pharmaceutical composition further comprises at least one additional pharmaceutically active agent, e.g., an additional adjuvant. The pharmaceutical composition may be prepared by combining an NP vaccine composition with a pharmaceutically acceptable carrier, excipient or diluent.

[0064] The terms “prophylactically effective amount,” “prophylactically effective dose,” "therapeutically effective amount," and "therapeutically effective dose" refer to an amount of the active ingredient(s) that is(are) sufficient, when administered, to deliver efficaciously the active ingredient(s) for the inhibition, prevention, or treatment of a disease or condition of interest to a subject in need thereof. The prophylactically or therapeutically effective amount / dose of such combination will vary depending upon the patient and the disease or condition being treated, the weight and age of the patient, the severity of the disease or condition, the manner of administration, and the like, which can readily be determined by one of ordinary skill in the art.

[0065] For any composition, the prophylactically or therapeutically effective amount / dose may be initially determined from animal models. A prophylactically or therapeutically effective amount / dose may also be determined from human data for compounds which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for parenteral administration. The applied dose may be adjusted based on the relative bioavailability and potency of the administeredcompound. Adjusting the amount / dose to achieve maximal efficacy based on such methods and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.

[0066] The terms "treat," "treating," "treatment," and the like refer to eliminating, reducing, or ameliorating a disease or condition and / or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. The term "treat" and synonyms contemplate administering a prophylactic or therapeutically effective amount / dose of a combination or composition described herein to a subject in need of such treatment. The treatment may be orientated symptomatically, for example, to suppress symptoms. It may be effected over a short period, be oriented over a medium term, or may be a long-term treatment, for example, within the context of maintenance therapy.

[0067] Generally, daily oral doses of a composition are from about 0.01 milligrams / kg per day to 1,000 milligrams / kg per day. Oral doses in the range of 0.5 to 50 milligrams / kg, in one or more administrations per day, can yield therapeutic results. Dosage may be adjusted appropriately to achieve the desired drug level, local or systemic, depending upon the mode of administration. For example, intravenous administration may vary from one order to several orders of magnitude lower dose per day. If the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of the compound.

[0068] The nanoparticle compositions may be typically administered in admixture with a pharmaceutical carrier to give a pharmaceutical composition selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and / or auxiliaries that facilitate the processing of the compound. The exact formulation, route of administration, and dosage of a pharmaceutical composition comprising an effective amount of the compound are determined by an individual physician in view of the diagnosed condition or disease. The dosage amount and interval may be adjusted individually to provide levels of the compound that are sufficient to maintain a prophylactic or therapeutic effect.

[0069] Toxicity and therapeutic efficacy of the combination may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the maximum tolerated dose (MTD) of a compound, which is defined as the highest dose that causes no toxicity in animals. The therapeutic index is the dose ratio between the maximum tolerated dose and therapeutic effects (e.g., inhibition of tumor growth). The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The determination of a therapeutically effective amount is well within the capability of those ordinarily skilled in the art, especially in light of the detailed disclosure provided herein.

[0070] A combination may be administered in an amount of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose. For example, the combination may be administered, per dose, in an amount of about 0.005, about 0.05, about 0.5, about 5, about 10, about 20, about 30, about 40, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 milligrams, including all doses between 0.005 and 500 milligrams.

[0071] As stated above, a composition described herein may be administered with one or more other prophylactically or therapeutically active agents.

[0072] It will be appreciated by persons skilled in the art that the present disclosure is not limited by what has been particularly shown and described herein above. Rather, the scope of the present disclosure includes both combinations and sub-combinations of the various features described hereinabove as well as variations and modifications that would occur to persons skilled in the art upon reading the specification and which are not in the prior art.EXAMPLES

[0073] The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention in any way.Preparation of nanoparticle vaccine composition by Flash NanoPrecipitation.

[0074] The shell components a diblock polymer (e.g., PCL-b-PEG), a diblock polymer-hapten conjugate (e.g., PCL - PEG - hapten conjugate), a surfactant / hydrophobic salt (DDAB), and dye were dissolved in different concentrations of hapten in organic solvent (e.g., THF) (see Table 1). The immunogenic protein (e.g., ovalbumin) was dissolved in the anti-solvent stream (e.g., RNAs-free water). The nanocarriers were developed by the known Flash NanoPrecipitation (FNP) process combined with the hydrophobic ion pairing technique using a confined impinging jet mixer (CIJ). The two streams were rapidly introduced, resulting in turbulent and homogeneous mixing within the CIJ’s mixing chamber. This rapid mixing led to the rapid precipitation of the components from the organic stream, driven by the sudden change in solvent quality. The sudden change in solvent quality led to particle assembly on the scale of milliseconds (Flash). Post-rapid homogenous mixing, the newly formed nanoparticles (NPs) were subjected to a 4 mL quench bath to minimize unimer exchange which directly affects size. All particle assembly was performed in the mixing chamber of the CIJ. The resulting carriers were kinetically frozen at a size of approximately 150 nm. The 5 mL of NPs were subjected to 24-hour dialysis against di water to remove any excess organic solvent to further eliminate any size change of NPs.Table 1Characterization of nanoparticles.

[0075] As illustrated in Fig. 3, NPs with differing amount of TNP on the surface, with a VitEAc core were formed in the range of 58.9 - 68.0 nm.

[0076] The nanoparticle’s size, poly dispersity index (PDI), and zeta potential were measured using Zetasizer Pro. Samples were diluted in Milli-Q® water for size and poly dispersity measurements using dynamic light scattering at 25°C. The samples were diluted in 20mMNaCl aqueous solution to measure the zeta potential of nanoparticles. Table 2 shows the size and PDI of the composition comprising PCL - PEG - TNP: 3 mg / mL, OVA: 3 mg / mL and DDAB: 3.32 mg / mL and Dil 0.1264 with 0%, 10%, and 100% of surface polymer chains conjugated with TNP.

[0077] NCs 13 l-147nm in diameter as illustrated in Fig. 4A, with a narrow PDI of 0.18-0.24 were formed. NC size was constant after dialysis and 6-fold concentration and did not change over at least 7 days as shown in Fig. 4B. Cryo-TEM of a concentrated sample of NCs indicated the presence of mostly-spherical nanoparticles approximately lOOnm in diameter, Fig. 4C.Table 2

[0078] Table 3 shows the dosing schedule for PCL-b-PEG-TNP NPs prepared with 0%, 10%, and 100% of surface polymer chains conjugated with TNP.Table 3The immunogenicity assay of ovalbumin- TNP (OVA-TNP) nanoparticle conjugate vaccines Method:

[0079] Young adult female BALB / c mice (8-10-week-old) were intramuscularly immunized with an OVA-TNP conjugate vaccine or OVA-containing nanoparticles with PCL-b-PEG-TNP-surface coverage that contained 0%, 1%, 10%, 50%, or 100% of surface polymer chains terminated with TNP. Each vaccine was normalized to contain 10 pg of OVA and 10 pg of MPL as an adjuvant. Mice were immunized on experimental days 0 and 14. Each vaccine dose was administered in 50 pL volumes. Day 0 immunizations were performed in the right hind leg, and day 14 was administered in the left hind leg. Serum was collected via submandibular vein punctures on day 35. The day 35 serum samples were measured for vaccine-induced OVA-TNP-, OVA-, and TNP- specific antibodies by ELISA.Results:

[0080] Monitored was vaccine-induced antibodies against the OVA-TNP protein conjugate antigen as an ELISA positive control because this antigen was utilized as the vaccine immunogen in the positive control immunization group (OVA-TNP conjugate vaccine). OVA-TNP antigen is constructed to contain ovalbumin as a protein carrier molecule that is chemically conjugated to the TNP hapten. It was expected that mice immunized with the OVA-TNP antigen to develop antibodies that recognize OVA-TNP and validate that this immunization and ELISA techniques are effective. Utilizing the OVA-TNP antigen in ELISA assays allowed the detection of antibodies that recognize the TNP hapten and the OVA carrier protein but did not allow for measuring antibodies specific for OVA or TNP alone. Therefore, unconjugated OVA and TNP-BSA as ELISA antigens were also included to measure OVA-specific and TNP-specific antibodies, respectively. Utilizing unconjugated OVA as an ELISA antigen ensured that we measured only antibodies that bind OVA without measuring antibodies that bind TNP. Likewise, TNP-BSA conjugate antigen was utilized to measure only TNP-specific antibodies without antibodies that recognize OVA. The TNP-BSA antigen is another TNP-conjugate antigen that combines the bovine serum albumin protein with TNP. The mice in this experiment have been immunized with vaccines containing TNP but not BSA. Therefore, the resulting antibody response detected using the TNP-BSA antigen ensures that the response to TNP was solely measured. The vaccine-inducedantibody responses against OVA-TNP, unconjugated OVA, and TNP-BSA (see Fig. 3) are listed below. a) Immunization with MPL-adjuvanted OVA-TNP protein conjugate vaccines induced elevated serum OVA-TNP- and TNP-specific IgG antibodies but did not induce detectable OVA-specific IgG antibodies. b) Mice immunized with 0% TNP and 1% TNP vaccines developed serum OVA-TNP- and OVA- specific serum IgG responses but did not demonstrate detectable TNP-specific serum IgG responses. c) Increasing the surface coverage of TNP to 10%, 50%, or 100% led to the induction of elevated serum OVA-TNP-, OVA-, and TNP-specific IgG antibodies in mice that received two IM immunizations with the OVA-TNP nanoparticle conjugate vaccines. d) OVA- TNP-specific serum IgG levels appear comparable between all vaccine groups. e) All mice immunized with OVA-encapsulated nanoparticles develop elevated OVA-specific serum IgG responses. Mice immunized with OVA-TNP protein conjugated vaccine do not develop detectable OVA-specific serum IgG. f) TNP-specific serum IgG antibodies levels are TNP-concentration dependent.

[0081] Mice were vaccinated by the intramuscular route on days 0 and 14 with different TNP-NP formulations that include different concentrations of TNP on their surface and free soluble ovalbumin as an antigen to provide T cell help. On Day 28, blood was collected, and serum tested for the presence of anti-TNP IgG by ELISA, as shown in Fig. 5. Kruskal-Wallis nonparametric test with Dunn’s multiple comparison post-test was used to determine differences in anti-TNP serum IgG compared to 0% TNP-NP, * p < 0.05, suggesting that FNP NPs may be an effective carrier for haptens.

[0082] Female BALB / c mice received two IM immunizations with MPL-adjuvanted OVA-TNP protein conjugated vaccines or MPL-adjuvanted OVA-encapsulated FNP-prepared nanoparticles with various densities of TNP surface coverage. Serum collected on Day 35 was measured for OVA-TNP-, TNP-, and OVA-specific IgG by ELISA, as shown in Figs. 6 and 7. A nonparametric Kruskal -Wallis test with the Dunn’s multiple comparison post-test was used to compare vaccine- induced antigen-specific serum IgG responses to mice immunized with OVA-TNP protein conjugate. **: p < 0.01, *: p < 0.05.

[0083] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

[0084] The term "about" can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.

[0085] The term "substantially" can allow for a degree of variability in a value or range, for example, within 90%, within 95%, or within 99% of a stated value or of a stated limit of a range.

[0086] The terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. In addition, the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section. The terms "including" and "having" are defined as comprising (i.e., open language).

[0087] It will be appreciated by persons skilled in the art that the present disclosure is not limited by what has been particularly shown and described herein above. Rather the scope of the present disclosure includes both combinations and sub-combinations of the various features described hereinabove as well as variations and modifications which would occur to persons skilled in the art upon reading the specification and which are not in the prior art.

[0088] Patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification are indicative of the level of skill of those in the art to which the disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

Claims

WE CLAIM:

1. A nanoparticle vaccine composition comprising:(i) a shell comprising (a) one or more amphiphilic molecules and (b) one or more hapten molecules, wherein at least one amphiphilic molecule is conjugated to the hapten molecule; and(ii) a substantially hydrophobic core comprising (a) an immunogenic protein, (b) a vaccine adjuvant, (c) one or more inert biocompatible materials, or (d) a combination of two or more thereof, wherein the core is encapsulated in the shell to form a nanoparticle.

2. The nanoparticle vaccine composition of claim 1, wherein one or more hapten molecule is selected from dinitrophenol, trinitrophenol (e.g., 2,4,6-trinitrophenol (TNP)), morphine, heroin, fentanyl, cocaine, malathion, sarin, methyl phosphonic acid, p-aminophenyl 1,2,2-trimethylpropyl diester, dimethyl methylphosphonate, phosphocholine, diisopropyl methylphosphonate, diethyl methylthiophosphonate, diethyl phenylthiophosphonate, diethyl phenylphosphorothioate, phenyl diphenylphosphinothioate, diethyl 3- (dimethylamino)propylphosphonate, diethyl chlorophosphonate, diisopropyl fluorophosphonate, diethyl cyanophosphonate, isopropyldodecylflouro phosphonate, phenylthiophosphonic acid O-ethyl S-[2- (diisopropylamino)ethyl] ester (PhX), pinacolylmethylphosphonic acid, diethyl N,N- diethylphosphoramidate, dicyclohexyl methylphosphonate, ethyl N,N- diethylphosphoramidocyanidate, chloroethyl phenylsulfide, bis(2-chloroethyl)sulfide, p- nitrophenyl diphenylphosphate, tabun, , soman GD, cyclosarin GF, VX, R-VX, parathion, parathion-methyl, paraoxon, diazinon, 4-nitrophenol, diethylchloro-thiophosphate, triazophos, fenthion, fenitrothion, chlorpyrifos, chlorpyrifos-methyl, atrazine, coumaphos, cyanophos, demeton, dichlorovos, dioxathion, glyphosate, fonofos, malaoxon, malathion, methamidophos, mevinphos, and oxydemeton-methyl.

3. The nanoparticle vaccine composition of claiml, wherein the vaccine adjuvant is a ligand that activates receptors of the innate immune system such as, but not limited to, toll-like receptor 4 ligands (monophosphoryl lipid A; MPLA) toll-like receptor 5 ligands (flagellin); toll-like receptor9 ligands (CpG oligodeoxynucleotides); toll-like receptor 2 / 1 ligands (lipopeptide PAM3CSK4); toll-like receptor 7 / 8 ligands (imidazoquinoline); toll-like receptors 3 ligands (Polyinosine- polycytidylic acid); stimulator of interferon genes, (STING) receptor ligands (cyclic dinucleotide 2’3’-cGAMP); C-type lectin ligands (Trehalose-6,6-dibehenate or trehalos 6,6’ dimycolate); compounds that inhibit immune inhibitory pathways (small molecule NF-%kappaB inhibitors), or hydrophobic salts thereof.

4. The nanoparticle vaccine composition of claim 1, wherein the immunogenic protein is a protein that induces an immune response such as, but not limited to, cRM197 (a genetically detoxified diphtheria toxin), genetically detoxified tenaus toxin, tetanus toxin heavy chain, influenza hemagglutinin, respiratory syncytial virus recombinant proteins (preF A and preF B recombinant proteins), other immunogenic proteins used in vaccines approved for human use, or hydrophobic salts thereof.

5. The nanoparticle vaccine composition of claim 2, wherein the hapten molecule is morphine, heroin, fentanyl, cocaine, or chloropyrifos.

6. The nanoparticle vaccine composition of claim 1, wherein one or more amphiphilic molecule is selected from a diblock polymer, an amphiphilic lipid, a surfactant, a polymer, a polymer-lipid and a combination of two or more thereof.

7. The nanoparticle vaccine composition of claim 6, wherein the diblock polymer comprises (a) at least one hydrophobic block selected from polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), polystyrene (PS), and polylactic acid (PLA); (b) at least one hydrophilic block selected from polyethylene glycol (PEG), polyacrylic acid (PAA), polylysine, and poly[2 (dimethylamino)ethyl methacrylate] (DMAEMA); or (c) a combination of (a) and (b).

8. The nanoparticle vaccine composition of claim 7, wherein the diblock polymer is poly(caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG).

9. The nanoparticle vaccine composition of claim 6, wherein the amphiphilic molecule is selected from tocopherol polyethylene glycol succinate, methoxypoly(ethylene glycol)ditetradecylacetamide (ALC-01 9), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), and a combination of two or more thereof.

10. The nanoparticle vaccine composition of any of claims 6-8, wherein the diblock polymer conjugated to hapten molecule on the shell is in a percentage of about 1% to about 100%.

11. The nanoparticle vaccine composition of any of claims 6-8, wherein the hapten molecule is conjugated to a hydrophilic section of the diblock polymer.

12. The nanoparticle vaccine composition of any claim 1, wherein the inert biocompatible material is a bulking agent comprising one or more of PCL, PLGA, Vitamin E, Vitamin E acetate, and PLA.

13. The nanoparticle vaccine composition of claim 1, wherein the vaccine adjuvant is a toll-like receptor 4 ligand (monophosphoryl lipid A; MPLA) toll-like receptor 5 ligand (flagellin); toll-like receptor 9 ligand (CpG oligodeoxynucleotides); toll-like receptor 2 / 1 ligand (lipopeptide PAM3CSK4); toll-like receptor 7 / 8 ligand (imidazoquinoline); toll-like receptors 3 ligand (Polyinosine-polycytidylic acid).

14. The nanoparticle vaccine composition of claim 1, wherein the immunogenic protein is selected from ovalbumin, CRM- 197, cholera toxin, keyhole limpet hemocyanin, or a salt of any of foregoing.

15. The nanoparticle vaccine composition of claim 1, wherein the immunogenic protein is ovalbumin or a hydrophobic salt thereof.

16. A pharmaceutical composition comprising the nanoparticle vaccine composition of any one of claims 1-6 and a pharmaceutically acceptable carrier, excipient, or diluent.

17. A method of inducing an immune response in a patient in need thereof, which method comprises administering to the patient a therapeutically effective amount of a nanoparticle vaccinecomposition of any one of claims 1-9 or a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient, or diluent.

18. The method of claim 17, wherein the nanoparticle vaccine composition is administered intramuscularly, intravenously, subcutaneously, intraperitoneally, intraocularly, intrathecally, intranasally, or intratracheal ly. Or sublingually or oral19. A method of preventing or treating an opioid or other drug use disorder by administering to a patient in need thereof a prophylactically or therapeutically effective amount of a nanoparticle vaccine composition of any one of claims 1-9 or a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient, or diluent .

20. A method of preparing a nanoparticle vaccine composition, which method comprises:(i) dissolving one or more components selected from (a) one or more amphiphilic molecules and (b) one or more hapten molecules, wherein at least one amphiphilic molecule is conjugated to the hapten molecule in an organic solvent to obtain an organic feed solution;(ii) mixing homogeneously the organic feed solution of step (i) rapidly with an aqueous feed solution, which contains (a) an immunogenic protein, (b) a vaccine adjuvant, (c) one or more inert biocompatible materials, or (d) a combination of two or more thereof, which leads to the rapid precipitation of the components from the organic feed solution; and(iii) allowing particles to assemble instantly by Flash NanoPrecipitation; whereupon the nanoparticle vaccine is formed.

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