Development of broad-spectrum Anti-opioid vaccines

Abstract

Provided herein are vaccine compositions comprising a hapten-conjugated to a capsid, wherein the capsid comprises wild type or native sequence, or at least one mutation. Also provided herein are vaccine compositions comprising a hapten conjugated to a capsid, wherein said capsid comprises at least one mutation, such as a non-natural mutation. Such compositions are useful in the treatment and / or prevention of opioid use disorders (OUDs), including opioid addiction and opioid overdose across a broad spectrum of opioids.

Description

[0001] DEVELOPMENT OF BROAD-SPECTRUM ANTI-OPIOID VACCINES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application serial number 63 / 601,913, filed November 22, 2023, and U.S. Provisional Patent Application serial number 63 / 617,555, filed January 4, 2024, the contents of each of which are incorporated by reference in their entirety.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under grant Al 146210 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] The problematic pattern of opioid use, otherwise referred to as opioid use disorder (OUD), has become a public health crisis. Serious consequences of OUD include disability, relapse, and death. On average there are over 250 deaths per day due to drug overdose, corresponding to nearly 100,000 death per year in the United States.1The number of deaths increased 50% since 2019, to a record high in 2022, taking tremendous social and economic tolls on families and society as a whole.2'5

[0008] A key approach to combat chronic OUD is medication-assisted treatment (MAT).6, 7Pharmacotherapies, such as, methadone, buprenorphine, and naltrexone, are crucial in the treatment of individuals recovering from OUD.8'11Unfortunately, these therapies have significant limitations including low patient compliance and adherence to medication, and poor retention in treatment regimens, especially in connection with the opioid antagonist naltrexone.12 13Consequently, there are substantial chances for relapse even with existing interventions and therapies.14, 15In addition, adverse events have been associated with longterm MAT, including significant hormonal effects as such medications enter into the brain to exert their biological activities.16The unintended consequences of such hormonal perturbations include growth retardation, affective instability, and increased fertility in adolescent girls. The significant increase in opioid overdose death in the past few years highlights the urgent need for novel treatment / prevention strategies beyond the current pharmacotherapies to stem the tide of OUD. An attractive complementary approach to address OUD is the development of antiopioid vaccines.17'21An effective vaccine would produce circulating antibodies that can bind with the opioid, thus lowering the free drug concentration in the blood and reducing the amount of drug that can enter the brain to interact with the p-opioid receptors (pORs). This sequestration of opioid can help reduce rewarding stimuli and mitigate the drugs addictive properties, lowering the chance of relapse for people recovering from OUD.22'24Most antibodies do not readily enter the brain or the endocrine system due to their large molecular weights (~ 150 kDa for IgG). Thus, in contrast to current pharmacotherapies (e.g., MATs) concerns of side effects are alleviated. Furthermore, vaccines can be administered by intramuscular injection in a clinic or physician’s office in a short period of time, representing a cost-effective and more readily-adhered-to therapy.

[0009] However, because opioid drugs are small molecules and are T-cell-independent antigens, they do not generate sufficient immune responses when administered alone. A general approach to overcome this hurdle in vaccine design is to link a drug-derived hapten to an immunogenic carrier.17'21The hapten-carrier conjugate can activate the B cells more efficiently compared to the hapten alone and produce anti-hapten IgG antibodies aided by the helper T (Th) cell epitopes from the carrier moiety. Several anti- addiction vaccines, including those against nicotine and cocaine, have been tested in human clinical trials.25'28While these vaccines did not show significant protection in the full subject cohort, proof of efficacy was observed in those who achieved the highest antibody titers.25'27These results highlight the need to design vaccines that are capable of producing high levels of antibodies.

[0010] BRIEF SUMMARY

[0011] Provided herein are vaccine compositions comprising a hapten conjugated to a capsid. In some embodiments, the vaccine composition comprises a hapten conjugated to a wild-type bacteriophage capsid. In some embodiments, the vaccine composition comprises a hapten conjugated to a wild type bacteriophage QP capsid. In some embodiments, the vaccine composition comprises an hapten conjugated to a bacteriophage QP capsid having a wild type or native sequence. In some embodiments, the vaccine composition comprises an hapten conjugated to a bacteriophage QP capsid having a wild type or natural sequence set forth in SEQ ID NO: 1. In some embodiments, the vaccine composition comprises a hapten conjugated to a capsid having at least one mutation relative to the wild-type capsid. In some embodiments, the vaccine composition comprises a hapten conjugated to a bacteriophage QP capsid having at least one mutation relative to the wild-type bacteriophage QP capsid. In some embodiments, the at least one mutation comprises a non-natural mutation. In some embodiments, the non-natural mutation comprises a non-natural amino acid mutation. In some embodiments, the non-natural mutations comprise a disulfide bond mutation. In some embodiments, the vaccine composition comprises a hapten conjugated to a bacteriophage QP capsid having at least three mutations. In some preferred embodiments, the at least three mutations are selected from A38K / A40C / D102C.

[0012] In some aspects of the invention, provided herein are vaccine compositions comprising a hapten conjugated to a capsid wherein the conjugated capsid comprises the formula: (X-Y)n-Z wherein X is a hapten; wherein Y is a linker; wherein Z is the capsid; and wherein n is at least 1.

[0013] In some embodiments, X is 6-AmHap or para-AmEtFenHap. In some such embodiments, Y is a polyethylene glycol (PEG) linker. In some such embodiments, the polyethylene glycol (PEG) linker comprises 1-20 ethylene glycol units. Preferably, the polyethylene glycol (PEG) linker comprises 2 ethylene glycol units. In other preferred embodiments, the polyethylene glycol (PEG) linker comprises 12 ethylene glycol units. In some embodiments n is 1-1000. More preferably, n is at least 100-700, 150-650, 200-600, or any intervening integer or rational number. Accordingly, n may be 1, 1.5, 2, 2.5, ...50, 50.5, 51, 50.5, ...100, 100.5, 101, 101.5, ...600, 600.5, 601, 601.5, ...999, 999.5, Or 1,000.

[0014] In some embodiments, Z is a bacteriophage QP capsid comprising at least three mutations selected from A38K / A40C / D102C.

[0015] In certain embodiments, X is 6-AmHap, Y is a polyethylene glycol (PEG) linker comprising 2 ethylene glycol units, and n is 200-650. In certain embodiments, X is para- AmEtFenHap, Y is a polyethylene glycol (PEG) linker comprising 12 ethylene glycol units, and n is 500-600.

[0016] In some embodiments, the vaccine compositions contemplated herein are monovalent. For example, such monovalent vaccines comprise a capsid conjugated to a plurality of a hapten contemplated herein. Alternatively, the vaccine compositions contemplated herein are multivalent, e.g., the vaccine comprises capsids that are conjugated with different haptens. Preferably, the vaccine is bivalent. Accordingly, in some aspects of the invention, provided herein is a bivalent vaccine composition comprising two conjugated capsids respectively comprising the formulas: (X^Y^n-Z : Formula I (X2-Y2)m-Z : Formula II wherein X1is 6-AmHap; wherein Y1is a is a polyethylene glycol (PEG) linker comprising 2 ethylene glycol units; wherein n is at least 1-1000; wherein X2is para-AmEtFenHap;

[0017] Y2is a polyethylene glycol (PEG) linker comprising 12 ethylene glycol units; wherein m is at least 1-1000; and wherein wherein Z is a capsid.

[0018] More preferably, n and / or m is at least 100-700, 150-650, 200-600, or any intervening integer or rational number. Accordingly, n and / or m may be 1, 1.5, 2, 2.5, ...50, 50.5, 51, 50.5, ...100, 100.5, 101, 101.5, ...600, 600.5, 601, 601.5, ...999, 999.5, or 1,000. In some embodiments, n is 200-650. In some embodiments, m is 500-600.

[0019] In some embodiments, the capsid of the bivalent vaccine composition comprises at least one, two, or three non-natural mutations. The at least one, two, or three non-natural mutations may comprise a disulfide bond mutation. In preferred embodiments the capsid is derived from bacteriophage Qp. More preferably, the capsid comprises at least three mutations selected from A40C / D102C / K13R or A38K / A40C / D102C. In some such embodiments, the capsid comprises at least three mutations selected from A38K / A40C / D102C.

[0020] In some embodiments, the vaccine compositions contemplated herein may further comprise an adjuvant. Such adjuvants may include adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, P-Glucan Peptide, CpG DNA, GPI- 0100, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D- isoglutamine, Pam3CSK4, quil A, Momordica saponin derivative II (VSA-2), monophosphoryl Lipid A (MPLA), double mutant heat-labile toxin (dmLT), Monophosphoryl-Lipid A (MPLA), Army Liposome Formulation (ALF), ALF adsorbed to aluminum hydroxide (ALFA), ALF containing QS21 saponin (ALFQ), or trehalose dimycolate. In some embodiments, the adjuvant is VSA-2, MPLA, dmLT, MPLA in ALFA, or ALFQ.

[0021] In some aspects, provided herein are methods of treating or preventing opioid addiction, comprising administering to a subject in need thereof a vaccine composition (e.g., a monovalent, bivalent, or multivalent vaccine composition) contemplated herein. The opioid addiction may be addiction to heroin, fentanyl, morphine, benzimidazole-derived new psychoactive substance opioids (BNO), codeine, oxycodone, hydrocodone, analogues and / or metabolites thereof, or any combination thereof.

[0022] Aspects of the invention contemplated herein include methods of treating or preventing an opioid overdose, comprising administering to a subject in need thereof a vaccine composition (e.g., a monovalent, bivalent, or multivalent vaccine composition) contemplated herein. The opioid overdose may be an overdose of heroin, fentanyl, morphine, BNO, codeine, oxycodone, hydrocodone, analogues and / or metabolites thereof, or any combination thereof.

[0023] In some aspects, provided herein are methods of preventing or reducing the analgesic effect of an opioid in a subject, comprising administering to a subject in need thereof a vaccine composition (e.g., a monovalent, bivalent, or multivalent vaccine composition) contemplated herein. The analgesic effect may be due to an opioid selected from heroin, fentanyl, morphine, BNO, codeine, oxycodone, hydrocodone, analogues and / or metabolites thereof, or any combination thereof.

[0024] Based on the disclosure provided herein, it will be appreciated that in the various aspects of the invention an individual hapten conjugate is effective against a plurality of opioids. Thus, aspects of the invention include a broad spectrum opioid vaccine suitable for the methods disclosed herein.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Fig. 1 depicts the structures of heroin, 6-acetyl morphine, morphine, and 6-AmHap.

[0027] Fig. 2 illustrates the experimental design for assessing efficacy of anti-opioid vaccine.

[0028] Fig. 3 depicts Scheme 1, a schematic representation of the synthesis of (A) mQP-6-AmHap; (B) TT-6-AmHap; and (C) BSA-6-AmHap conjugates.

[0029] Fig. 4 depicts high-resolution mass spectrometry (HRMS) spectra for (A) mQP; (B) mQP-6- AmHap vaccine conjugate; and (C) the Matrix- Assisted Laser Desorption Ionization Thne-of- Flight Mass Spectroscopy (MALDLTOF MS) spectrum of the BSA-6-AmHap conjugate.

[0030] Fig. 5 depicts vaccine induced anti-6-AmHap serum IgG antibody levels for (A) C57BL / 6 mice and (B) BALB / C mice immunized with mQP-6-AmHap, TT-6-AmHap, mQp / 6-AmHap admix or mQP only on day 35 post prime immunization. Panel (C) shows the persistent antibody responses with significant IgG titers that remain 600 days after the prime immunization with mQP-6-AmHap. Arrows indicate days of immunization (prime injection VI on day 0 with boosters V2 and V3 on days 14 and 28). Statistical analysis was performed via a two tailed student t test. * p < 0.05; **** p < 0.0001.

[0031] Fig. 6 shows the titers of anti-6-AmHap IgG subtypes from mice vaccinated with mQP-6- AmHap with (A) MPLA; (B) ALFA; (C) dmLT; (D) VSA-2 adjuvant, or (E) without an adjuvant.

[0032] Fig. 7 depicts (A) Anti-6-AmHap IgG titer values induced by the mQP-6-AmHap vaccine formulated with various adjuvants or without any adjuvant on day 35. Each symbol represents an individual mouse serum (n=5) with the group mean indicated by the horizontal line. Statistical analysis was performed via student t test. * p < 0.05. Panel (B) depicts the assessment of antibody persistence induced by mQP-6-AmHap with pooled serum from each group. All mice received subcutaneous injections of mQP-6-AmHap with various adjuvants on days 0, 14, and 28. Another group of mice (n = 5) received injections of mQP-6-AmHap without any adjuvant.

[0033] Fig. 8 depicts the average body weight of mice following vaccination.

[0034] Fig. 9 depicts the chemical structures of the drugs used in the competition ELISA.

[0035] Fig. 10 illustrates (A) the study design for testing mQP-6-AmHap vaccine in mice, depicting the timeline and procedures conducted during the experiment. Groups of C57B16 mice (n = 10 each) were fully immunized with mQP-6-AmHap + MPLA (adjuvant) or admix of mQp / 6- AmHap + MPLA (adjuvant) respectively weeks 0, 2, and 4. Blood samples were collected at weeks -1, 1, 3, and 5. The mice were then challenged with morphine (10 mg / kg) at weeks 15 and 16. Vaccine efficacy was assessed using the locomotion and hot plate anti-nociception assays. At week 60, mice were euthanized, and blood and brain samples were collected by cardiac puncture. Locomotion assays for (B) ambulation counts, and (C) total distance were performed at week 15. One week after the hyperlocomotion assay (week 16), the mice were subjected to (D) the hot plate anti-nociception assay. Each symbol in the figure represents one mouse. The ratio of morphine concentrations in brain vs blood, 30 minutes after intraperitoneal injection of morphine, is shown in panel (E). The values presented are the mean ± SEM of 10 mice per group. Lor (B) and (C), a two-way repeated measure ANOVA was completed (independent variables were vaccine and drug (morphine) treatment). This was followed by Post-hoc Lisher’s LSD test for statistical significances, ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Lor (D) and (E), statistical analyses were performed by paired student t test. * p < 0.05; **** p < 0.0001. Fig. 11 depicts the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of mQP(A38K, A40C, D102C), displaying Marker ladder in lane 1 and mQP(A38K, A40C, D102C) in lane 2 for monomer (14kDa). Fast protein liquid chromatography (FPLC) analysis of mQP(A38K, A40C, D102C). It was performed on a GE AKTA Explorer (Amersham Pharmacia) instrument equipped with a Superose-6 column.

[0036] Fig. 12 depicts Scheme 2, a schematic representation of the synthesis of (A) mQP-6-AmHap; (B) mQP-para-AmEtFenHap; (C) TT-para-AmEtFenHap; and (D) BSA-para-AmEtFenHap conjugates. Chemical structures of haptens para-AmEtFenHap and 6-AmHap are depicted in (E) and (F), respectively.

[0037] Fig. 13 depicts the vaccine induced anti-hapten serum IgG antibody levels for (A) anti-para- AmEtFenHap, (B) anti-6-AmHap and (C) anti-BNO mice immunized with mQP-para- AmEtFenHap , 2R mQB-para-AmEtFenHap , TT- para-AmEtFenHap, bivalent vaccine (mQP-6-AmHap + mQP-para-AmEtFenHap ) and saline on day 35 post prime immunization. ELISA was performed using BSA-para-AmEtFenHap, BSA-6-AmHap and BSA-BNO as the coating antigens. (D) mQP-conjugated immunization induced persistent antibody responses with significant IgG titers remaining 30 weeks after the prime immunization. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA, followed by Tukey’s post hoc test for multiple comparisons, p < 0.05 was considered statistically significant. Differences between groups are indicated by asterisks (*), where p < 0.05 is noted as *, p < 0.01 as **, p < 0.001 as *** and p < 0.0001 as ****.

[0038] Fig. 14 depicts the evaluation of hyperlocomotion in mice administrated with target and other opioids performed similarly to the study design for testing mQP-6-AmHap vaccine in mice described herein. At weeks 12, 14, 16 and 26, mice were challenged with (A) fentanyl (0.3mg / kg), (B) heroin mixture with fentanyl ( 2 mg / kg+9% fentanyl w / w ), (C) heroin ( 2 mg / kg) and (D) etonitazene ( 0.075mg / kg) to evaluate vaccine efficacy via locomotion assays. Data are expressed as mean ± SEM for 10 mice per group. Statistical analyses were performed using two-way repeated measures ANOVA [with vaccine and drug treatment as independent variables], followed by a Fisher’s LSD posthoc test, ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0039] Fig. 15 depicts the thermal antinociception analysis of mice administered different opioids. Following baseline nociception assessment, mice received doses of (A) fentanyl (0.3mg / kg), (B) heroin mixture with fentanyl (2 mg / kg+9% fentanyl w / w ), (C) heroin ( 2 mg / kg) and (D) etonitazene ( 0.075mg / kg) with nociceptive latency measured 15 minutes using hot-plate test. Bars indicating means ± SEM across all plots. Sample sizes: n = 10 per group. Statistical significance, derived from two-way repeated measures ANOVA [with vaccine and drug treatment as independent variables], followed by a Fisher’s LSD posthoc test, ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0040] DETAILED DESCRIPTION

[0041] General

[0042] Vaccination can provide an attractive complementary strategy to combat OUD. A key factor in enhancing vaccine efficacy is to induce high levels of antibodies specific against the drug of abuse. For example, in the body, the O-acetyl moieties of heroin are quickly hydrolyzed, converting into metabolites, such as, 6-acetyl morphine (6-Am) and morphine (Fig. 1) respectively.29Existing vaccines utilizing haptens based on various derivatives of morphine / heroin, have been reported to generate antibodies that can target morphine, 6-AM, as well as heroin.9’30'37Without being bound by theory or methodology, a hydrolytically more stable heroin mimetic, 6-AmHap, has been developed and conjugated to tetanus toxoid (TT), which, according to Sulima et al., has shown promising results in preclinical studies.38However, the use of such protein carriers comes with drawbacks. The hapten-conjugate may induce high anti-carrier antibody responses which may suppress the generation of the haptenspecific antibodies. Such carriers typically have limited surface area. Thus, antigen presentation tends to be less dense and ordered, qualities that are relevant to crosslinking B cell receptors and potent B cell activation.

[0043] Virus-like particles (e.g., caspids) have emerged as effective immunogenic carriers for vaccine design. By way of example and without being bound by theory, the virus-like particle (VLP) of bacteriophage Qbeta (Qubevirus durum), also referred to as Qbeta or QP, is capable of eliciting strong IgG antibody responses when used as an antigen carrier. However, QP can also induce significant anti-carrier antibodies. Provided herein are mutants of QP (mQP), which generate much lower levels of antibodies against the carrier itself and are used as carriers for the haptens contemplated herein.

[0044] Another challenge in prevention of opioid misuse related death through vaccines is that the illicit opioids taken are often mixtures of multiple compounds. With the dissimilar chemical structures of fentanyl, benzimidazole-derived new psychoactive substance (NPS) opioids (BNO also referred to as nitazene), and heroin, it has been generally accepted that tackling multiple opioids such as heroin and fentanyl at the same time will likely require the development of a combination vaccine (e.g., multivalent vaccine) containing various haptens, each resembling the particular opioid to be targeted. While combination vaccines targeting multiple protein or polysaccharide antigens have been widely used in infectious disease prevention, applying this approach to small molecules like opioids is relatively new. The unique challenges of creating vaccines for drugs of abuse lie in the complex chemistry needed to create these molecules and the potential competition of multiple constructs to interact with the immune system. Thus, there is a long-felt and unmet need for a vaccine that provides protection and / or therapeutic effect against a plurality of opioids, or a broad spectrum of opioids. The complexity in manufacturing the multiple conjugates needed is another significant drawback of this approach. Provided herein is a fentanyl hapten (para- AmEtFenHap), which when conjugated to a carrier, such as, tetanus toxoid (TT) or bacteriophage mQP, can induce high levels and long-lasting antibodies that are effective in reducing the effects of multiple classes of opioid drugs of abuse including fentanyl, heroin and nitazene.

[0045] Accordingly, disclosed herein, for the first time, is the generation of anti-opioid vaccines that display antigen on a well-organized VLP mutant bacteriophage QP (mQP) with significantly reduced anti-QP antibody responses, and eliciting superior anti-opioid antibody responses. The mQP-hapten conjugates (e.g., mQP-6-AmHap and mQP para-AmEtFenHap) were able to induce significantly higher levels of IgG antibodies against the target hapten than mice immunized with a corresponding tetanus toxoid-hapten conjugate in head-to-head comparison studies in multiple strains of mice. The IgG antibody responses were persistent with high titers 600 days after immunization. These antibodies exhibited strong binding towards multiple heroin / morphine derivatives that have the potential to be abused, while binding weakly to medications used for OUD treatment and pain relief. Furthermore, vaccination effectively reduced the impacts of morphine on mice in both ambulation and antinociception assays, highlighting the translational potential of the mQP-hapten conjugate to mitigate the harmful effects of drugs of abuse.

[0046] Definitions

[0047] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0048] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. The term “administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.

[0049] As used herein, the term “subject” or “recipient” means a human or non-human animal selected for treatment or therapy. As such, a “subject” or “recipient” can include a human subject for medical purposes, such as for the treatment of an existing disease, disorder, condition or the prophylactic treatment for preventing the onset of a disease, disorder, or condition or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, guinea pigs, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with, suspected of being afflicted with, or at risk of being afflicted with a disease, disorder, or condition. Thus, the terms “subject” and “patient” are used interchangeably herein. Subjects also include animal disease models (e.g., rats or mice used in experiments, and the like).

[0050] The term “subject in need thereof’ means a subject identified as in need of a therapy or treatment.

[0051] The phrases “therapeutically effective amount” and “effective amount” as used herein means the amount of an agent compound, material, or composition which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit / risk ratio applicable to any medical treatment.

[0052] “Treating” a disease in a subject or “treating” a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug and / or a vaccine composition as described herein, such that at least one symptom of the disease is decreased or prevented from worsening.

[0053] As used herein, “bacteriophage” refers to viruses that infect and replicate within bacterium. In certain embodiments, the bacteriophage is selected from, but not limited to, the group consisting of bacteriophage QP; (b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage Mi l; (h) bacteriophage MX1; (i) bacteriophage NL95; (j) bacteriophage f2; (k) bacteriophage PP7; (1) bacteriophage AP205; and (m) bacteriophage P22. As used herein, “bacteriophage QP” (also referred to as “bacteriophage Qb”,“QP” and “Qbeta”) is one of many small RNA bacteriophages infecting Escherichia coli.

[0054] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Thus, “pharmaceutically- acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or poly anhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. “Pharmaceutically acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds.

[0055] Capsids

[0056] Provided herein are vaccine compositions comprising an opioid-associated antigen (e.g., a hapten) conjugated to a capsid (e.g., a virus-like particle (VLP)). In some embodiments, the vaccine composition comprises a hapten conjugated to a wild-type capsid or a mutant capsid. In some embodiments, the vaccine composition comprises a hapten conjugated to a wild type bacteriophage QP capsid or mutant QP (mQP). In some embodiments, the vaccine composition comprises a hapten conjugated to a bacteriophage QP capsid having a wild type or native sequence. In some embodiments, the vaccine composition comprises a hapten conjugated to a bacteriophage QP capsid having a wild type or natural sequence set forth in SEQ ID NO: 2 and understood in SEQ ID NO: 1. In some embodiments, the vaccine composition comprises a hapten conjugated to a capsid having at least one mutation from the wild type capsid. In some embodiments, the vaccine composition comprises a hapten conjugated to a bacteriophage QP capsid having at least one mutation from the wild type bacteriophage QP capsid. In some embodiments, the at least one mutation comprises a non-natural mutation. In some embodiments, the non-natural mutation comprises a non-natural amino acid mutation. In some embodiments, the vaccine compositions provided herein comprises an antigen (e.g., T-cell independent antigens including haptens, small molecules, and opioid mimics) conjugated to a capsid (e.g., bacteriophage QP), wherein said capsid comprises at least one mutation (e.g., at least one point mutation, or at least one non-natural disulfide bond). In some embodiments, the capsid are fragments or a portion of the capsid amino acid sequence of sufficient length, that when conjugated to the antigen, can elicit an enhanced and strong immune response. In certain embodiments, the capsid polypeptide also includes amino acids that do not correspond to the naturally occurring capsid amino acid sequence (e.g., comprising at least one point mutation, or at least one non-natural disulfide bond mutation, or a fusion protein comprising a capsid amino acid sequence and an amino acid sequence corresponding to a non-capsid protein or polypeptide).

[0057] In some embodiments, the capsid is derived from bacteriophage. In certain embodiments, the bacteriophage is selected from the group consisting of:

[0058] (a) bacteriophage QP;

[0059] (b) bacteriophage R17;

[0060] (c) bacteriophage fr;

[0061] (d) bacteriophage GA;

[0062] (e) bacteriophage SP;

[0063] (f) bacteriophage MS2;

[0064] (g) bacteriophage Mi l;

[0065] (h) bacteriophage MX1;

[0066] (i) bacteriophage NL95;

[0067] (j) bacteriophage f2;

[0068] (k) bacteriophage PP7 ; (l) bacteriophage AP205; and

[0069] (m) bacteriophage P22.

[0070] In some embodiments, the capsid has a sequence set forth in coat protein Table A.

[0071] Table A: Listing of capsid and associated GenBank numbers

[0072]

[0073] In some embodiments, the bacteriophage QP capsid comprises 180 copies of a monomeric coat protein containing 132 amino acid residues derived from the sequence set forth in SEQ ID NO: 1.

[0074] (M)AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKR VTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFT QYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY (SEQ ID NO: 1)

[0075] Notably, it is well understood in the art that the experimentally determined lengths of bacteriophage capsid proteins (e.g., from analysis of mRNA transcript) are always one residue shorter than the actual proteins because the N-terminal methionine is cleaved off in infected E. coli cells. Accordingly, those of skill in the relevant art would appreciate and understand that in the 133-amino acid sequence set forth in SEQ ID NO: 1, amino acid position 1 refers to the alanine that follows the cleaved methionine residue represented in parentheses “(M)”. Thus, the amino acid sequence set forth SEQ ID NO: 2 represents said cleaved monomeric coat protein having 132 amino acid residues, beginning with alanine (A at position 1). Therefore, following this numbering convention, SEQ ID NO: 2 (and as described above, SEQ ID NO: 1) also depicts alanine at position 38 (A38) and position 40, and aspartic acid at position 102 (D102).

[0076] AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKR VTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFT QYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY (SEQ ID NO: 2)

[0077] For exemplary purposes and without being bound by theory, the structure-guided rational design of QP mutants (mQPs) is described in international publication WO 2019045791 and U.S. Patent No. 11,576,957, each of which are hereby incorporated by reference in their entirety. Thus, in some embodiments, the bacteriophage QP capsid comprises at least one mutation set forth in Table B.

[0078] Table B. QP mutants reported that assemble to form the capsid.

[0079] *Yield: “+ + ” = >80 mg / L; “+” = >20-80 mg / L; = <20 mg / L; “na” = not applicable.

[0080] “7a” corresponds to Fiedler, J et al. Biomacromolecules 2012, 13 (8), 2339-2348;

[0081] “42a” corresponds to Prasuhn, D et al. JACS 2008, 130 (4), 1328-1334; “42b” corresponds to Udit, A et al. ChemBioChem 2009, 10 (3), 503-510.

[0082] “43” corresponds to Hovlid, M. L et al. The Scripps Research Institute, La Jolla, 2014.

[0083] In certain embodiments, the QP capsid comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty mutations. In some embodiments, the mutation is a nonnatural disulfide bond mutation. In some embodiments, the capsid comprises, but not limited to, the following mutations (Table C).

[0084] Table C: Listing of bacteriophage QP capsid mutations

[0085] Additional single point mutations may comprise, but not limited to, point mutations at K2, L4, V7, N10, K13, D14, K16, Q18, L20, A38, G40, E46, V67, T75, V85, Q99, E103, Al 17, Pl 19, L122, or D127 set forth in SEQ ID NO: 2 and understood in SEQ ID NO: 1, or combinations thereof.

[0086] Thus, in some preferred embodiments, the mutant bacteriophage capsid comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0087] AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQA'GCVPALEKR VTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFT QYSTCEERAFVRTELAALLASPLLIDAIDQLNPAY (SEQ ID NO: 3)

[0088] In some embodiments, the bacteriophage QP capsid comprises a polypeptide comprising amino acid sequences at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NOs: 1-3. In some embodiments of the methods, compositions, and kits provided herein, the QP mutant has at least one mutation set forth in Table C. In some embodiments, the bacteriophage QP capsid comprises a wild type or native sequence. In some embodiments, the bacteriophage QP capsid comprises a sequence consisting essentially of the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the QP mutant has an amino acid sequence that consists essentially of the mutations set forth in SEQ ID NOs: 2 or 3, or set forth in Table C. In some embodiments, the capsid comprises a polypeptide comprising amino acid sequences at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in the GenBank numbers set forth in Table A.

[0089] In some embodiments of the methods, compositions and kits provided herein, the vaccine composition comprises a capsid having an amino acid sequence that consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130 of SEQ ID Nos: 1-3, or biologically active variant thereof, or combinations thereof, or consecutive amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence for a capsid (e.g., SEQ ID NOs: 1-3, or any of the GenBank numbers set forth in Table A). In some embodiments, the bacteriophage QP capsid comprises a wild type or native sequence. In some embodiments, the bacteriophage QP capsid comprises a sequence consisting essentially of the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0090] As is well-known to those skilled in the art, polypeptides having substantial sequence similarities can cause identical or very similar immune reaction in a host animal. Accordingly, in some embodiments, a derivative, equivalent, variant, fragment, or mutant of the QP capsid, or fragment thereof, can also be suitable for the methods, compositions and kits provided herein.

[0091] In some embodiments, the altered polypeptide may have an altered amino acid sequence, for example by conservative substitution, yet still elicits an enhanced immune response, and are considered functional equivalents. As used herein, the term “conservative substitution” denotes the replacement of an amino acid residue by another, biologically similar residue. It is well known in the art that the amino acids within the same conservative group can typically substitute for one another without substantially affecting the function of a protein. According to certain embodiments, the derivative, equivalents, variants, or mutants of the QP are at least 85% homologous to a sequence set forth in SEQ ID NOs: 1-3, or biologically active variant thereof, or combinations thereof. In some embodiments, the homology is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the bacteriophage QP capsid comprises a wild type or native sequence. In some embodiments, the bacteriophage QP capsid comprises a sequence consisting essentially of the sequence set forth in EQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0092] Vaccine Compositions and Pharmaceutical Compositions / Formulations of Same

[0093] Provided herein are vaccine compositions comprising an opioid-associated antigen (e.g., a hapten) conjugated to a capsid. In some embodiments, the vaccine composition comprises said antigen conjugated to a wild-type capsid. In some embodiments, the vaccine composition comprises said antigen conjugated to a wild-type bacteriophage QP capsid. In some embodiments, the vaccine composition comprises said antigen conjugated to a bacteriophage QP capsid having a wild type or native sequence. In some embodiments, the vaccine composition comprises said antigen conjugated to a bacteriophage QP capsid having a wild type or natural sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the vaccine composition comprises said antigen conjugated to a capsid having at least one mutation from the wild-type capsid. In some embodiments, the vaccine composition comprises said antigen conjugated to a bacteriophage QP capsid having at least one mutation from the wild-type bacteriophage QP capsid. In some embodiments, the at least one mutation comprises a non-natural mutation. In some embodiments, the non-natural mutation comprises a non-natural amino acid mutation. In some embodiments, the vaccine compositions provided herein comprises said antigen (e.g., an opioid-derived hapten, such as and without limitation, haptens derived from heroin, morphine, fentanyl, codeine, dihydrocodeine, oxycodone, hydrocodone, benzimidazole opioids, or any other natural, synthetic, or semi-synthetic opioid-of-abuse known and appreciated by those of skill in the art) conjugated to a capsid (e.g., bacteriophage QP), wherein said capsid comprises at least one mutation (e.g., at least one point mutation, at least one non-natural amino acid mutation, or at least one non-natural disulfide bond mutation). The capsid is described herein, supra. In some embodiments, the hapten are fragments, portions, or derivatives of an opioid (or metabolite thereof), that when conjugated to the capsid with an appropriate linker, can elicit an enhanced and strong immune response. The capsid may be conjugated to a plurality of multiple haptens that are the same or different haptens. In certain embodiments, the hapten is derived from opioids selected from, but not limited to, heroin, morphine, fentanyl, and benzimidazole-derived new psychoactive substance opioids (BNOs).

[0094] In some embodiments, the hapten is conjugated to the capsid as set forth in the Examples, infra. In some embodiments, the vaccine composition is a QP mutant conjugated to 6-AmHap (e.g., Fig. 3A and Fig. 12A). In some embodiments, the vaccine composition is a QP mutant conjugated to para-AmEtFenHap (e.g., Fig. 12B). In other embodiments, the hapten (e.g., 6-AmHap or para-AmEtFenHap) is conjugated to TT.

[0095] Accordingly, aspects of the invention include vaccine compositions comprising a hapten conjugated to a capsid. Such vaccine compositions may comprise a capsid comprising at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty non-natural mutations. The non- natural mutations may comprise at least one disulfide bond mutation. In some embodiments, the capsid is a bacteriophage capsid. In some embodiments, the capsid is derived from a bacteriophage selected from the group consisting of (a): bacteriophage QP; (b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage Mi l; (h) bacteriophage MX1; (i) bacteriophage NL95; (j) bacteriophage f2; (k) bacteriophage PP7; (1) bacteriophage AP205; and (m) bacteriophage P22. In some preferred embodiments, the capsid is derived from bacteriophage Qp.

[0096] In some such embodiments, said capsid comprises at least one mutation selected from N10K, A38K, A40C, A40S, T75K, D102C, D102S, or A117K, or combination thereof. In certain embodiments, said capsid comprises at least two mutations selected from A40C / D102C, A40S / D102S, or A43C / Q98C. In other embodiments, said capsid comprises at least three mutations selected from A40C / D102C / K13R or A38K / A40C / D102C. In certain preferred embodiments, said capsid comprises at least three mutations selected from A38K / A40C / D102C.

[0097] In some embodiments of the invention, the hapten (z.e., the hapten conjugated to the capsid contemplated herein) is a derivative of a natural, synthetic, or semi- synthetic opioid. For example the hapten may be derived from heroin, morphine, fentanyl, codeine, dihydrocodeine, oxycodone, hydrocodone, benzimidazole opioids, analogs thereof, or metabolites thereof. In some such embodiments, the hapten is a derivative of an opioid selected from the group consisting of fentanyl, heroin, morphine, 6-acetylmorphine, and morphine-3-P-glucuronide (M-3G). In some preferred embodiments, the hapten is 6-AmHap. In other preferred embodiments the hapten is para-AmEtFenHap.

[0098] In some aspects of the invention, the conjugated capsid contemplated herein comprises the formula:

[0099] (X-Y)n-Z wherein X is a hapten contemplated herein; wherein Y is a linker; wherein Z is a capsid contemplated; and wherein n is at least 200.

[0100] In some embodiments, Y is a polyethylene glycol (PEG) linker. Said PEG linkers may comprise at least 1-20 ethylene glycol units. Thus, PEG linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene glycol units. In some preferred embodiments, the PEG linker comprises at least 2 ethylene glycol units. In other preferred embodiments the PEG linker comprises at least 12 ethylene glycol units. In some embodiments, Z is a bacteriophage QP capsid comprising at least three mutations selected from A38K / A40C / D102C. Alternatively, in the contemplated embodiments, Z is tetanus toxoid (TT). In some embodiments, X is 6-AmHap or para-AmEtFenHap. In certain embodiments, X is 6-AmHap, Y is a polyethylene glycol (PEG) linker comprising at least 2 ethylene glycol units, and n is at least 200-650. For example, the capsid (Z) may be conjugated to at least 212 hapten-linker units (e.g., (X-Y)2i2). Similarly, in other embodiments, n is at least 620. In other embodiments, X is para-AmEtFenHap, Y is a polyethylene glycol (PEG) linker comprising at least 12 ethylene glycol units, and n is at least 500-600. Thus, in some embodiments the capsid (Z) may be conjugated to at least 540 hapten-linker units (e.g., (X-Y)s4o). In certain embodiments, Z is tetanus toxoid (TT). Thus, X may be 6-AmHap, Y may be a polyethylene glycol (PEG) linker comprising at least 2 ethylene glycol units, and n is at least 30-40. Preferably, n is at least 34. Similarly, X may be para-AmEtFenHap, Y may be a polyethylene glycol (PEG) linker comprising at least 2 ethylene glycol units, and n is at least 30-40. Preferably, n is at least 34. In other embodiments, the vaccine composition comprises a second conjugated capsid comprising the formula:

[0101] (X2-Y2)m-Z wherein X2is a second hapten contemplated herein, Y2is a polyethylene glycol (PEG) linker comprising at least 1-20 ethylene glycol units (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene glycol units), and m is at least 1-1000, including any intervening integer or rational number, such as, 1, 1.5, 2, 2.5, ...50, 50.5, 51, 50.5, ...100, 100.5, 101, 101.5, ...600, 600.5, 601, 601.5, ...999, 999.5, or 1,000; more preferably m is at least 200-650.

[0102] In some aspects of the invention, provided herein is a broad spectrum vaccine composition capable of eliciting an immune response to a plurality of opioids following administration to a subject, said composition comprising a conjugated capsid comprising the formula:

[0103] (X-Y)n-Z wherein X is a hapten; wherein Y is a linker; wherein Z is a capsid or tetanus toxoid (TT) carrier; and wherein n is at least 1-1000.

[0104] More preferably, n is at least 100-700, 150-650, 200-600, or any intervening integer or rational number. Accordingly, n and / or m may be 1, 1.5, 2, 2.5, ...50, 50.5, 51, 50.5, ...100, 100.5, 101, 101.5, ...600, 600.5, 601, 601.5, ...999, 999.5, or 1,000. In some embodiments, n is 200-650. In some embodiments, n is 500-600. In some embodiments, X is 6-AmHap or para-AmEtFenHap. In some embodiments, Y is a polyethylene glycol (PEG) linker. The polyethylene glycol (PEG) linker may comprise 1-20 ethylene glycol units or any intervening integer or rational number. The polyethylene glycol (PEG) linker may comprise 2 ethylene glycol units. The polyethylene glycol (PEG) linker may comprise 12 ethylene glycol units. In some embodiments, Z is a bacteriophage QP capsid comprising at least three mutations selected from A38K / A40C / D102C.

[0105] In some embodiments, X is 6-AmHap, Y is a polyethylene glycol (PEG) linker comprising 2 ethylene glycol units, and n is at least 200-650. In some embodiments, X is para- AmEtFenHap, Y is a polyethylene glycol (PEG) linker comprising 12 ethylene glycol units, and n is at least 500-600. In some embodiments, Z is TT, X is 6-AmHap, Y is a polyethylene glycol (PEG) linker comprising 2 ethylene glycol units, and n is at least 30-40. In some embodiments, Z is TT, X is para-AmEtFenHap, Y is a polyethylene glycol (PEG) linker comprising 2 ethylene glycol units, and n is at least 30-40. In some embodiments, the vaccine composition comprises a monovalent vaccine.

[0106] In some embodiments, the vaccine compositions contemplated herein are monovalent. For example, such monovalent vaccines comprise a capsid conjugated to a plurality of a hapten contemplated herein. Alternatively, the vaccine compositions contemplated herein are multivalent, e.g., the vaccine comprises capsids that are conjugated with different haptens. Without being bound by theory or methodology, such multivalent vaccines comprise an admixture of capsids, each conjugated to a different hapten. Preferably, the vaccine is bivalent. Thus, in some aspects of the invention, provided herein is a bivalent vaccine composition comprising two conjugated capsids, said capsids respectively comprising the formulas: (X^Y^n-Z : Formula I (X2-Y2)m-Z : Formula II wherein X1is 6-AmHap; wherein Y1is a is a PEG linker comprising at least 2 ethylene glycol units; wherein n is at least 200-650; wherein X2is para-AmEtFenHap; wherein Y2is a PEG linker comprising at least 12 ethylene glycol units; wherein m is at least 500-600; and wherein wherein Z is a capsid contemplated herein.

[0107] More preferably, n and / or m is at least 100-700, 150-650, 200-600, or any intervening integer or rational number. Accordingly, n and / or m may be 1, 1.5, 2, 2.5, ...50, 50.5, 51, 50.5, ...100, 100.5, 101, 101.5, ...600, 600.5, 601, 601.5, ...999, 999.5, or 1,000. In some embodiments, n is 200-650. In some embodiments, m is 500-600.

[0108] In other embodiments, Z of Formula I, Formula II, or both is TT. In some such embodiments, when Z is TT in Formula I and / or Formula II, Y is a polyethylene glycol (PEG) linker comprising at least 2 ethylene glycol units, and n is at least 30-40. Preferably, n is at least 34.

[0109] In some embodiments, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more vaccine compositions (e.g., one or more Qb wild type, or QP mutant, hapten-conjugate as described supra), formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. In another aspect, the compositions can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with other therapies, such as medication-assisted treatment (MAT) for opioid abuse disorders / addiction that will be known and appreciated by those of skill in the art. Conjunctive therapy thus includes sequential, simultaneous and separate, or co-administration of the composition, wherein the therapeutic effects of the first administered has not entirely disappeared (e.g., is still effective in the body) when the subsequent compound is administered (e.g., at least two agents are simultaneously effective in the subject, which may include synergistic effects of the at least two agents). In some embodiments, at least one vaccine compositions (e.g., one or more TT, QP wild type, or QP mutant, hapten conjugate as described above) may be provided to the subject alone or in combination with at least one medication-assisted treatment, including but not limited to buprenorphine, naloxone, methadone, naltrexone, or any combination thereof, e.g., administered prior to, concomitantly with, conjointly with, or following MAT

[0110] In some embodiments, the preparation and / or pharmaceutical composition may further comprise an adjuvant. As used herein, the term “adjuvant” broadly refers to an immunological or pharmacological agent that modifies or enhances the immunological response to a composition in vitro or in vivo. For example, an adjuvant might increase the presence of an antigen (e.g., a hapten) over time, help absorb an antigen-presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines. By changing an immune response, an adjuvant might permit a smaller dose of the immune interacting agent or preparation to increase the dosage effectiveness or safety. For example, an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent or preparation. Examples of adjuvants include, but are not limited to, an immune modulatory protein, adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, P-glucan, CpG DNA, GPI-0100, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, Army Liposome Formulations (ALFs), ALF adsorbed to aluminum hydroxide (ALFA), ALF containing QS21 saponin (ALFQ), and trehalose dimycolate. By way of example, the vaccine compositions contemplated herein may comprise an the adjuvant, wherein the adjuvant is adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, P-Glucan Peptide, CpG DNA, GPLOIOO, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, Momordica saponin derivative II (VSA-2), monophosphoryl Lipid A (MPLA), double mutant heat-labile toxin (dmLT), Monophosphoryl-Lipid A (MPLA), Army Liposome Formulation (ALF), ALF adsorbed to aluminum hydroxide (ALFA), ALF containing QS21 saponin (ALFQ), or trehalose dimycolate. In some embodiments, the adjuvant is VSA-2, MPLA, dmLT, MPLA in ALFA, or ALFQ.

[0111] Methods of preparing the formulations or compositions disclosed herein include the step of bringing into association an agent described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0112] Compositions of the invention contemplated herein suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacterio stats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0113] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0114] Regardless of the route of administration selected, the agents of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

[0115] Therapeutic Methods

[0116] Aspects of the invention provided herein include the treatment or prevention of OUD comprising administering to a subject in need thereof a composition contemplated herein. In certain aspects of the invention, provided herein are methods of treating or preventing opioid addiction, comprising administering to a subject in need thereof a vaccine composition contemplated herein. In some such embodiments, the methods contemplated herein include treatment or prevention of OUD and addiction for at least two or more opioids, at least 3 or more opioids, at least 4 or more opioids, at least 5 or more opioids, 6 or more opioids, 7 or more opioids, 8 or more opioids, 9 or more opioids, or 10 or more opioids. In some embodiments, the opioid addiction is to heroin, fentanyl, morphine, benzimidazole-derived new psychoactive substance opioids (BNO), codeine, oxycodone, hydrocodone, analogues and / or metabolites thereof, or any combination thereof.

[0117] In some aspects, provided herein are methods of treating or preventing an opioid overdose, comprising administering to a subject in need thereof a vaccine composition contemplated herein. In some such embodiments, the methods contemplated herein include treatment or prevention of overdose from at least two or more opioids, at least 3 or more opioids, at least 4 or more opioids, at least 5 or more opioids, 6 or more opioids, 7 or more opioids, 8 or more opioids, 9 or more opioids, or 10 or more opioids. In some embodiments, the opioid overdose is an overdose of heroin, fentanyl, morphine, BNO, codeine, oxycodone, hydrocodone, analogues and / or metabolites thereof, or any combination thereof.

[0118] Other aspects of the invention include methods of preventing or reducing the analgesic effect of an opioid in a subject, comprising administering to a subject in need thereof a vaccine composition contemplated herein.

[0119] In some embodiments, the level of antibodies generated in response to the administered vaccine composition is sufficient to alter the opioid's (or plurality of opioid’s) distribution or its behavioral effects in the subject. In certain embodiments, the antibodies generated in response to the administered vaccine composition may be isolated from the subject, e.g., from the blood of the subject as sera or purified antibodies. For example, without being bound by theory and without limitation, the subject may be an animal host (e.g., a mammal) or a human donor. Said sera and / or purified antibodies may be used to detect the presence of an opioid in a blood sample by methods known in the art. In some embodiments, the subject is undergoing medication-assisted treatment (MAT). The vaccine composition may be administered prior to, concomitantly with, conjointly with, or following MAT. In some such embodiments, the vaccine composition may be administered conjointly with buprenorphine, naloxone, methadone, naltrexone, or any combination thereof. As described supra, and without limitation, the at least one vaccine compositions (e.g., one or more TT, QP wild type, or QP mutant, hapten conjugate described herein) may be provided to the subject alone or in combination with at least one medication-assisted treatment, including but not limited to buprenorphine, naloxone, methadone, naltrexone, or any combination thereof, e.g., administered prior to, concomitantly with, conjointly with, or following MAT

[0120] In some embodiments, the vaccine compositions contemplated herein are administered systemically. The systemic administration may be selected from the group consisting of oral, intravenous, intradermal, intraperitoneal, subcutaneous, and intramuscular administration.

[0121] In some embodiments, the subject is a mammal. In some such embodiments, the mammal is human.

[0122] Actual dosage levels of the active ingredients in the compositions provided herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0123] The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0124] In some embodiments, the methods provided herein further comprise treating the identified subject using a therapeutic method provided herein (e.g., by administering to the subject a pharmaceutical composition provided herein).

[0125] EXAMPLES

[0126] Example 1: Hapten and Vaccine Design

[0127] Compared to heroin, 6-AmHap contains two amides instead of acetoxy (OAc) groups at the 3,6-positions, which are more hydrolytically stable and can mimic heroin. In addition a 3-thio-propanamide linker was incorporated into the C-3 position of 6-AmHap to enable bioconjugation. (See Fig. 1.) In order to elicit a sufficient antibody response against 6- AmHap, the hapten was conjugated to an immunogenic carrier derived from mutant bacteriophage QP (mQP) virus-like particle.39The QPmutantA38K-A40c-DJ02ccomprises a well- organized 3D structure and reduced carrier-antibody response. The mQP carrier was functionalized with the bifunctional linker succinimidyl-[(N-maleimidopropionamido)- diethyleneglycol] ester (SM(PEG)2) to introduce maleimide groups onto the mQP surface. Upon removal of unconjugated linker, the maleimide-functionalized mQP was incubated with 6-AmHap (Fig. 3A). The conjugate was characterized by LC-MS, which showed an average of 212 copies of 6-AmHap per particle (Fig. 4). For the first time, mQP was applied to antiopioid vaccine development.

[0128] The generality of the vaccine response to the mQP-AmHap conjugate was tested in two strains of mice. C57BL / 6 and Balb / c mice were each immunized subcutaneously with either mQP-6-AmHap conjugate or tetanus toxoid (TT) -6-AmHap control conjugate containing 0.83 pg of 6-AmHap (n=5 per group) with the Army Liposome Formulation adjuvant (ALFA).40The immunization protocol involved the administration of a prime injection followed by two boosters, with a two-week interval between each injection.

[0129] A further control group of mice received an admixture of mQP and 6-AmHap comprising the same amounts of the component mQP and 6-AmHap present in the administered mQP-6-AmHap conjugate. On days 0, 7, and 35, blood was collected from all mice and the levels of antibodies produced were assessed through enzyme-linked immunosorbent assay (ELISA). To avoid any potential interference from anti-mQP or anti-SM(PEG)2 linker antibodies, 6-AmHap was conjugated with BSA through an acrylic ketone linker41(Fig. 3C) and the BSA-6-AmHap was used as the ELISA coating antigen.

[0130] Results

[0131] Immunization with mQP-6-AmHap elicited high levels of anti-6-AmHap IgG in both C57BL / 6 and BALB / C mice (mean IgG titers of 2.7 million and 8.0 million ELISA units, respectively) on day 35 (Figs. 5A and 5B) with low titers of IgM antibodies (< 2,000 ELISA units). Without being bound by any particular theory or model, this suggested that the vaccination successfully activated helper T cells, presumably through the T cell epitopes within QP, leading to affinity maturation and isotype switching to IgG.42,43In comparison to TT-6-AmHap, mice immunized with mQP-6-AmHap exhibited significantly higher titers (2.9 and 5.8 folds higher in C57BL / 6 and BALB / C strains respectively) of anti-6- AmHap IgG antibodies. The IgG responses were monitored over time (Fig. 5C). After 600 days significant IgG titers remained, suggesting persistent antibody responses were induced against 6-AmHap. These results highlight the potential of mQP as a powerful OUD vaccine carrier. The control group of mice immunized with the admixture of mQP and 6-AmHap only had baseline levels (< 2,000 ELISA units) of anti-6-AmHap IgG antibodies on day 35 (Figs. 5A and 5B) suggesting the covalent conjugation between mQP and the hapten 6-AmHap is critical for generating a robust immune response.

[0132] Example 2: Adjuvanted Antisen-specific Humoral Response

[0133] Antigen- specific humoral response was also compared following vaccination with four different adjuvants, z.e., ALFA, monophosphoryl Lipid A (MPLA),44dmLT (a double mutant R192G / L211A of heat-labile toxin),45and Momordica saponin derivative II (VSA-2).46MPLA is a ligand for Toll-like receptor 4 (TLR-4), with ALFA being a liposomal formulation of MPEA with reduced reactogenicity in humans.40Adjuvant dmET is the most advanced and clinically relevant protein from a family of adjuvant derived from heat-labile enterotoxins from E. coli. VSA2 is a novel synthetic adjuvant, which is an analog of QS-21. Four groups of C57BE / 6 mice (n=5) were immunized with the mQP-6-AmHap conjugate containing 0.83 pg of 6-AmHap formulated with either MPEA, AEFA, or dmET, according to the aforementioned biweekly schedule. In addition, another group of mice received the vaccine without any exogenous adjuvant.

[0134] Results

[0135] ELISA analysis of day 35 sera showed that all adjuvanted groups gave high anti-6- AmHap IgG titers, which were significantly higher than those from the non-adjuvanted group (Fig. 7A). In addition, all adjuvanted groups gave persistent anti-6- AmHap IgG antibody responses (Fig. 7B). To better understand the antibody responses, the IgG subtypes being produced were analyzed. As shown in Fig. 6, all the major subtypes of IgG including IgGl, IgG2b, IgG2c, and IgG3 were produced, suggesting balanced humoral responses.

[0136] The vaccines were well tolerated and safe. There were no injection site adverse reactions observed for any of the vaccines, except for some minor hair loss in the group that received the ALFA mixture with mQP-6-AmHap. The body weights of the mice were monitored weekly following vaccination (Fig. 8). The average weight gains between the groups with and without adjuvants were similar suggesting the vaccines were biocompatible. Further studies proceeded with MPLA due to its ready availability. Example 3: Binding Specificity of Antibodies induced b m() B-6-AmHap

[0137] Because heroin is rapidly metabolized in the body, for a vaccine to be effective it is important that the antibodies resulting from immunization recognize heroin as well as metabolites and analogs that can similarly be potentially abused. Furthermore, it is crucial to ascertain that the antibodies generated do not exhibit cross -reactivity with medication- assisted treatment, e.g., methadone, buprenorphine, and naltrexone, which are commonly employed in opioid abuse therapies. Competition ELISA studies were conducted to determine the IC50 values (corresponding to the analyte concentration that produces 50% inhibition of antibody binding to 6-AmHap) for both abused drugs and therapeutic medications (Fig. 9 and Table 1). This was used as a surrogate measure of the affinity of antibodies towards the target drug.

[0138] Results

[0139] The mQP-6-AmHap vaccine generated antibodies in mice that exhibited low IC50 values (low pM) against a variety of heroin metabolites, including 6-acetyl morphine, morphine, and morphine-3-P-glucuronide (M-3G), as well as multiple other prescription opioid drugs that can be abused, such as codeine, oxycodone, hydrocodone, and hydromorphone (Table 1). Importantly, the antibodies demonstrated no binding to drugs utilized in opioid abuse therapy or acute overdose rescue including buprenorphine, naltrexone, or naloxone (ICso > 1,000 pM). Opioids with structures unrelated to heroin such as tramadol and meperidine, did not bind with the antibodies induced by the vaccine either under the competition ELISA conditions. The IC50 values of several drugs to TT-6-AmHap induced antibodies were also measured by competition ELISA (Table 2). Similar trends of binding were observed, which corroborated the prior reports.38

[0140] Table 1: IC50 values for various opioids or drugs used for MAT or pain treatment measured using the competition ELISA from pooled sera for each group using triplicate measurements.

[0141] *ICso was defined as the drug concentration that produced 50% inhibition of maximal antibody and hapten binding as calculated from normalized competition curves using log(inhibitor) vs normalized response-variable slope regression.

[0142] Example 4: Reduction of Morphine Activities In Vivo

[0143] The mQP-6-AmHap vaccine was tested for its capacity to reduce the effects of opioids on mouse locomotor activity and sensitivity to pain following morphine administration. This assessment was performed using locomotion behavioral assays and the hot plate antinociception test.47'50(See Fig. 10A.)

[0144] C57B16 mice (n = 10) were fully immunized with mQP-6-AmHap (+ MPLA adjuvant) with the 1 prime and 2 boost protocol described above (z.e., weeks 0, 2, and 4), and the control group of mice (n = 10) was immunized with the admixture of mQP and 6-AmHap (+ MPLA adjuvant). Blood samples were collected at weeks -1, 1, 3, and 5. The mice were then challenged with morphine (10 mg / kg) at weeks 15 and 16, administered intraperitoneally (IP) to each mouse. Twenty-four hours prior to challenge the two groups of mice were administered saline (saline day) to establish the base line for locomotion. Vaccine efficacy was assessed using locomotion assays (week 15).

[0145] To prevent the development of morphine analgesic tolerance, a one-week interval was maintained between locomotion and analgesic assays. Thus, on week 16 post prime immunization, the anti-nociception assay was performed to test response to painful stimuli, and mice from both the mQP-6-AmHap immunized group and the admixture group were placed on hot plates. The time from the onset of the heat to showing a pain response (latency to withdrawal) was measured and the latency data (anti-nociception) were converted to percentage of maximum possible effect (%MPE). At week 60, mice were euthanized, and blood and brain samples were collected by cardiac puncture. Results

[0146] As shown in Fig. 10B, mice receiving the admixture had significantly higher ambulation counts stimulated by morphine vs those on saline day. Compared to the admixture group, the group of mice vaccinated with mQP-6-AmHap had significantly lower ambulation counts presumably due to morphine sequestration by anti-6-AmHap antibodies. To confirm this behavioral effect, open field tests were performed by measuring total distance traveled by mice. Following morphine administration, the admixture immunized mice were much more active and moved much longer total distances than those receiving the mQP-6-AmHap conjugate (Fig. IOC).

[0147] As depicted in Fig. 10D, morphine-induced antinociception was significantly reduced with much lower MPE values in the mQP-6-AmHap group compared to the mQP / 6-AmHap admix vaccinated control mice.

[0148] Example 5: Reduction of Free Drug Reachins the Brain

[0149] To confirm the effect of the mQP-6-AmHap vaccine, the level of morphine in the brain vs. the level circulating in the blood following drug administration was assessed. Sixty weeks after the initial immunization of two groups of mice, one with mQP-6-AmHap conjugate the other with the admixture, they were given a dose of morphine (10 mg / kg) IP. Thirty minutes after drug administration, mice were euthanized, their blood collected, and their brains extracted following extensive perfusion to thoroughly remove the blood from the brain. The amounts of morphine in the blood and the brain were quantified by LC-MS.

[0150] Results

[0151] As shown in Fig. 10E, mQP-6-AmHap conjugate vaccinated mice had significantly lower levels of brain / blood ratio of morphine as compared to mice that received the admixture, suggesting that immunization with mQP-6-AmHap reduced the amounts of morphine reaching the brain.

[0152] Example 6: Summary of mO 0-6-AmHap Conjugate

[0153] Drugs of abuse (e.g., schedule I drugs, including narcotics, such as, heroin and morphine) are typically small molecules, which have low inherent immunogenicity. To enhance the immune responses haptens that mimic the drug of abuse are covalently linked to an immunogenic carrier. Pre-clinical studies of such conjugate vaccine candidates have shown that vaccine efficacy correlates with serum antibody titers. Thus, it is important vaccination induces high levels of antibodies against the drug of abuse.

[0154] Carriers such as KLH,30, 31CRM-19751and TT38, 52have been investigated for anti- OUD vaccine studies. Compared to these more traditional carriers, virus-like particles, as represented by bacteriophage QP, can be attractive alternatives for immune-potentiation.53, 54The QP capsid has a highly ordered three-dimensional structure39, 55and can display the antigen in an organized array, which is important for crosslinking B cell receptors and potent B cell activation.56The mutant QP is associated with reduced levels of anti-carrier antibody production and can further improve the antibody responses against the conjugated hapten.39The ability of the conjugate of mQP-6-AmHap to induce high levels and long lasting anti-6- AmHap antibody responses and reduce the morphine effect suggests virus-like particles can be another class of attractive carrier for OUD vaccine development.

[0155] For anti-OUD vaccines, it is also important that the antibodies elicited by immunization have high selectivity for the drug of abuse, which was observed in mQP-6-AmHap immunized mice (Table 1). Without being bound by theory, this selectivity can be rationalized by the structure of 6-AmHap. For example, upon conjugation to a carrier through the free sulfhydryl group, the C-3 and C-6 face of hapten 6-AmHap may be less sterically accessible as it is on the same side as the linker facing toward the mQP surface. On the other hand, the N-17 area of 6- AmHap is likely projected away from the mQP carrier, which may be a critical part of the epitopes for antibody recognition. Thus, compounds such as naloxone and naltrexone, with their much larger substituents at N-17 (compared to 6-AmHap; see Fig. 9), are not well recognized by the antibodies generated by mQP-6-AmHap immunization. Presumably it is the steric hinderance from the N-17 substituents that allows for selective recognition of heroin derivatives with N-17 methyl (e.g., including codeine, oxycodone, and hydrocodone) over these compounds (e.g., naloxone and naltrexone). Thus, vaccinated patients can potentially benefit from both the vaccine and their prescribed pharmacotherapy without compromising the effectiveness of either intervention. Besides heroin and its metabolites, the cross -recognition of several other commonly abused opioids such as codeine, oxycodone, and hydrocodone by the antibodies induced by mQP-6-AmHap demonstrated that mQP-6-AmHap has the potential to serve as a broad opioid vaccine, capable of targeting a range of opioids.

[0156] Heroin is rapidly deacetylated to 6-acetyl morphine and morphine in the body. Monoclonal antibodies specific to 6-acetyl morphine appear to be less efficacious in reducing the impact of heroin.66Thus, polyclonal responses against heroin and its multiple pharmacologically active metabolites may be attractive. Notably, the antibodies elicited by mQP-6-AmHap can bind with morphine and various heroin metabolites. The binding to heroin, morphine, and 6-AmHap were the strongest, having IC50 values around 2 pM; followed by binding to 6-acetyl morphine, codeine, hydrocodone, and hydromorphone (IC50 - 6.5 pM). (See Table 1.) These compounds contain a hydrogen atom at C-14. However, oxycodone and oxymorphone bear an OH group at C-14 instead, and they had reduced apparent affinities and higher IC50 values (29 pM), suggesting C-14 may be part of the epitope involved in antibody recognition of heroin and morphine. The antibodies induced by TT-6-AmHap exhibited a similar relative affinity trend (Table 2) as those by mQP-6-AmHap, but the IC50 values for oxycodone and oxymorphone were significantly higher at 550 pM and 94 pM respectively, which were similar to known values.38The higher IC50 values for oxycodone and oxymorphone from TT-6-AmHap induced antibodies may be due to higher dependence of C-14 for antibody recognition. To provide more comprehensive protection, it is important that a vaccine can protect against multiple types of drugs. Multiple antigens can be potentially combined on the mQP platform. This is a future direction to pursue and establish whether the efficacy of the individual vaccine would maintain the efficacy in a multivalent mQP construct.

[0157] Table 2: IC50 values for various opioids or drugs used for MAT or pain treatment measured using the competition ELISA from pooled sera from TT-6-AmHap immunized mice with triplicate measurements. The conjugate of mQP with the heroin derivative 6-AmHap has been shown to be able to elicit powerful IgG antibody responses against 6-AmHap following the prime-boost immunization protocol. The anti-6-AmHap IgG antibodies produced were persistent, with significant titers even after 600 days post-immunization. The covalent linker between mQP and 6-AmHap in the mQP-6-AmHap conjugate is critical for the high immune response as the physical mixture of mQP and 6-AmHap was not effective in antibody production. Compared to the TT-6-AmHap conjugate entering human clinical trials, mQP-6-AmHap immunization was able to elicit significantly higher anti-6-AmHap IgG antibodies.

[0158] The immunogenicity of mQP-6-AmHap could be boosted with a variety of adjuvants as compared to the non-adjuvanted formulation. The antibodies generated by the vaccine bound well with heroin, morphine as well as a range of heroin metabolites and multiple other commonly abused opioids. At the same time, they do not recognize common drugs used for MAT and non-morphine-based medications used for pain management, suggesting the high structural selectivity of the antibodies and alleviating the concern of cross -reactivity adversely impacting pain relieve. Mice immunized with mQP-6-AmHap conjugate had much reduced responses to morphine as reflected in the hyperlocomotion assays for both ambulation and total distance traveled as well as the anti-nociception assay. The mQP-6- AmHap conjugate can be an attractive addition to the toolbox complementing current MAT to stem the rising tide of OUD.

[0159] Example 7: Materials and Methods

[0160] Expression and Characterization of the Carrier Protein Bacteriophage mQB mQ / 3 (A38K, A40C, D102C) Expression

[0161] A single colony of BL21(DE3) E. coli with the QP mutant-A3S , A40C, D102C plasmid was selected to inoculate a 25 mL starting culture of Super Optimal Broth (SOB) media containing 20 pg / mL kanamycin.

[0162] The starter culture was incubated overnight at 37°C, 230 rpm. The following morning, the cloudy starter culture was transferred into IL SOB culture medium with 20 pg / mL kanamycin and cultured at 37°C, 230 rpm until the OD600 was between 0.7 and 1.0. At that point, 1 mL of 1 M isopropyl P-D-l -thiogalactopyranoside (IPTG) was added to induce protein expression and cultured overnight at 30°C, 230 rpm. The cells were then pelleted at 7000 rpm for 30 min, culture media was discarded, and pelleted cells were resuspended in 30 mL 0.1 M potassium phosphate buffer (KPBS) buffer, pH 7, and stored at -20 °C until lysis. mQB (A38K, A40C, D102C) Purification

[0163] Cell pellets collected after IPTG-induced protein expression were lysed by mixing with 2 mL lx cell lytic detergent (Sigma) per gram of pelleted cells. An equal volume of 0.1 M KPBS buffer plus lysozyme (2 mg / mL final concentration) was added and stirred while adding 50 pL of Dnase I (5000 U), and stirring was continued for 1 h at room temperature. Lysed cells were centrifuged at 14,000 rpm for 20 min using a Liberlite L21-8x50y, and the supernatant with the capsid protein was collected into a 50 mL centrifuge tube containing 5 g of PEG-8000 to a final concentration of 10% (w / v) and placed on a nutating mixer overnight at 4 °C to enable complete protein precipitation. The precipitate was pelleted at 14,000 rpm for 20 min, resuspended in 10 mL 0.1 M KPBS, pH 7, added to 10 mL of 1:1 (v / v) chloroform / n-butanol solution, and mixed until the solution became colloidal. The colloidal mixture was centrifuged at 7000 rpm for 60 min in glass centrifuge tubes to separate the layers. The aqueous (top) layer was collected, layered onto freshly prepared linear sucrose gradients (10-40%, w / v), and centrifuged using a swing bucket rotor (SW32) at 28,000 rpm for 4.5 h. The protein band was visualized using LED light directed at the top of the tube and collected in fractions to separate aggregates from capsids. mQ / > (A38K, A40C, D102C) Characterization

[0164] The fractions were analyzed for purity using fast protein liquid chromatography (FPLC), performed on a GE AKTA Explorer (Amersham Pharmacia) instrument equipped with a Superose-6 column. Fractions showing a single peak at around 10-15 mL elution were determined to be mQP capsid, and any aggregated capsid eluted between 8 and 10 mL (Fig. 11). All fractions containing pure mQP were combined and concentrated to ~1 mL for quantification by the Bradford assay (BSA). The purified capsid had the ratio of absorbance values of 260 nm vs 280 nm around 2, suggesting the encapsulation of RNA inside the capsid. After dithiothreitol (DTT) addition, the protein molecular weight was determined by LCMS QTOF ESI mass spectrometry (Fig. 4A). The multiple charge mass spectrum was transformed to a single charge by Maximum Entropy deconvolution algorithm (Max Ent).

[0165] Preparation of protein - hapten immunoconjugates (mQfi and BSA conjugate)

[0166] The amount of antigen added was based on the ratio of antigen per accessible surface amines on mQP. Viral particle mQP is composed of 180 identical subunits with five accessible surface amines per subunit. As a result, the total number of surface accessible amines available for conjugation is 900.

[0167] The haptens provided herein, such as but not limited to, 6-AmHap, were coupled to the carrier protein mQP by incubating mQP with the SM-(PEG)2 cross-linker (e.g.. 50 equivalents per amine) for 2 hours at 37°C. The conjugate was purified using Amicon Ultra (10,000 MW cutoff) centrifugal filtration against 0.1 M PBS, pH 7.4 (5 x 0.5 mL). After multiple washes and buffer exchange through membrane filtration for excess reagents removal, the protein content was quantified by the Bradford assay using bovine serum albumin (BSA) standards. The exemplified mQP-6-AmHap bioconjugation was achieved by dropwise addition of the hapten (25 equivalents per amine) to the solution of mQP-SM-(PEG)2 intermediate, followed by incubation at 37°C for 2 hours. Excess reagents were removed through multiple washes and buffer exchanged using Amicon Ultra (10,000 MW cutoff) centrifugal membrane filtration. The mQP-hapten conjugates were quantified by the Bradford assay using bovine serum albumin (BSA) standards. The number of heroin hapten attached to the mQP molecule was determined by LCMS QTOF ESI mass spectrometry to be an average of 212 per mQP capsid (Fig. 4B). The following equation was used to determine the hapten loading on mQP capsid:

[0168] Total loading on the capsid = n*180(# of subunits for each mQ6 capsid)

[0169] • "n" is the average loading on one subunit of mQP capsid.

[0170] • relativepeak intensities of MS peaks of mQP monomer with varying number of the haptens attached. IQ refers to the intensity of the peak with MS value corresponding to the unconjugated subunit with 0 hapten I refers to the intensity of the peak with MS value corresponding to the mQP monomer with one copy of (linker+hapten), etc.

[0171] Similarly, BSA was coupled to 6-AmHap by incubating BSA with an acrylic ketone linker (15 equivalents per amine) in PBS for 2 hours at 37 °C. After removing the excess linker, the BSA-linker conjugate was incubated for 2 hours with 6-AmHap (50 equivalents per amine), following a similar procedure to mQP-6-AmHap. The BSA-6-AmHap conjugate was membrane filtered and quantified by the Bradford assay using bovine serum albumin (BSA) standards. The number of haptens attached to the BSA was measured by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, resulting in an approximate count of 25 haptens per BSA molecule (Fig. 4C).

[0172] Vaccine formulation

[0173] The immunoconjugates were stored at a temperature of -80°C until the day of immunization, ensuring their preservation. The vaccine was made prior to each immunization. Adjuvants were added to the immunoconjugates and allowed to mix for ten minutes at room temperature under sterile conditions. The vaccines were formulated with specific adjuvant doses per mouse, including 100 pg of VSA-2, 20 pg of MPLA, 1 pg of dmLT, and 20 pg of MPLA in ALFA. Each formulation achieved a final vaccine concentration of 13 pg of mQP protein, which contained approximately 0.83 pg of 6- AmHap. The adjuvants were mixed with PBS at a pH of 7.4, and then the mQ conjugate protein was slowly added. For the TT conjugation vaccine formulation, a similar procedure was followed. The ratio of approximately 34 haptens per TT molecule corresponds to an approximate dosage of 0.8-0.9 pg of 6-AmHap per dose. Behavioral assays

[0174] Animal studies

[0175] All animal care procedures and experimental protocols were approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC) (Protocol number: 202200444). Female BALB / c and CB57BL / 6 mice, aged 7-10 weeks, were selected for the carrier protein comparison and adjuvant tests with a group size of 5 mice per group. For the in vivo studies of vaccine constructs, female CB57BL / 6 mice, aged 7-10 weeks, were used, with a group size of 10 mice per group. Subcutaneous injections of 0.2 mL vaccine were administered at the scruff of the neck on day 0, followed by booster injections on days 14 and 28 subcutaneously. Serum samples were collected from the mice on days 0 (before immunization), 7, 21, and 35 for analysis. Additionally, the weights of the mice were recorded as well as injection site reactions were monitored every other week throughout the study.

[0176] Locomotion Tests

[0177] The mice were first acclimated to the locomotion chamber for 60 minutes to familiarize them with the environment and reduce exploratory behavior caused by novelty. On the first day of testing, female mice were given an intraperitoneal (IP) injection of saline and locomotor activity was monitored for 60 minutes to establish the baseline locomotor behavior. On the following day, morphine (10 mg / kg) was administered, and locomotor activity was again monitored for 60 minutes. In the first cohort, locomotor activity was measured in conditioned place preference boxes (San Diego Instruments) and calculated as total beam breaks (4 x 16 (x-y) photobeam array). In the second cohort, locomotor activity was assessed in 38 cm x 38 cm open field boxes using video-tracking software (TopScan Suite, CleverSys).1Statistical differences in locomotor activity (beam breaks or distance traveled) were determined using a two-way repeated measures ANOVA (Graphpad Prism) followed by a Fischer’s LSD post-hoc test when appropriate.

[0178] Hot Plate Test

[0179] The hot plate assay was conducted one week after the locomotion testing. 16 weeks after the initial immunization with mQP-6-AmHap, the animals underwent a cumulative morphine response test primarily in the supraspinal region using a hot plate. This test aimed to evaluate the efficacy of the vaccine following a morphine challenge. The test was performed following the reported protocol on literature. Briefly, the hot plate (Harvard Apparatus, Holliston, MA, USA) was set to a temperature of 54 °C. To establish a baseline, the latency to the thermal stimulus was measured by placing the mice on the hot plate and recording the time it took for the mice to lick or lift their hind paws or jump. After an intraperitoneal injection with morphine (10 mg / kg, dissolved in saline), the mice were retested on the hot plate 30 minutes later. To prevent burns, a cutoff time of 60 seconds was set.

[0180] The antinociception data obtained from the tests were transformed into percent maximum possible effect (%MPE), calculated using the following formula: %MPE = [(test - baseline) / (cutoff - baseline)] x 100.

[0181] ELISA and competitive Elisa procedure

[0182] A Nunc MaxiSorp flat-bottom 96-well plate was coated with BSA-6-AmHap conjugate (10 pg / mL, 100 pL / well) in NaHCO3 / Na2CO3 / NaN3 coating buffer (0.05 M, pH 9.6) and incubated overnight at 4 °C. The coated plate was washed four times with PBS / 0.5% Tween 20 (PBST), followed by the addition of 1% (w / v) BSA in phosphate-buffered saline (PBS) to each well and incubated at room temperature for 1 h. The plate was washed again four times with PBST (4 x 200 pL). Serial dilutions of mouse sera in 0.1% BSA / PBS (100 pL) were added to each well and incubated for 2 h at 37 °C. The wells were then washed with PBST (4 x 200 pL) and a 1:2,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson Immune Research Laboratory catalog #115-005-062) in 0.1% BSA / PBS (100 pL) was added to each well and incubated for 1 h at 37 °C. The enzymatic substrate was prepared by dissolving 3,3 ',5,5 '-tetramethylbenzidine (TMB, 5 mg) in DMSO (2 mL) and citric acid buffer (18 mL) in a 50 mL centrifuge tube covered with aluminum foil. H2O2 (20 pL) was added and vortexed to homogeneity. The plate was washed with PBST (4 x 200 pL) and enzymatic substrate was added (200 pL), color was allowed to develop for 15 min, and then 0.5 M H2SO4 (50 pL) was added to quench the reactions. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad). Titers were determined by regression analysis with loglO dilution plotted against optical density. The titer was reported as the highest fold dilution giving an optical absorbance value of 0.1 over pre immune control sera (OD = 0.2). The samples were tested in triplicate. The relative affinities of various drugs for serum 6-AmHap specific antibodies were measured by competition ELISA, which differs from the above ELISA assay in two ways. 1) Serum titers from day 35 measured using ELISA described above was diluted to a fixed 70% maximal binding (ELISA absorbance of ~ 1.5) in the absence of competitor and 2) mixed with competitors with concentrations ranging between 0.01 and 1000 pM. Relative affinity was determined by the 50% inhibitory concentration (IC50 value) of each compound calculated from its normalized ELISA curve for pooled sera from each group.

[0183] Blood-brain biodistribution

[0184] The blood-brain biodistribution was determined according to literature procedure2'4with minor modifications. A calibration curve for using standard solutions of morphine was constructed based on the intensities of the LC-MS signals.

[0185] 60 weeks from last boost, vaccine groups and nonvaccinated control mice (n = 10 and 9, respectively) were injected intraperitoneally (10.0 mg / kg) with morphine dissolved in saline. At 30 minutes following injection the animals were fully anesthetized and then blood sampling (500 pL) was performed by heart puncture with a syringe containing 80 pL ice-cold NaF (final concentration 4 mg / mL to inhibit enzymatic activity and maintain the integrity of the sample) dissolved in heparin (100 lE / mL). The blood was transferred to a microcentrifuge tube, and aliquots of 100 pL were taken out into plastic tubes and diluted (1:1, v / v) in ice-cold ammonium formate buffer (5 mM, pH 3.1) containing NaF (final concentration 4 mg / mL). The blood samples were immediately frozen in liquid N2 and stored at -20°C until analysis.

[0186] After blood sampling, brain perfusion without brain fixation is performed. In brief, the animal’s brain was flushed with PBS to fully clear the blood from the brain's blood vessels then the brain was quickly removed and washed in ice-cold ammonium formate buffer containing NaF (final concentration 4 mg / mL), blotted on a filter paper. Then brain tissue was immediately flash frozen using a dry ice / acetone bath with 1 mL of formate buffer (5 mM, pH 3.1). The brain tissue was homogenized with small sonicate in ice-cold ammonium formate buffer (5 mM, pH 3.1) to a final concentration of 0.33 g tissue / mL homogenate.

[0187] Samples were stored at -20°C. The brain homogenate was thawed and frozen once before analysis to break intact cells then centrifuged at 2,500 rpm for ten minutes. A 100 pL aliquot of the homogenate or plasma was added to 100 pL of spiked morphine concentrations (for standard curve, made up in 85:15 ACN:MeOH) or 100 pL of 85:15 ACN:MeOH (for samples), 100 pL of t / a-morphinc (1 pg / mL in ACN) and 300 pL of ice-cold acetonitrile / methanol (85:15). The mixture was vortexed for 30 s and stored in the -20 °C freezer for twenty minutes, followed by centrifugation at 2500 rpm for ten minutes. A 450 pL aliquot was transferred to another test tube and the samples was evaporated using GENEVAC. The dried sample was taken up in acetonitrile, centrifuged at 10,000 rpm for five minutes, and then transferred to vials for LCMS analysis.

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[0295] Described herein is a novel fentanyl hapten (para-AmEtFenHap) that retains the intact fentanyl scaffold and uses the para position of the phenethyl ring for linker attachment for the formulation of a monovalent mQP-para-AmEtFenHap and a bivalent vaccine combining mQP- 6-AmHap and mQP-para-AmEtFenHap. The mQP-para-AmEtFenHap vaccine induced high- affinity antibodies not only against fentanyl and its highly potent analogues, heroin and its metabolites, but also to the nitazene family compounds.

[0296] The monovalent mQP-para-AmEtFenHap vaccine, as well as bivalent mQP-6-AmHap / -para-AmEtFenHap vaccine provided protection in mice against heroin and fentanyl and etonitazene induced effects. The following represents the first immunological study of the AmEtFenHap hapten; the first time mQP VLP has been used in a bivalent opioid vaccine; as well as the first report of a single fentanyl vaccine where in vivo and in vitro data showed protective cross-reactivity with three potent opioids using a monovalent vaccine formulation. These findings can potentially lead to a new era in immunopharmacotherapy for opioid-use disorders, offering a long-lasting, preventative solution to the opioid crisis.

[0297] Conjugation of Hapten to Carrier Proteins

[0298] Bioconjugation 6-AmHap was performed as described above (see Fig. 3A and 12A). Similarly, the mQP-para-AmEtFenHap mQP particle was functionalized using the bifunctional linker succinimidyl-[(N-maleimidopropionamido)-diethylene glycol] ester (SM(PEG)i2) to introduce maleimide groups onto its surface (Fig. 12). Given the hydrophobic nature of the fentanyl hapten, to improve the overall stability and dispersibility of conjugate in an aqueous environment, twelve (12) ethylene glycol units were used to make the conjugation. After removing any unconjugated linker, the maleimide-functionalized mQP was incubated with para-AmEtFenHap (see Fig. 12B).

[0299] LC-MS analysis revealed an average of 540 copies of para-AmEtFenHap and 620 copies of 6-AmHap per particle. To compare the performance of the mQP conjugate, a para- AmEtFenHap conjugate with TT was also prepared using the same SM(PEG)2 linker, with an average of 30-35 copies of para-AmEtFenHap per carrier TT molecule (Fig. 12C), as quantified by MALDI-TOF MS (data not shown). Similarly, BSA-6-AmHap and BSA-para- AmEtFenHap conjugates were synthesized for ELISA evaluation using an alternative linker to minimize the detection of potential antibodies induced against the linker itself (see Fig. 12D. Example 9: Evaluation of Vaccine Responses In Vivo

[0300] Five groups (n = 10 per group) of C57BL / 6 mice were immunized subcutaneously with either mQP-para-AmEtFenHap, TT-para-AmEtFenHap, or mQP-6-AmHap + mQP-para- AmEtFenHap conjugates, each containing 10 pg of para-AmEtFenHap in the monovalent vaccines, and 10 pg each of para-AmEtFenHap and 6-AmHap in the bivalent vaccine (20 pg total). The vaccines were formulated with the army liposome adjuvant ALFQ.10(at 800 pg / ml 3D-PHAD and 400 pg / ml QS-21).

[0301] The immunization protocol consisted of a prime dose followed by two booster shots at two- week intervals. A control group received only saline, as the lack of immunogenicity for either the mQP and hapten admixture or mQP alone has been established.9’11Blood samples were collected from all groups on days 0, 7, and 35, and antibody levels were measured using ELISA with BSA-para-AmEtFenHap and BSA-6-AmHap as the coating antigens. To further assess vaccine efficacy with a reduced dosage, an additional group of animals (n = 10) received only two doses of the mQP-para-AmEtFenHap / ALFQ vaccine, consisting of a prime injection followed by a single booster on day 28, instead of the standard three-dose regimen.

[0302] Results

[0303] Immunization with mQP-conjugates generated high levels of anti-para- AmEtFenHap and anti-6-AmHap IgG responses. By day 35, the mean IgG titers reached 10 million, 8.5 million, and 3.5 million ELISA units in monovalent three regiment and two regiment mQP conjugates and TT conjugates, respectively. Additionally, sera from the bivalent vaccine group exhibited IgG endpoint titers of 7.6 million against para-AmEtFenHap and 7.1 million against 6-AmHap, compared to unvaccinated mice (Fig. 13) and a low average IgG titers (<2000 ELISA units) in controls. The high levels of anti-hapten titer indicate successful activation of helper T cells, likely through T-cell epitopes within QP, leading to affinity maturation and isotype switching to IgG19'21. Notably, these high titers were achieved with just a single prime immunization in both the three-dose and two-dose regimens of the mQP- para-AmEtFenHap vaccine, as well as in the bivalent mQP-6-AmHap + mQP-para- AmEtFenHap vaccine. The mQP-conjugate vaccines produced significantly higher titers, with an over 100,000 fold increase after the prime dose, and the booster effect in enhancing anti-hapten IgG levels was insignificant.

[0304] IgG responses in C57BL / 6 mice were sustained over time, with significant titers still present after 26 weeks, underscoring the potential of mQP as a robust vaccine carrier for opioid use disorder. Sera from both three- and two-regiment mQP-para-AmEtFenHap vaccine group had an endpoint titer of 2.7 and 2.1 million ELISA units against the BSA-6-AmHap ELISA coating agent, while sera from TT-para-AmEtFenHap vaccine group had an endpoint titer of 1.1 million ELISA units against the BSA-para-AmFenHap coating agent. Moreover, sera from mQP-para-AmEtFenHap demonstrated an average anti-BNO IgG titer of 1.6 ELISA units against the BSA-BNO (benzimidazole-derived new psychoactive substance (NPS) opioids (BNO)). Thus the mQP carrier protein is superior in activating and presenting antigens to the immune system, leading to the production of polyspecific antibodies (polybodies) with broad cross-reactive properties among opioids.

[0305] Current research of opioid vaccines target both the drug and its analogues15'20, in an effort to provide broad- spectrum defense against a range of substances. However, thus far, there have been no reports of a monovalent vaccine capable of protecting against a variety of opioids in vivo and in vitro, including synthetically high potent drugs such as fentanyl and etonitazene. Described herein is a monovalent mQP-para-AmEtFenHap that shows high binding affinity toward fentanyl and its analogs, as well as heroin, including its metabolites, and benzimidazole-derived NPS opioids (BNO). Importantly, the neutralizing antibodies do not cross-react with medications commonly used in opioid abuse treatment, such as methadone, buprenorphine, and naltrexone.

[0306] To assess this, competition ELISA studies were performed to measure IC50 values, representing the concentration of analyte required to inhibit 50% of antibody binding to para- AmEtFenHap and 6-AmHap. These values serve as a proxy for the affinity of antibodies toward the target drug. Low IC50 values indicate high-affinity hapten-induced antibodies toward the target drug. Generally, competition ELISA IC50 values tend to overestimate the actual Kd (dissociation constant) of the antibodies by approximately 1,000-fold21. Thus, IC50 values in the pM range from competition ELISA likely correspond to Kd values in the nM range.22A few of the most seized fentanyl analogues, including carfentanil, cyclopropyl fentanyl, parafluor fentanyl, furanyl fentanyl, and a fentanyl metabolite norfentanyl were selected, and the IC50 values for these drugs were measured. See Table 3.

[0307] Notably, the antibodies showed no binding to medications used in opioid addiction treatment or overdose reversal, such as buprenorphine, naltrexone, naloxone or methadone (IC50 > 1000 pM). Drug structures for buprenorphine, naltrexone, naloxone or methadone, used in the ELISA, are depicted in Fig. 9. The structures of haptens para-AmEtFenHap and 6- AmHap are depicted in Fig. 12E and Fig. 12F, respectively

[0308] In addition, measurements of IC50 values for the mQP-para-AmEtFenHap vaccine exhibited low IC50 values (low pM) against heroin and a variety of heroin metabolites, including 6-acetyl morphine, morphine, and morphine-3-P-glucuronide (M-3G), as well as multiple other prescription opioid drugs that can be abused, such as codeine, oxycodone, hydrocodone, and hydromorphone. This is a surrogate measure of the affinity of antibodies toward the target drug, comparable binding affinity was detected against heroin and metabolites using antibodies generated from mQP-para-AmEtFenHap and mQP -conjugated bivalent vaccine. Using similar approach both mono- and bi-valent mQP-conjugate vaccine generated antibodies in mice that exhibited low IC50 values (low pM) against a variety of benzimidazolederived NPS opioids (BNO) Table 3.

[0309] Table 3: IC50values for Various Opioids Measured Using Competition ELISA

[0310] Mixture of Activities In Vivo

[0311] The efficacy of all vaccine groups was evaluated, including two- and three-dose regimens of monovalent mQP-para-AmEtFenHap, TT-para-AmEtFenHap, and the bivalent mQP-para-AmEtFenHap + mQP-6-AmHap in reducing opioid effects on mouse locomotion and thermal nociception following various opioid administrations. This assessment was conducted using the locomotor activity assays and hot-plate antinociception test.

[0312] Locomotor activity: Fentanyl

[0313] Four groups of C57B16 mice (n = 10) were fully immunized with mQP-para- AmEtFenHap, TT-para-AmEtFenHap, the bivalent (mQP-para-AmEtFenHap + mQP-6- AmHap), and saline. The saline group received equivalent injections protocol with saline to mimic potential behavioral effects observed in the immunized groups, following a prime and two-boost protocol. An additional group (n = 10) received a two-dose regimen of mQB-para- AmEtFenHap (2R mQB-para-AmEtFenHap). Twelve weeks post-prime, all five groups were administered saline (saline day) to establish a baseline for locomotion. One day later, each mouse received 0.3 mg / kg fentanyl intraperitoneally (IP) to assess hyperlocomotion. As indicated by the results on Fig. 14A, control mice had significantly higher ambulation counts stimulated by fentanyl vs those on saline day. Compared to the control group, the vaccinated groups of mice had significantly lower ambulation counts.

[0314] Locomotor activity: Heroin + 9% Fentanyl

[0315] At week 14, all five groups were challenged with a mixture of heroin and fentanyl, with a baseline recorded the day prior. A dose of 2 mg / kg of heroin containing 9% fentanyl was administered. This 1:10 fentanyl-to-heroin ratio was chosen to reflect typical adulteration in illicit markets.23'25Results indicated that the control group did not inhibit the effect of the drug mixture, suggesting the lack of sequestering anti -hapten antibodies. In contrast, vaccinated groups continued to demonstrate protection in hyperlocomotion activity. Notably, the TT-para- AmEtFenHap group showed reduced protection in heroin and fentanyl mixture (Fig. 14B).

[0316] Locomotor activity: Heroin

[0317] A week-long interval between opioid behavioral studies allows the drug to be cleared from the animals' systems, minimizing residual effects, preventing tolerance, and allowing withdrawal symptoms to subside, ensuring each study reflects the drug's acute effects rather than cumulative or lingering influences from previous doses26, 27. Therefore, with a week between each behavioral study at week 16, all five groups were tested with a dose of 2mg / kg of heroin. A baseline measure was recorded the day before administering heroin. Fig. 14C demonstrates the superiority of mQP -conjugated vaccines in protection against hyperlocomotion caused by heroin administration.

[0318] Locomotor activity: benzimidazole-derived NPS opioids (BNO), Etonitazene

[0319] To evaluate the efficacy of the mQP-para-AmEtFenHap vaccine against potent synthetic drugs increasingly found in street drugs, all five animal groups were challenged with 75 pg / kg of etonitazene at week 26. A saline baseline was recorded one day prior to etonitazene administration. Results showed that mice receiving three-dose vaccinations with mQP-para-AmEtFenHap and the bivalent mQP vaccine exhibited significantly lower ambulation counts compared to the control group, underscoring the ability of mQP- conjugated vaccines to elicit high-affinity antibodies effective against potent drugs like etonitazene. See Fig. 14D.

[0320] A notable observation emerged from the foregoing studies. Mice vaccinated with mQP- para-AmEtFenHap demonstrated a significantly greater ability to sequester all opioids tested in the study compared to TT-para-AmEtFenHap, which serves as a standard in the immunization field.

[0321] Example 12: Thermal Nociception Assays

[0322] Fentanyl and Heroin elicited with Fentanyl

[0323] Alternating weekly between challenges at week 13, all five groups of mice indicated above received 0.3 mg / kg of fentanyl; and at week 15, drug mixture comprising 2 mg / kg of heroin containing 9% fentanyl (w / w) via intraperitoneal (IP) injection. Fifteen minutes after each dose, thermal nociception was measured using a hotplate test. The latency from heat exposure to pain response (withdrawal time) was recorded. Latency measurements (antinociception) were expressed as a percentage of the maximum possible effect (% MPE). As shown in Fig. 15A and Fig. 15B, the mQP mono and bivalent groups displayed significantly reduced drug-induced antinociception, with notably lower % MPE values compared to the unvaccinated control mice. In contrast, the TT-para-AmEtFenHap group exhibited considerably less protection against the drug mixture.

[0324] Heroin

[0325] Similarly, at week 17, all animals were challenged with 2 mg / kg heroin. As shown in Fig. 15C, the mQP-conjugate groups significantly reduced withdrawal time in the drug- induced pain assay with an average % MPE that is 77%-fold lower than the control group and 30%-fold lower than the TT-para-AmEtFenHap group emphasizing mQP-conjugate vaccine dominance in protecting against heroin.

[0326] Benzimidazole-derived NPS Opioids (BNO), Etonitazene

[0327] One week after the locomotion study with etonitazene (week 27), all animals were administered 75 pg / kg etonitazene following a baseline latency measurement. As shown in Fig. 15D, the mQP-conjugate groups exhibited significantly lower latency compared to the control group.

[0328] Example 13: Summary of Broad Spectrum Opioid Vaccines

[0329] The research described herein demonstrates that the mutant form of the bacteriophage QP, associated with lower anticarrier antibody production, effectively boosted antibody responses when conjugated with an opioid- specific hapten (e.g., heroin). Additionally, the encapsulated RNA within QP VLPs (e.g., m QP) serves as a toll-like receptor ligand, enhancing T helper responses and boosting immunogenicity, thereby leveraging the VLP’s inherent adjuvant properties.5Building on this, the monovalent fentanyl vaccine, mQP-para- AmEtFenHap, was developed and demonstrated cross-protection against multiple types of opioids, alongside a bivalent vaccine combining mQP-para-AmEtFenHap and mQP-6- AmHap. Given that carriers like KLH, CRM- 197, and TT have been widely studied in anti- OUD vaccines, a side-by-side comparison of the mQP-para-AmEtFenHap vaccine with TT- para-AmEtFenHap was conducted to assess relative efficacy. Notable observations include: a) Immunization with the monovalent vaccine mQP-para-AmEtFenHap, TT-para AmEtFenHap and bivalent vaccine containing mQP-para-AmEtFenHap and mQP-6- AmHap generated high anti-hapten IgG titers. b) The resulting serum IgG from monovalent vaccine mQP-para-AmEtFenHap showed strong affinity binding to heroin, and metabolites and fentanyl and its analogs and etonitazene and its analogs and other drugs of abuse (table...) in vitro. c) Immunization protected mice from thermal antinociception and hyperlocomotion induced by challenges with heroin, fentanyl, and a heroin + 9% fentanyl (w / w) mixture as well as etonitazene. d) The two-dose mQP-para-AmEtFenHap immunization protocol achieved comparable results to the three-dose protocol in in vivo thermal antinociception and hyperlocomotion challenges.

[0330] As described herein, hapten-specific IgG endpoint titers exceeding 8 million ELISA units for both two- and three-dose regimens of the monovalent mQP-para-AmEtFenHap vaccine we observed after the initial prime shot. In contrast, TT-para-AmEtFenHap gradually achieved a titer value around 2 million ELISA units only after the complete three-dose immunization regimen. Immunization with the bivalent vaccine similarly induced high hapten- specific IgG endpoint titers. Combining two distinct antigens in a bivalent vaccine has been shown to result in significantly lower IgG endpoint titers compared to monovalent vaccines, as reported by Barrientos et al,28'30and with Pravetoni et al. demonstrating improvements by normalizing protein content.31A bivalent formulation was used, comprising 10 pg each of fentanyl and heroin vaccines (20 pg total) without normalizing protein content. The data similarly indicated a slightly lower antibody response in the bivalent group (Fig. 13A).

[0331] Cross -reactivity was observed between antibodies from the monovalent vaccines to multiple types of opioids, sufficient to impact in vivo outcomes when mice were challenged with heroin, fentanyl and etonitazene. The protective cross-reactivity of mQP-para- AmEtFenHap-induced antibodies with heroin and etonitazene may be attributed to a shared binding pocket accommodating aromatic rings. Without being bound by theory, the structures of fentanyl’s aromatic phenethyl group, heroin’s benzene rings within its morphine backbone, and etonitazene’s benzyl ring system all facilitate 7t-7t stacking and hydrophobic interactions at opioid receptors, forming a structural basis that enhances cross-reactivity potential. On the other hand, both heroin and etonitazene have ether or carbonyl linkages which can mimic each other’s stereochemistry and spatial distribution, affecting antibody binding and receptor affinity. These findings suggest that mQP-para-AmEtFenHap induces the production of antibodies with unique multi-recognition capabilities.

[0332] The hapten linker attachment site is crucial, as it determines the orientation of the molecule's face presented to the immune system, thus directing IgG specificity.32By exposing the intact fentanyl scaffold in para-AmEtFenHap fentanyl hapten, the immune system is sensitized to small changes in the N-alkyl, phenyl, and piperidine regions, enhancing antibody binding to fentanyl and its analogues, such as cyclopropyl fentanyl, furanyl fentanyl, para- fluorofentanyl, cis-3-methylfentanyl, and carfentanil. Given the potency and associated risks of these analogues, effective antibodies should cross-react with variations in these structural areas.

[0333] The inactive metabolite norfentanyl, missing phenyl ring c, demonstrated reduced / increased serum binding, highlighting rings a, b, and c’s critical roles in antibody binding. These findings align with the facial recognition model, suggesting that antibodies may rely on hydrophobic pockets for binding fentanyl’s rings a and c. Substitutions at fentanyl's key sites, such as N-alkyl groups, have minimal impact on binding, while modifications in the piperidine and phenyl rings significantly alter IgG affinity. Changes like the phenyl ring orientation in carfentanil and oxygen positioning in cis-3-methylfentanyl affect IgG interactions. Similarly, fluorine substitution on para-fluorofentanyl’ s phenyl ring reduces binding, highlighting structural features critical for antibody recognition.

[0334] As disclosed herein, the structure of 6-AmHap in mQP-6-AmHap influences selective antibody recognition. Upon conjugation, the C-3 and C-6 faces are oriented near the mQP surface, while the N-17 region faces outward, serving as a key epitope for antibody binding. This spatial configuration limits recognition of compounds with bulky N-17 groups, like naloxone and naltrexone, due to steric hindrance, allowing effective opioid pharmacotherapy without impacting vaccine efficacy. Simultaneously, this design enabled cross -recognition of other commonly abused opioids, such as codeine, oxycodone, and hydrocodone.

[0335] Both the monovalent and bivalent vaccine formulations showed a similar trend in recognizing a range of opioids of abuse (Table 3). Notably, the bivalent vaccine exhibited comparable cross-reactivity with heroin and its metabolites and demonstrated a broad reactivity profile across other opioids, aligning with the monovalent formulation indicating a subtle difference in binding strength across the opioid spectrum.

[0336] Hyperlocomotion and hot plate nociception assays were employed as surrogate metrics of vaccine efficacy. These assays were chosen to evaluate the vaccine's ability to attenuate opioid-induced effects in both vaccinated and unvaccinated groups. The observed crossreactivity of the antibodies suggested the potential for generating specific antibody responses not only to fentanyl but also to other compounds. Inspired by this possibility, we challenged the mice with heroin, heroin mixtures, and etonitazene to explore the in vivo sequestration power of these antibodies against off-target opioids. The immunized mice showed a strong reduction in fentanyl-induced (0.3 mg / kg) antinociception in the hot plate assay. The antinociception assay with the heroin-fentanyl mixture (2 mg / kg of heroin containing 9% fentanyl) demonstrated the superiority of mQP-para-AmEtFenHap (both two- and three-dose regimens) over TT-para-AmEtFenHap. The bivalent mQP conjugate vaccine showed comparable effects to the monovalent mQP-para-AmEtFenHap, with both significantly reducing withdrawal time compared to the TT-conjugate vaccine in heroin mixture challenge. Building on these findings, immunized mice displayed strong protection against challenges with heroin (2 mg / kg) and etonitazene (0.030 mg / kg), highlighting the enhanced in vivo efficacy of mQP-para-AmEtFenHap against potent drugs of abuse. The three-dose mQP-para- AmEtFenHap group demonstrated slightly greater protection than the two-dose group, but both surpassed the TT-para-AmEtFenHap group. The bivalent vaccine exhibited particularly strong results in the heroin-only and etonitazene challenges, suggesting that the mQP-6-AmHap component may contribute to the generation of heroin- specific antibodies, expanding the vaccine's protective scope.

[0337] To evaluate vaccine efficacy, all groups underwent challenge tests with fentanyl, a fentanyl-heroin mixture, heroin alone, and etonitazene, followed by assessments of opioid- induced hyperlocomotion. Baseline locomotion, measured prior to each challenge, confirmed that vaccinated groups displayed a strong reduction in fentanyl-induced hyperlocomotion compared to controls. Interestingly, the TT-para-AmEtFenHap vaccine group showed no protection when challenged with a higher dose of the heroin-fentanyl mixture (2 mg / kg heroin containing 9% fentanyl). With a lower dose (0.7 mg / kg heroin containing 9% fentanyl), some protection was observed in this group, but the lack of a full drug effect in the mixture limits the reliability of this result (data not shown). Both two- and three-dose mQP-para-AmEtFenHap groups, as well as the mQP-conjugate bivalent group, showed a significant decrease in ambulation counts on challenge days, indicating robust protection compared to controls.

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[0352] 31. Hwang, C. S.; Smith, L. C.; Natori, Y; Ellis, B.; Zhou, B.; Janda, K. D., Improved

[0353] Admixture Vaccine of Fentanyl and Heroin Hapten Immunoconjugates:

[0354] Antinociceptive Evaluation of Fentanyl-Contaminated Heroin. ACS Omega 2018, 3 (9), 11537-11543.

[0355] Incorporation by Reference

[0356] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0357] Equivalents

[0358] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

What is claimed is:

1. A vaccine composition comprising a hapten conjugated to a capsid.

2. The vaccine composition of claim 1, wherein the capsid comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty non-natural mutations.

3. The vaccine composition of claim 1 or 2, wherein the non-natural mutations comprise a disulfide bond mutation.

4. The vaccine composition of any one of claims 1-3, wherein the hapten is a derivative of a natural, synthetic, or semi-synthetic opioid.

5. The vaccine composition of claim 4, wherein the opioid is heroin, morphine, fentanyl, codeine, dihydrocodeine, oxycodone, hydrocodone, benzimidazole opioids, or metabolites thereof.

6. The vaccine composition of claim 4 or 5, wherein the hapten is a derivative of an opioid selected from the group consisting of fentanyl, heroin, morphine, 6-acetylmorphine, and morphine-3-P-glucuronide (M-3G).

7. The vaccine composition of any one of claims 1-6, wherein the hapten is 6-AmHap.

8. The vaccine composition of any one of claims 1-6, wherein the hapten is para- AmEtFenHap.

9. The vaccine composition of any one of claims 1-8, wherein the capsid is a bacteriophage capsid.

10. The vaccine composition of claim 9, wherein the bacteriophage is selected from the group consisting of (a): bacteriophage QP; (b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage Mi l; (h) bacteriophage MX1; (i) bacteriophage NL95; (j) bacteriophage f2; (k) bacteriophage PP7; (1) bacteriophage AP205; and (m) bacteriophage P22.

11. The vaccine composition of claim 10, wherein the bacteriophage is bacteriophage QP.

12. The vaccine composition of claim 11, wherein said capsid comprises at least one mutation selected from N10K, A38K, A40C, A40S, T75K, D102C, D102S, or A117K, or combination thereof.

13. The vaccine composition of claim 11, wherein said capsid comprises at least two mutations selected from A40C / D102C, A40S / D102S, or A43C / Q98C.

14. The vaccine composition of claim 11, wherein said capsid comprises at least three mutations selected from A40C / D102C / K13R or A38K / A40C / D102C.

15. The vaccine composition of claim 11, wherein said capsid comprises at least three mutations selected from A38K / A40C / D102C.

16. The vaccine composition of any one of claims 1-15, wherein the conjugated capsid comprises the formula:(X-Y)n-Z wherein X is the hapten; wherein Y is a linker; wherein Z is the capsid; and wherein n is at least 1.

17. The vaccine composition of claim 16, wherein Y is a polyethylene glycol (PEG) linker18. The vaccine composition of claim 16 or 17, wherein the polyethylene glycol (PEG) linker comprises 1-20 ethylene glycol units.

19. The vaccine composition of claim 18, wherein the polyethylene glycol (PEG) linker comprises 2 ethylene glycol units.

20. The vaccine composition of claim 18, wherein the polyethylene glycol (PEG) linker comprises 12 ethylene glycol units.

21. The vaccine composition of any one of claims 16-20, wherein Z is a bacteriophage QP capsid comprising at least three mutations selected from A38K / A40C / D102C.

22. The vaccine composition of claim 16-21, wherein X is 6-AmHap or para- AmEtFenHap.

23. The vaccine composition of claim 22, wherein X is 6-AmHap, Y is a polyethylene glycol (PEG) linker comprising 2 ethylene glycol units, and n is at least 1-1,000.

24. The vaccine composition of claim 22, wherein X is para-AmEtFenHap, Y is a polyethylene glycol (PEG) linker comprising 12 ethylene glycol units, and n is at least 1- 1,000.

25. The vaccine composition of any one of claims 1-24, wherein said composition comprises a monovalent vaccine.

26. The vaccine composition of claim 23, further comprising a second conjugated capsid comprising the formula:(X2-Y2)m-Z wherein X2is para-AmEtFenHap, Y2is a polyethylene glycol (PEG) linker comprising 12 ethylene glycol units, and m is at least 1-1,000.

27. The vaccine composition of any one of claims 1-26, further comprising an adjuvant.

28. The vaccine composition of claim 27, wherein the adjuvant is adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, P-glucan, CpG DNA, GPI- 0100, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D- isoglutamine, Pam3CSK4, quil A, Momordica saponin derivative II (VSA-2), monophosphoryl Lipid A (MPLA), double mutant heat-labile toxin (dmLT), Monophosphoryl-Lipid A (MPLA), Army Liposome Formulation (ALF), ALF adsorbed to aluminum hydroxide (ALFA), ALF containing QS21 saponin (ALFQ), or trehalose dimycolate.

29. The vaccine composition of claim 27, wherein the adjuvant is VSA-2, MPLA, dmLT, MPLA in ALFA, or ALFQ.

30. A broad spectrum vaccine composition capable of eliciting an immune response to a plurality of opioids following administration to a subject, said composition comprising a conjugated capsid comprising the formula:(X-Y)n-Z wherein X is a hapten; wherein Y is a linker;wherein Z is a capsid or tetanus toxoid (TT) carrier; and wherein n is at least 1-1000.

31. The vaccine composition of claim 30, wherein Y is a polyethylene glycol (PEG) linker32. The vaccine composition of claim 30 or 31, wherein the polyethylene glycol (PEG) linker comprises 1-20 ethylene glycol units.

33. The vaccine composition of claim 32, wherein the polyethylene glycol (PEG) linker comprises 2 ethylene glycol units.

34. The vaccine composition of claim 32, wherein the polyethylene glycol (PEG) linker comprises 12 ethylene glycol units.

35. The vaccine composition of any one of claims 30-34, wherein Z is a bacteriophage QP capsid comprising at least three mutations selected from A38K / A40C / D102C.

36. The vaccine composition of claim 30-35, wherein X is 6-AmHap or para- AmEtFenHap.

37. The vaccine composition of claim 36, wherein X is 6-AmHap, Y is a polyethylene glycol (PEG) linker comprising 2 ethylene glycol units, and n is at least 1-650.

38. The vaccine composition of claim 36, wherein X is para-AmEtFenHap, Y is a polyethylene glycol (PEG) linker comprising 12 ethylene glycol units, and n is at least 1-600.

39. The vaccine composition of claim 34, wherein Z is TT, X is 6-AmHap, Y is a polyethylene glycol (PEG) linker comprising 2 ethylene glycol units, and n is at least 1-40.

40. The vaccine composition of claim 34, wherein Z is TT, X is para-AmEtFenHap, Y is a polyethylene glycol (PEG) linker comprising 2 ethylene glycol units, and n is at least 1-40.

41. The vaccine composition of any one of claims 1-38, wherein said vaccine composition comprises a monovalent vaccine.

42. A bivalent vaccine composition comprising two conjugated capsids, said conjugated capsids respectively comprising the formulas: Formula I(X2-Y2)m-Z : Formula II wherein X1is 6-AmHap; wherein Y1is a is a polyethylene glycol (PEG) linker comprising 2 ethylene glycol units; wherein n is at least 1-1000; wherein X2is para-AmEtFenHap;Y2is a polyethylene glycol (PEG) linker comprising 12 ethylene glycol units; wherein m is at least 1-1000; and wherein wherein Z is a capsid.

43. The bivalent vaccine composition of claim 42, wherein the capsid comprises at least one, two, or three non-natural mutations.

44. The bivalent vaccine composition of claim 43, wherein the at least one, two, or three non-natural mutations comprise a disulfide bond mutation.

45. The bivalent vaccine composition of any one of claims 42-44, wherein the capsid is derived from bacteriophage Qp.

46. The bivalent vaccine composition of claim 45, wherein said capsid comprises at least three mutations selected from A40C / D102C / K13R or A38K / A40C / D102C.

47. The bivalent vaccine composition of claim 45 or 46, wherein said capsid comprises at least three mutations selected from A38K / A40C / D102C.

48. The bivalent vaccine composition of any one of claims 42-47, further comprising an adjuvant.

49. The bivalent vaccine composition of claim 48, wherein the adjuvant is adjuvant 65, a- GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, P-glucan, CpG DNA, GPI-0100, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L- alanyl-D-isoglutamine, Pam3CSK4, quil A, Momordica saponin derivative II (VSA-2), monophosphoryl Lipid A (MPLA), double mutant heat-labile toxin (dmLT), Monophosphoryl-Lipid A (MPLA), Army Liposome Formulation (ALF), ALF adsorbed to aluminum hydroxide (ALFA), ALF containing QS21 saponin (ALFQ), or trehalose dimycolate.

50. The bivalent vaccine composition of claim 48, wherein the adjuvant is VSA-2, MPLA, dmLT, MPLA in ALFA, or ALFQ.

51. A method of treating or preventing opioid addiction, comprising administering to a subject in need thereof the vaccine composition of any one of claims 1-50.

52. The method of claim 51, wherein the opioid addiction is an addiction to one or more of heroin, fentanyl, morphine, benzimidazole-derived new psychoactive substance opioids (BNO), codeine, oxycodone, hydrocodone, analogues and / or metabolites thereof, or any combination thereof.

53. A method of treating or preventing an opioid overdose, comprising administering to a subject in need thereof the vaccine composition of any one of claims 1-50.

54. The method of claim 53, wherein the opioid overdose is an overdose of one or more of heroin, fentanyl, morphine, BNO, codeine, oxycodone, hydrocodone, analogues and / or metabolites thereof, or any combination thereof.

55. A method of preventing or reducing the analgesic effect of an opioid in a subject, comprising administering to a subject in need thereof the vaccine composition of any one of claims 1-50.

56. The method of any one of claims 51-55, wherein the level of antibodies generated in response to the administered vaccine composition is sufficient to alter the distribution or behavioral effects of one or more opioids in the subject.

57. The method of any one of claims 51-56, wherein the subject is undergoing medication-assisted treatment (MAT).

58. The method of any one of claims 51-57, wherein the vaccine is administered conjointly with buprenorphine, naloxone, methadone, naltrexone, or any combination thereof.

59. The method of any one of claims 51-58, wherein the vaccine composition is administered systemically.

60. The method of claim 59, wherein the systemic administration is selected from the group consisting of oral, intravenous, intradermal, intraperitoneal, subcutaneous, and intramuscular administration.

61. The method of any one of claims 51-60, wherein the composition is administered prior to, concomitantly with, conjointly with, or following MAT.

62. The method of any one of claims 51-61, wherein the subject is a mammal.

63. The method of claim 62, wherein the mammal is human.

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