Bacteriophage virus-like particle vaccines against xylazine

Abstract

The present invention is directed to virus-like particles (VLPs) preferably derived from Qbeta bacteriophage which are engineered to conjugate to xylazine and derivatives thereof. The xylazine and xylazine derivatives are conjugated often at high density to the virus-like particles to achieve long-lasting and high titer antibodies to the drugs of interest. Methods of treatment and vaccines are also described.

Description

[0001] BACTERIOPHAGE VIRUS-LIKE PARTICLE VACCINES AGAINST XYLAZINE Field of the Invention

[0001] The present invention is directed to virus-like particles (VLPs) preferably derived from Qbeta bacteriophage which are engineered to allow conjugation to xylazine and xylazine derivatives to treat the effects of such agents and related conditions and disease states related to xylazine use and addiction. In this invention, the compound xylazine and its derivaties are conjugated at high density to the virus-like particles. These conjugated virus- like particles are assessed for immunogenicity (e.g. in mice) over a range of doses and immunization schedules to assess [1] the titers of antibodies elicited by the vaccines, [2] the longevity of the antibody response, [3] the optimal dosing and immunization schedule to achieve long-lasting and high titer antibodies to the drugs of interest. Related Applications and Grant Support

[0002] This application claims the benefit of priority of provisional application s.n. US63 / 626,134, filed January 29, 2024, the entire contents of said application being incorporated by reference in its entirety.

[0003] This invention was made with government support under grant no. F31DA059236 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. Background and Overview of the Invention

[0004] The drug overdose crisis in the United States has been a public health issue of increasing urgency throughout the last several decades. The profile of substances causing overdoses has shifted over time to include cocaine, prescription opioids, heroin, and recently fentanyl and fentanyl analogs. Polysubstance drug use and the increasing prevalence of contaminating drug additives are exacerbating drug overdose rates. The inclusion of multiple drug classes in street drugs is done with the purpose of mitigating negative side effects of the primary drug or increasing positive reinforcement. Inclusion of multiple drugs in a street drug formulation leads to an increase of the risk of overdose, particularly with opioids. Xylazine, also known by its street name “Tranq”, is emerging as an increasingly popular addictive in the US drug supply.

[0005] Xylazine ((2,6-dimethylphenyl)-5,6-dihydro-4H-1,3-thiazin-2-amine) is a non-opiate alpha-2 adrenergic receptor agonist. Agonistic activity at this receptor causes decreased release of norepinephrine and epinephrine causing sedation, muscle relaxation anti- nociception, and respiratory depression. Xylazine was initially investigated as

[0006] an antihypertensive agent in humans but was not cleared for human use due to excessive adverse effects. Today, the compound is not a scheduled substance (i.e. there are no additional DEA restrictions on its purchase) and is FDA approved for uses in veterinary practices as a sedative or analgesic agent marketed as Rompun, Anased, Sedazine, and Chanazine. Standard side effects in animals include hypotension, anti-nociception, transient hypertension, and respiratory depression. Notably respiratory depression is the underlying causes of opioid overdoses, emphasizing the risk in simultaneous exposure to an opioid with xylazine. In addition to the central nervous system (CNS) impacts of xylazine, the drug can cause severe skin ulcerations or lesions as a result of decreased skin perfusion when injected, leading to increased risk of infections in persons who inject drugs (PWID).

[0007] Xylazine was first identified as an additive in illicit drugs in the early 2000s in Puerto Rico and subsequently spread throughout communities on the U.S. east coast throughout the 2010s. The drug continued to spread through the U.S. and the DEA reported seizure of xylazine in nearly all 50 states in 2021, warranting a public safety alert regarding the growing issue. Xylazine has spread into the western U.S. giving rise to a 750% increase in xylazine positive overdoses and a 112% increase in xylazine positive laboratory identified samples.

[0008] Over the past 5 years, six xylazine deaths were reported in New Mexico in young adults. As a result, the New Mexico Department of Health released a statement in April 2023 alerting the public about the growing risk of xylazine and its presence in the community. Currently, there is little research into treatments for xylazine toxicity and it is well- established that naloxone, the standard for opioid overdose intervention, is not efficacious in reducing xylazine toxicity due to the differences in receptor site of action.

[0009] Over the last several years, vaccines have been investigated as novel interventions for substance use disorders, with a focus on opioid use disorder (OUD). These strategies have primarily utilized opioid hapten targets conjugated onto large immunogenic protein carriers such as keyhole limpet hemocyanin (KLH), tetanus toxoid (TT), and diphtheria toxin cross- reactive material (CRM197). The overall approach of vaccines against opioids is to generate high-titer serum antibodies that limit the concentration of free drug that crosses the blood brain barrier to access opioid receptors in the CNS. These vaccines have shown promising preclinical success and several have entered phase-I clinical trials for investigation in patients. While these strategies have shown promise in animal models for blocking the effects of opioids, there remain significant challenges in translating this approach into a viable treatment of OUD and preventing overdose in humans.

[0010] Two major challenges facing opioid vaccines are: (1) needing multiple immunizations over a period of weeks to months to achieve effective antibody titers, and (2) antibody titers not being sufficiently high to protect against overdose. These challenges limit the usefulness of opioid vaccines for OUD and overdose prevention. We hypothesized that using Qβ bacteriophage virus-like particle (VLPs) as an immunogenic platform for displaying opioid drugs would address these two challenges and have received an NIH / NIDA UG3 grant to fund those studies. As shown preliminary data, we can conjugate opioid drug targets to Qβ VLPs to achieve high-titer antibodies against oxycodone or heroin with a single, intramuscular immunization without exogenous adjuvant in mice. Furthermore, we showed that in two doses, our Qβ VLP based vaccine protects against heroin-induced antinociception. The present invention is directed to determining whether the results for opioid vaccines could possibly translate to a vaccine for xylazine and its untoward effects. Brief Description of the Invention

[0011] Bacteriophage virus-like particles (VLPs) are highly immunogenic vaccine platforms that are multivalent platforms that can be used to dramatically increase the immunogenicity of molecules that are normally poorly immunogenic. The present invention is directed to virus-like particles (VLPs) derived from bacteriophages, especially Qbeta or AP205 bacteriophage which are engineered to provide VLPs to allow conjugation to xylazine and its derivatives. The xylazine compounds are conjugated at high density to the virus-like particles. The xylazine compounds are conjugated principally to lysine residues which exist on the surface of the VLP.

[0012] The present invention provides immunotherapeutic and prophylactic Qbeta bacteriophage viral-like particles (VLPs) conjugated to xylazine and xylazine derivatives which are useful in the treatment and prevention of xylazine dependency, xylazine overdose, xylazine toxicity and xylazine side effects which often manifest after use of xylazine in patients in need. Related compositions (e.g. immunogenic compositions, including vaccines) and therapeutic methods are also provided. VLPs and related compositions of the invention induce high titer antibody responses to protect against xylazine dependency, hydralazine overdose, xylazine toxicity and associated symptoms and side effects in subjects in need. VLPs, VLP-containing compositions, and therapeutic methods of the invention induce an immunogenic response against xylazine and xylazine derivatives, confer immunity against, protect against and reduce the likelihood of xylazine dependency, xylazine overdose, xylazine toxicity and related symptoms and side effects of xylazine overuse as disclosed herein caused by xylazine use, especially including xylazine overuse.

[0013] To that end, the inventors propose an approach, using Qbeta or AP205 VLPs, to elicit high-titer and long-lasting antibodies more rapidly, potentially after only one dose, and without the use of exogenous adjuvants. Notably, there is a well-established clinical pathway for bacteriophage VLP-based vaccines. As of late 2022, a number of Qbeta bacteriophage VLP-based vaccines had entered clinical trials, including several that had moved into phase II / IIa trials. These include VLP-based vaccines for Alzheimer’s Disease, allergy, and hypertension that target amyloid-beta, dust mite allergen, and angiotensin II, among others, respectively. These trials also demonstrated that VLP-based vaccines are highly immunogenic in humans.

[0014] For these reasons, VLPs can be used as a platform to elicit rapid, high titer, and long- lasting antibody responses to xylazine and xylazine derivatives. These features are required for effective vaccine-based treatment for xylazine overuse and / or overdose and the symptoms and side effects elicited. The present vaccines provide an unexpectedly quick immunogenic response to the vaccines of the present invention which represents an unexpected result.

[0015] In embodiments, the bacteriophage coat protein used to form the VLPs is a coat protein derived from Qbeta or AP205 bacteriophage, preferably a coat protein derived from Qbeta bacteriophage.

[0016] In embodiments, the bacteriophage coat protein used to form the VLPs is a Qβ, MS2, or AP205 bacteriophage single coat protein, preferably a Qβ bacteriophage single coat protein which self-assembles into VLPs. In embodiments, the single coat protein is presented as a dimer. In embodiments, often 90 single coat protein dimers or 180 bacteriophage single coat proteins self-assemble into a VLP onto the surface of which immunogenic haptens comprising xylazine or xylazine derivatives are conjugated. In embodiments, the xylazine haptens are conjugated to the surface of the VLP. In embodiments, Xylazine-PEG2-(Gly)4-Cys is conjugated onto the surface of the VLP (often Qβ-VLP) to a surface-exposed lysine through a crosslinker as described herein, often SMPH.

[0017] In embodiments, in the composition according to the present invention the xylazine conjugate determinant is displayed at one or more nucleophilic or electrophilic amino acid residues on the surface of the bacteriophage, preferably at a plurality of lysine residues on the surface of the VLP. In embodiments, the xylazine conjugate is displayed on the bacteriophage at the lysine residues by covalently binding a xylazine molecule to the lysine residues through a linker group. In embodiments, the linker group comprises a 4 to 15 mer, preferably a 4 to 10 mer oligopeptide covalently bonded to a crosslinker as described herein.

[0018] In embodiments of the present invention, the oligopeptide of the xylazine conjugate is covalently bonded to an electrophilic or nucleophilic group of the xylazine molecule (often an exocyclic amine group) which optionally has been modified to facilitate the binding of the oligopeptide to the hydrazine molecule and the crosslinker is bonded to the nucleophilic or electrophilic amino acid residues, preferably lysine residues on the surface of the bacteriophage through the crosslinker. In embodiments, the oligopeptide of the linker is a 4 to 15 mer, preferably a 4 to 10 mer oligopeptide comprising neutral amino acid residues bonded to nucleophilic or electrophilic sites, often an exocyclic amine group on the xylazine molecule. In embodiments, on one end of the oligopeptide, often the carboxyl terminus, the oligopeptide comprises a cysteinyl group or other amino acid which may be used to link the oligopeptide to the crosslinker. The amino end of the oligopeptide may optionally be conjugated to the xylazine molecule through the use of a short amide linker (e.g. a C1-C4 alkyl amide group which forms a urea or urethane group with the xylazine radical) or a phenol group, among others.

[0019] In embodiments, the neutral amino acid residues are selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, methione, proline, serine and mixtures thereof. In embodiments, the neutral amino acids often are selected from the group consisting of glycine, serine and mixtures thereof, more often glycine.

[0020] In embodiments, the xylazine conjugate comprises a xylazine radical as depicted below.

[0021] In embodiments, xylazine forms a conjugate with a pegylated oligopeptide, to form a conjugate, often Xylazine-PEG2-(Gly4)-Cys, which can be conjugated or covalently bonded to the surface of the VLP at an electrophilic or hydrophilic site, such as a lysine, through a crosslinker to form a xylazine conjugated VLP.

[0022] In embodiments, the present invention is directed to a population of virus-like particles as otherwise described herein.

[0023] In embodiments, the present invention is directed to a pharmaceutical composition comprising a population of virus-like particles as described herein in combination with a pharmaceutically acceptable carrier, additive and / or excipient. In embodiments, the composition is formulated for administration to a subject or patient as a vaccine. In embodiments the pharmaceutical composition or vaccine comprises an adjuvant (e.g., Advax, MF 59, CPG 1018, AS01B, AS03, AS04, etc.).

[0024] In embodiments, the present invention is directed to a method for enhancing an immune response against a xylazine compound in a patient or subject in need comprising introducing a pharmaceutical composition comprising a population of VLPs as otherwise described herein to said subject or patient, wherein an enhanced immune response against said xylazine compound is produced in said patient or subject. In embodiments, the present invention is directed to a method for reducing the likelihood of xylazine-induced dependency, overdose, toxicity, symptoms and side effects in a patient or subject in need, including high blood sugar, low blood pressure, low heart rate, blurry vision, coma, contracted pupils (miosis), disorientation, drowsiness, shallow breathing, central nervous system depression, staggering and disfiguring and potentially life-threating ulcers, including necrotic ulcers. In embodiments, the present invention is directed to a method wherein the composition is prophylactic for a xylazine induced disorder or for reducing the likelihood of dependency, overdose, toxicity, symptoms or side effects of xylazine overuse .

[0025] In embodiments, the present invention is directed to a method of inducing an immunogenic response in a patient or subject comprising administering a composition comprising an effective amount of a population of xylazine conjugated VLPs as otherwise described herein to said patient or subject.

[0026] In embodiments, the present invention is directed to a method for treating or inhibiting xylazine dependency, xylazine overdose, or a disorder, symptom or side effect thereof in a patient or subject in need comprising administering to said patient or subject a composition comprising an effective amount of a population of xylazine conjugated VLPs as otherwise described herein to said patient or subject.

[0027] In embodiments, the disorder is xylazine dependency. In embodiments, the disorder is hydrazine overdose or a symptom or side effect of xylazine overuse. In embodiments, the symptom or side effect is one or more of high blood sugar, low blood pressure, low heart rate, blurry vision, coma, contracted pupils (miosis), disorientation, drowsiness, shallow breathing, central nervous system depression, staggering and disfiguring and potentially life-threating ulcers, including necrotic ulcers.

[0028] In embodiments, the present invention is directed to a method for treating or reducing the likelihood of a xylazine overdose or inhibiting or reducing the likelihood of a symptom thereof in a patient or subject in need comprising administering to said patient a composition comprising an effective amount of a population of xylazine conjugated VLPs as otherwise described herein to said patient or subject. In embodiments, the symptom is high blood sugar, low blood pressure, low heart rate, blurry vision, coma, contracted pupils (miosis), disorientation, drowsiness, shallow breathing, central nervous system depression, staggering and disfiguring and potentially life-threating ulcers, including necrotic ulcers.

[0029] The present invention is therefore directed to vaccines which target xylazine and xylazine derivative drugs as otherwise disclosed herein for prophylactic and / or therapeutic purposes. Brief Description of the Figures

[0030] FIGURES 1A-C shows the chemical conjugation of Xylazine-PEG2-(Gly)4-Cys to Qβ-VLPs. (A) shows the Structure of Qβ-VLP with surface-exposed lysines (small light dots). (B) Drug conjugation to Qβ-VLP using the crosslinker SMPH. (C) Coomassie-stained SDS-PAGE showing successful conjugation as indicated by the increase in molecular weight and laddering pattern.

[0031] FIGURE 2 shows that immunization with Qβ-xylazine generates high titer antibody responses in mice. Endpoint dilution anti-xylazine IgG titers determined by ELISA at the indicated time points. Intramuscular immunizations (20μg / 50μL) were conducted on days 0 and 21. n=8, male / female BALB / c.

[0032] FIGURE 3 shows the Dose response of respiratory frequency measured in breaths per minute (bpm) after subcutaneous injection with 50, 25, 10 or 5mg / kg xylazine in male (A) or female (B) mice. Naïve animals (n=4) were acclimated in whole body plethysmography chambers for 30 minutes followed by xylazine injection at t=30min and monitored for an additional 1.5hr. Detailed Description of the Invention

[0033] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, 2001, "Molecular Cloning: A Laboratory Manual"; Ausubel, ed., 1994, "Current Protocols in Molecular Biology" Volumes I-III; Celis, ed., 1994, "Cell Biology: A Laboratory Handbook" Volumes I-III; Coligan, ed., 1994, "Current Protocols in Immunology" Volumes I-III; Gait ed., 1984, "Oligonucleotide Synthesis"; Hames & Higgins eds., 1985, "Nucleic Acid Hybridization"; Hames & Higgins, eds., 1984,"Transcription And Translation"; Freshney, ed., 1986, "Animal Cell Culture"; IRL Press, 1986, "Immobilized Cells And Enzymes"; Perbal, 1984, "A Practical Guide To Molecular Cloning."

[0034] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise,

[0035] between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0037] It must be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

[0038] Furthermore, the following terms shall have the definitions set out below.

[0039] The term “patient” or “subject” is used throughout the specification within context to describe an animal, generally a mammal and preferably a human, to whom treatment, including prophylactic treatment (prophylaxis), with the immunogenic compositions and / or vaccines according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. In most instances, the patient or subject of the present invention is a human patient of either or both genders.

[0040] The term “effective” is used herein, unless otherwise indicated, to describe a number of VLP’s or an amount of a VLP-containing composition which, in context, is used to produce or effect an intended result, whether that result relates to the prophylaxis and / or therapy of xylazine dependency and / or xylazine use or overdose as otherwise described herein. The term effective subsumes all other effective amount or effective concentration terms (including the term “therapeutically effective”) which are otherwise described or used in the present application.

[0041] As used herein, the term xylazine or a “xylazine derivative” refers to the compound according to the chemical structure or a derivative of such chemical structure as depicted herein below. Xylazine is a drug used for sedation, anesthesia, muscle relaxation and analgesia in veterinary applications, typically in animals such as horses, cattle, and other non-human mammals. Xylazine is an analog of clonidine and is an agonist at the α2class of adrenergic receptor.

[0042] As used herein, the term “opioid”, “opiate” or “opioid drug” is used to describe a compound, distinguishable from xylazine, which interacts with an opiate receptor to produce a pharmacological response, often analgesia. Opioid compounds may be found co- administered with xylazine in drug users. These agents typically produce dependency and related symptoms among other symptoms as described herein often occur when the drug is no longer administered. These drugs are chemically conjugated with VLPs according to the present invention in order to produce an immunogenic response in a subject administered same against opiates as otherwise described herein. Typical opiates include, for example, codeine, fentanyl, hydrocodone, hydromorphone, meperidine, methadone, morphine, oxycodone and diacetylmorphine (heroin), hydroxylmorphine, 2,4-dinitrophenylmorphine, 6- methyldihydromorphine, 6-methylenedihydrodesoxymorphine, 6-acetyldihydromorphine, chloranaltrexamine, chloroxymorphamine, dexomorphine, dihydromorphine, ethyldihydromorphine, hydromorphinol, methyldesorphine, morphine methyl bromide, n- phenylnordesomorphine, N-phenylnormorphine, 6-nicotinoyldihydromorphine, acetylpropionylmorphine, 3,6-dibutanoylmorphine, dibutyrylmorphine, dibenzoylmorphine, diformylmorphine, diacetyl morphine (herone), dipropanooymorphine, nicomorphine, 6- monoacetylecodeine, benzylmorphine, codeine methylbromide, desocodeine, dimethylmorphine, ethyldihydromorphine, heterocodeine, dihydrocodeine, isocodeine, morpholinylethylmorphine, myrophine, transisocodeine, acetylcodone, acetylmorphone, dihydrocodeine, hydroxycodeine, codeinone, hydrocodone, hydromorphone, morphinol and morphinone, among others.

[0043] The term “xylazine conjugate” refers to a xylazine or xylazine derivative molecule which is conjugated to the external surface of a VLP, often a Qβ or AP205 bacteriophage, often a Qβ bacteriophage through a linker molecule to a nucleophilic amino acid on the surface of the bacteriophage. In embodiments, the nucleophilic amino acid is often a lysine residue on the surface of the bacteriophage. The xylazine is conjugated to the bacteriophage through a linker molecule. Often the linker molecule comprises a 4-15 mer, often a 4-12 mer, a 4-10 mer, a 4-8 mer a 4-6 mer or a 4 mer oligopeptide (preferably comprising neutral amino acid residues) which is covalently bonded to a crosslinker molecule as described herein to form the linker. The oligopeptide is covalently linked at one end to the xylazine radical hapten often through a nucleophilic functional group on the xylazine (an exocyclic amine group which is optionally further linked by a PEG group to the oligopeptide or other group, and on the other end to the crosslinker, which links the VLP to the oligopeptide and the xylazine hapten. This is shown in FIGURE 1B. In FIGURE 1B, xylazine is linked to the oligopeptide (in this case (Gly)4Cys) through a PEG2(di(ethylene glycol)) group to form a conjugated xylazine group which is further crosslinked to the surface of the VLP using a crosslinker (as depicted in FIGURE 1B SMPH) which links the distil end of the oligopeptide (as depicted, a cysteinyl group) to an amine group in the side chain of a lysine residue on the surface of the VLP, often a Qβ VLP.

[0044] The term “crosslinker” or “crosslinking agent” refers to a chemical compound used to covalently bind, or conjugate, biomolecules together, such as an oligopeptide to a VLP or an oligopeptide to a xylazine hapten. The term "protein crosslinking" refers to utilizing protein crosslinkers to conjugate peptides or proteins together. Crosslinking agents for use herein possess reactive moieties specific to various electrophilic or nucleophilic functional groups (e.g., sulfhydryls, amines, carbohydrates, carboxyl groups, hydroxyl groups, carbonyls, etc.) on proteins, peptides, or other molecular complexes or molecules such as opioids as described herein. The atoms separating a crosslinker agent’s reactive groups, and eventually the conjugated oligopeptide / VLP or oligopeptide / xylazine form the “spacer arm”. A zero- length crosslinker refers to protein crosslinkers that join two molecules without adding additional spacer arm atoms. Homobifunctional crosslinker reagents have the same reactive group on both ends of the spacer arm (i.e., Amine Reactive-Amine Reactive); while heterobifnctional crosslinkers have different reactive groups on each end of a spacer arm (i.e., Sulfhydryl Reactive-Amine Reactive). It is noted that in addition to the following crosslinking agents, additional short-chain crosslinking agents such as short-chain PEG groups (from 1-6 ethylene glycol units in length, often two ethylene glycol units in length), short-chain alkyl amides (CH2)iC(O)NH2, (CH2)iC(O), C(O)(CH2)iC(O), NHC(O)(CH2)iC(O) or NHC(O)(CH2)iC(O)NH groups where i is from 0 to 4, often 1 to 4, can be used to link a xylazine hapten to an oligopeptide or a crosslinker to a lysine group on the VLP. The following crosslinking agents are exemplary for use in the present invention with SMPH often being used: ANB-NOS (N-5-Azido-2-nitrobenzoyloxysuccinimide) BMPS N-(ß-Maleimidopropyloxy)succinimide ester EMCS (N-[e-Maleimidocaproyloxy]succinimide ester) GMBS (N-[Gamma-Maleimidobutyryloxy] Succinimide) LC-SPDP Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate) MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester) PDPH (3-[2-Pyridyldithio]propionyl hydrazide) SBA (N-Succinimidyl bromoacetate) SIA (N-Succinimidyl iodoacetate) Sulfo-SIA N-Sulfosuccinimidyl iodoacetate) SMCC (Succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate) SMPB (N-Succinimidyl 4-[4-maleimidophenyl]butyrate) SMPH (Succinimidyl-6-[ß-maleimidopropionamido]hexanoate) SPDP (N-Succinimidyl 3-[2-pyridyldithio]-propionate) Sulfo-LC-SPDP Sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate Sulfo-MBS (m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester) Sulfo-SANPAH (N-Sulfosuccinimidyl-6-[4'-azido-2'-nitrophenylamino] hexanoate) sulfo-SMCC (Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate) BS2G (Bis[Sulfosuccinimidyl] glutarate) BS3 (Bis[sulfosuccinimidyl] suberate) DSG (Disuccinimidyl glutarate) DSP (Dithiobis[succinimidyl propionate]) DSS (Disuccinimidyl suberate) DSSeb (Disuccinimidyl sebacate) DST (Disuccinimidyl tartrate) DTSSP (3,3´-Dithiobis[sulfosuccinimidylpropionate]) EGS (Ethylene glycolbis(succinimidylsuccinate) Sulfo-EGS Ethylene glycolbis(sulfosuccinimidylsuccinate) CDI (N,N'-Carbonyldiimidazole) DCC (N,N'-dicyclohexylcarbodiimide) EDC-HCl 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) NHS (N-hydroxysuccinimide) and Sulfo-NHS (N-hydroxysulfosuccinimide).

[0045] Preferred crosslinkers for use in the present invention are heterobifunctional agents which are capable of linking Amine-to-Sulfhydryl groups. Exemplary crosslinking agents include:SIA (succinimidyl iodoacetate)SBAP (succinimidyl 3-(bromoacetamido)propionate) SIAB (succinimidyl (4-iodoacetyl)aminobenzoate)Sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl)aminobenzoate)AMAS (N-α-maleimidoacet-oxysuccinimide ester) BMPS (N-β-maleimidopropyl-oxysuccinimide ester) GMBS (N-γ-maleimidobutyryl-oxysuccinimide ester)MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester)SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) EMCS (N-ε-malemidocaproyl-oxysuccinimide ester)Sulfo-GMBS (N-γ-maleimidobutyryl-oxysulfosuccinimide ester)Sulfo-MBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester)Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate)Sulfo-EMCS (N-ε-maleimidocaproyl-oxysulfosuccinimide ester)Sulfo-SMPB (sulfosuccinimidyl 4-(N-maleimidophenyl)butyrate)SMPB (succinimidyl 4-(p-maleimidophenyl)butyrate)SMPH (Succinimidyl 6-((beta-maleimidopropionamido)hexanoate))LC-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6- amidocaproate)) andSulfo-KMUS (N-κ-maleimidoundecanoyl-oxysulfosuccinimide ester).

[0046] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single- stranded DNA and RNA. A polynucleotide may include nucleotide sequences having different functions, such as coding regions, and non-coding regions such as regulatory sequences (e.g., promoters or transcriptional terminators). A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment.

[0047] As used herein, the term "polypeptide" refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term "polypeptide" also includes molecules which contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e g., dimers, tetramers). Thus, the terms peptide, oligopeptide, and protein are all included within the definition of polypeptide and these terms may be used interchangeably in context. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. Often, in the present invention, oligopeptide linkers are used to link a xylazine or xylazine derivative through a separate crosslinker to the surface of a VLP.

[0048] The term “single-chain dimer” refers to a normally dimeric protein whose two subunits of coat polypeptide of a RNA bacteriophage have been genetically (chemically, through covalent bonds) fused into a single polypeptide chain. Specifically, in the present invention single-chain dimer versions of bacteriophage are often constructed. Each of these proteins is naturally a dimer of identical polypeptide chains. In certain of the bacteriophages coat protein dimers the N-terminus of one subunit lies in close physical proximity to the C- terminus of the companion subunit. Single-chain coat protein dimers were produced using recombinant DNA methods by duplicating the DNA coding sequence of the coat proteins and then fusing them to one another in tail to head fashion. The result is a single polypeptide chain in which the coat protein amino acid appears twice, with the C-terminus of the upstream copy covalently fused to the N-terminus of the downstream copy. Normally (wild- type) the two subunits are associated only through noncovalent interactions between the two chains. In the single-chain dimer these noncovalent interactions are maintained, but the two subunits have additionally been covalently tethered to one another. This greatly stabilizes the folded structure of the protein and confers to it its high tolerance of peptide insertions as described above.

[0049] The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired function is retained by the polypeptide. NH2refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.

[0050] The term “valency” is used to describe the density of the opioid conjugates displayed on VLPs according to the present invention. Valency in the present invention may range from low valency to high valency (“high density”), from less than 1 to more than about 180, often 10 to 25 to more than 180, often 90 to 180 or more (e.g between 180-720, or between 1 and 4 conjugates per coat protein in the VLP). Immunogenic compositions according to the present invention comprise VLPs which are preferably high valency and comprise VLPs which display at least 50-60 up to about 180 or more, often 90-720 or more, often 180 to 720 or more crosslinked conjugated xylazine per VLP as otherwise described herein. In embodiments, at least 90 xylazine conjugates are “high density” because the display of 90 copies of antigen / hapten on the surface of the VLP produces high titer antibodies.

[0051] The term “coding sequence” is defined herein as a portion of a nucleic acid sequence which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by a ribosome binding site (prokaryotes) or by the ATG start codon (eukaryotes) located just upstream of the open reading frame at the 5’- end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3’- end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0052] A "heterologous" region of a recombinant cell is an identifiable segment of nucleic acid within a larger nucleic acid molecule that is not found in association with the larger molecule in nature.

[0053] An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.

[0054] A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

[0055] In bacteria, transcription normally terminates at specific transcription termination sequences, which typically are categorized as rho-dependent and rho-independent (or intrinsic) terminators, depending on whether they require the action of the bacterial rho-factor for their activity. These terminators specify the sites at which RNA polymerase is caused to stop its transcription activity, and thus they largely define the 3’-ends of the RNAs, although sometimes subsequent action of ribonucleases further trims the RNA.

[0056] An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

[0057] An “antibiotic resistance gene” refers to a gene that encodes a protein that renders a bacterium resistant to a given antibiotic. For example, the kanamycin resistance gene directs the synthesis of a phosphotransferase that modifies and inactivates the drug. The presence on plasmids of a kanamycin resistance gene provides a mechanism to select for the presence of the plasmid within transformed bacteria. Similarly, the chloramphenicol resistance gene allows bacteria to grow in the presence of the drug by producing an acetyltransferase enzyme that inactivates the antibiotic through acetylation.

[0058] The term “PCR” refers to the polymerase chain reaction, a technique used for the amplification of specific DNA sequences in vitro. The term “PCR primer” refers to DNA sequences (usually synthetic oligonucleotides) able to anneal to a target DNA, thus allowing a DNA polymerase (e.g. Taq DNA polymerase) to initiate DNA synthesis. Pairs of PCR primers are used in the polymerase chain reaction to initiate DNA synthesis on each of the two strands of a DNA and to thus amplify the DNA segment between two primers. Representative PCR primers which used in the present invention are those which are presented in the examples section hereof.

[0059] A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid, which normally replicate independently of the bacterial chromosome by virtue of the presence on the plasmid of a replication origin. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.

[0060] A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

[0061] It should be appreciated that also within the scope of the present invention are nucleic acid sequences encoding the polypeptide(s) of the present invention, which code for a polypeptide having the same amino acid sequence as the sequences disclosed herein, but which are degenerate to the nucleic acids disclosed herein. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid.

[0062] As used herein, "epitope" refers to an antigenic determinant of a polypeptide. An epitope could comprise 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 4 such amino acids, and more often, consists of at least 5-10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.

[0063] As used herein, the term "coat protein(s)" refers to the protein(s) of a bacteriophage or a RNA-phage capable of being incorporated within the capsid assembly of the bacteriophage or the RNA-phage. These include, but are not limited to Q^, AP205, PP7, MS2, AP205, R17, SP, PP7, GA, M11, MX1, f4, Cb5, Cb12r, Cb23r, 7s and f2 RNA bacteriophages. Preferred coat proteins which are used in the present invention include coat proteins from bacteriophages include Q^, AP205, PP7 and MS2. Preferably, Q^ or AP205, most often Q^ coat polypeptides are used to create conjugated VLPs according to the present invention. Often these are formed from monomeric coat polypeptides which self-assemble (180 units) to from a VLP.

[0064] As used herein, a “coat polypeptide” as defined herein is a polypeptide of the full length coat protein of the bacteriophage, a polypeptide fragment of the coat protein that possesses coat protein function and additionally encompasses the full length coat protein as well or single-chain variants thereof.

[0065] As used herein, the term "immune response" refers to a humoral immune response and / or cellular immune response leading to the activation or proliferation of B- and / or T- lymphocytes and / or antigen presenting cells. In some instances, however, the immune responses may be of low intensity and become detectable only when using at least one substance in accordance with the invention. "Immunogenic" refers to an agent used to stimulate the immune system of a living organism, so that one or more functions of the immune system are increased and directed towards the immunogenic agent. An "immunogenic xylazine" is a conjugated xylazine that elicits a cellular and / or humoral immune response as described above, whether alone or linked to a carrier in the presence or absence of an adjuvant. Preferably, antigen presenting cell may be activated.

[0066] As used herein, the term "vaccine" refers to a formulation which contains the composition of the present invention and which is in a form that is capable of being administered to an animal, often a human patient or subject.

[0067] As used herein, the term "virus-like particle of a bacteriophage" refers to a virus-like particle (VLP) resembling the structure of a bacteriophage, being non-replicative and noninfectious, and lacking at least the gene or genes encoding for the replication machinery of the bacteriophage, and typically also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host. In embodiments of the present invention Qβ-VLPs are used.

[0068] This definition should, however, also encompass virus-like particles of bacteriophages, in which the aforementioned gene or genes are still present but inactive, and, therefore, also leading to non-replicative and noninfectious virus-like particles of a bacteriophage.

[0069] VLP of RNA bacteriophage coat protein: The capsid structure formed from the self- assembly of one or more subunits of RNA bacteriophage coat protein and optionally containing host RNA is referred to as a "VLP of RNA bacteriophage coat protein". In a particular embodiment, the capsid structure is formed from the self assembly of 90 coat protein single-chain dimers or 180 coat protein monomers. In the case of Qβ or AP205 VLPs 180 coat protein monomers typically self-assemble into the VLP.

[0070] A nucleic acid molecule is "operatively linked" to, or “operably associated with”, an expression control sequence when the expression control sequence controls and regulates the transcription and translation of nucleic acid sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the nucleic acid sequence to be expressed and maintaining the correct reading frame to permit expression of the nucleic acid sequence under the control of the expression control sequence and production of the desired product encoded by the nucleic acid sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene. Xylazine Dependency, Immunogenicity, and Prophylactic Efficacy

[0071] “Xylazine dependency”, “xylazine Use” or “xylazine Overdose” includes, but is not limited to, the disorders, symptoms and side effects identified in this application which are caused by xylazine use, includingxylazine overuse. Immunogenicity and prophylactic efficacy (e.g. whether a composition is prophylactic for xylazine induced disorders such as dependency or overdose) or other symptoms and / or side effects of xylazine use may be evaluated either by the techniques and standards mentioned in this section, or through other methodologies that are well-known to those of ordinary skill in the art.

[0072] To assess immunogenicity (e.g. whether a composition has induced a high titer antibody responses against xylazine), an anti-xylazine geometric mean titer (GMT) can be measured by ELISA, e.g. after a few weeks of treatment (e.g.3 or 4 weeks) and after administration of a few dosages (e.g.3 or 4). The percentage of subjects who seroconverted for xylazine antigenicity (OA) after a few weeks of treatment (e.g.3 or 4 weeks) and after administration of a few dosages (e.g.3 or 4) can also be determined to assess immunogenicity.

[0073] To assess efficacy assessment of sequestration of opiates in the blood (not getting to site of action in the brain) and the ability of the vaccine to block the effects of xylazine is performed.

[0074] Production of Virus-Like Particles

[0075] The present invention is directed to virus-like phage particles as well as methods for producing these particles in vivo as well as in vitro. As used herein, producing virions "in vitro" refers to producing virions outside of a cell, for instance, in a cell-free system, while producing virions "in vivo" refers to producing virions inside a cell, for instance, an Escherichia coli or Pseudomonas aeruginosa cell or a yeast cell among others.

[0076] Bacteriophages

[0077] The VLPs described herein consist of assemblies of the coat proteins of single-strand RNA bacteriophage [RNA Bacteriophages, in The Bacteriophages. Calendar, RL, ed. Oxford University Press.2005]. The known viruses of this group attack bacteria as diverse as E. coli, Pseudomonas and Acinetobacter. Each possesses a highly similar genome organization, replication strategy, and virion structure. In particular, the bacteriophages contain a single-stranded (+)-sense RNA genome, contain maturase, coat and replicase genes, and have small (<300 angstrom) icosahedral capsids. These include but are not limited to Q^, AP205, PP7, MS2, R17, SP, PP7, GA, M11, MX1, f4, Cb5, Cb12r, Cb23r, 7s and f2 RNA bacteriophages. Q^ and AP205 RNA bacteriophages are preferred, Q^ is most preferred. Q^ and AP205 RNA bacteriophages form self-assembled VLPs from 180 monomeric coat polypeptide units. Methods for producing these coat polypeptides are well known in the art. See, for example Freivalds, et al., J Biotechnol., 2006 May 29;123(3):297- 303.

[0078] The information required for assembly of the icosahedral capsid shell of this family of bacteriophage is contained entirely within coat protein itself. For example, purified coat protein can form capsids in vitro in a process stimulated by the presence of RNA [Beckett et al., 1988, J. Mol Biol 204: 939-47]. Moreover, coat protein expressed in cells from a plasmid assembles into a virus-like particle in vivo [Peabody, D.S., 1990, J Biol Chem 265: 5684- 5689].

[0079] The preferred VLP for use in the present invention is a Qbeta or Qβ VLP. These VLPs are typically made by transformation of E. coli with a plasmid expressing the Qbeta coat protein as a monomer under a lac promoter. Colonies are selected on kanamycin Luria Broth (LB) agar plates. A single colony is used to inoculate LB broth and grown overnight at 37 degrees C. This is then used to inoculate a larger culture. Cultures are shaken at 37 degrees C for several hours until OD600 reaches 0.8. Then the expression of the Qbeta coat protein is induced with Isopropyl β-d-1-thiogalactopyranoside (IPTG) and incubated another 3 hours. Then cells are pelleted and frozen at -20 degrees C. Lysis of bacteria is then performed in isotonic buffer with sonication. VLPs are isolated by size exclusion chromatography and endotoxin is depleted with sequential Triton-X-100 phase extraction. There are many alternative methods to isolate the VLPs, which are well known in the art.

[0080] RNA Bacteriophage Coat Polypeptide

[0081] The coat polypeptides useful in the present invention also include those having similarity with one or more of the coat polypeptides described above. The similarity is referred to as structural similarity. Structural similarity may be determined by aligning the residues of the two amino acid sequences (i.e., a candidate amino acid sequence and the amino acid sequence) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate amino acid sequence can be isolated from a single stranded RNA virus, or can be produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, two amino acid sequences are compared using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI), or the Blastp program of the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMS Microbial Lett 1999, 174:247-250), and available at http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / bl2.html. Preferably, the default values for all BLAST 2 search parameters are used, including matrix =BLOSUM62; open gap penalty = 11, extension gap penalty = 1, gap xdropoff = 50, expect = 10, wordsize = 3, and optionally, filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as "identities." Preferably, a coat polypeptide also includes polypeptides with an amino acid sequence having at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, or at least 95% amino acid identity to one or more of the amino acid sequences disclosed above. Preferably, a coat polypeptide is active. Whether a coat polypeptide is active can be determined by evaluating the ability of the polypeptide to form a capsid and package a single stranded RNA molecule. Such an evaluation can be done using an in vivo or in vitro system, and such methods are known in the art and routine. Alternatively, a polypeptide may be considered to be structurally similar if it has similar three-dimensional structure as the recited coat polypeptide and / or functional activity.

[0082] The Xylazine Conjugate

[0083] As described herein, in certain embodiments the xylazine conjugate may be present (covalently linked) to the VLP in the A-B loop, at the N-terminus or the carboxy terminus of a coat polypeptide. Preferably, the xylazine conjugate is covalently linked on the outer surface of the capsid. In particularly preferred embodiments, the approach is to attach / conjugate the xylazine conjugates to the lysines present on the surface of Qbeta VLPs. Attached Figure 1 shows this strategy.

[0084] Qbeta VLPs lysine positions:

[0085] The lysine residues which are available for conjugation on the coat polypeptide of the Qbeta VLPs of the present invention are set forth in the enclosed coat polypeptide sequence (SEQ ID NO:1) herein below: 1 akletvtlgn igkdgkqtlv lnprgvnptn gvaslsqaga vpalekrvtv svsqpsrnrk 61 nykvqvkiqn ptactangsc dpsvtrqaya dvtfsftqys tdeerafvrt elaallaspl 121 lidaidqlnp ay

[0086] Lysine amino acid residues are indicated above in bold. They are at amino acid positions 2, 13, 16, 46, 60, 63, and 67 of the monomeric coat polypeptide.

[0087] In embodiments, the present invention is directed to A-B loop, N-terminal or C- terminal presentation / conjugation of xylazine conjugates on VLPs including PP7 and MS2. These VLP-CXs can be used singly or as a combination vaccine. In embodiments, the present invention is preferably directed to VLPs formed from monomeric single coat polypeptides of Qbeta VLPs as described herein. The inventors show protection against xylazine dependency, overdose, symptoms and side effects of xylazine use / overuse using a vaccine consisting of a Qbeta VLP conjugated to xylazine as described herein.

[0088] In an embodiment, the coat polypeptide is a single-chain dimer containing an upstream and downstream subunit. Each subunit contains a functional coat polypeptide sequence. The xylazine conjugate may be conjugated to the upstream and / or downstream subunit at the sites mentioned herein above, e.g., the A-B loop, the N-terminus or a carboxyl terminus. In a particular embodiment, the coat polypeptide is a single chain of a Q^ bacteriophage (which self-assembles from 180 monomeric coat polypeptides), or a single chain dimer of PP7 or MS2 coat polypeptide, preferably a Q^ coat polypeptide, although a number of bacteriophage coat polypeptides may be used. Preparation of Transcription Unit

[0089] The transcription unit of the present invention comprises an expression regulatory region, (e.g., a promoter), a sequence encoding a coat polypeptide and transcription terminator. The RNA polynucleotide may optionally include a coat recognition site (also referred to a “packaging signal”, “translational operator sequence”, “coat recognition site”). Alternatively, the transcription unit may be free of the translational operator sequence. The promoter, coding region, transcription terminator, and, when present, the coat recognition site, are generally operably linked. “Operably linked" or “operably associated with” refer to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to, or “operably associated with”, a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence. The coat recognition site, when present, may be at any location within the RNA polynucleotide provided it functions in the intended manner.

[0090] The invention is not limited by the use of any particular promoter, and a wide variety of promoters are known. The promoter used in the invention can be a constitutive or an inducible promoter. Preferred promoters are able to drive high levels of RNA encoded by me coding region encoding the coat polypeptide Examples of such promoters are known in the art and include, for instance, the lac promoter, T7, T3, and SP6 promoters.

[0091] The nucleotide sequences of the coding regions encoding coat polypeptides described herein are readily determined. These classes of nucleotide sequences are large but finite, and the nucleotide sequence of each member of the class can be readily determined by one skilled in the art by reference to the standard genetic code. Furthermore, the coding sequence of an RNA bacteriophage single chain coat polypeptide comprises a site for covalent binding of an xylazine conjugate. In a particular embodiment, the site for insertion of the xylazine conjugate at an appropriate amino acid residue exposed on the surface of the VLP (i.e., an amino acid which contains a functional group capable of conjugation to the xylazine conjugate) and the xylazine conjugate is conjugated onto amino acid sidechains (often lysine sidechains) on the surface of the VLP.

[0092] In an embodiment, the coding region encodes a single-chain monomer of the coat polypeptide of Qbeta (Qβ) bacteriophage. In a most particular embodiment, the coding region encodes a single-chain coat polypeptide monomer, where the conjugation of the xylazine conjugate may occur on electrophlic or nucleophilic sidechains of amino acids on the surface of the VLP, most often lysine sidechains. The transcription unit may contain a bacterial promoter, such as a lac promoter or it may contain a bacteriophage promoter, such as a T7 promoter.

[0093] Synthesis

[0094] The VLPs of the present invention may be produced in vivo by introducing transcription units into bacteria, especially if transcription units contain a bacterial promoter. Alternatively, it may be synthesized in vitro in a coupled cell-free transcription / translation system.

[0095] The preferred VLP for use in the present invention is a Qbeta or Qβ VLP. These VLPs are typically made by transformation of E. coli with a plasmid expressing the Qbeta coat protein as a monomer under a lac promoter. Colonies are selected on kanamycin Luria Broth (LB) agar plates. A single colony is used to inoculate LB broth and grown overnight at 37 degrees C. This is then used to inoculate a larger culture. Cultures are shaken at 37 degrees C for several hours until OD600 reaches 0.8. Then the expression of the Qbeta coat protein is induced with Isopropyl β-d-1-thiogalactopyranoside (IPTG) and incubated another 3 hours. Then cells are pelleted and frozen at -20 degrees C. Lysis of bacteria is then performed in isotonic buffer with sonication. VLPs are isolated by size exclusion chromatography and endotoxin is depleted with sequential Triton-X-100 phase extraction. There are many alternative methods to isolate the VLPs, which are well known in the art. Assembly of VLPs

[0096] As noted above, the VLPs of the present invention conjugate xylazine or xylazine derivatives on the surface of the VLPs. These VLPs may also be assembled in combination with another substance, such as an adjuvant. Specifically, purified coat protein subunits are obtained from VLPs that have been disaggregated with a denaturant (usually acetic acid). The adjuvant is mixed with coat protein, which is then reassembled in its presence. In a particular embodiment, the substance has some affinity for the interior of the VLP and is preferably negatively charged.

[0097] In another embodiment, the adjuvant is passively diffused into the VLP through pores that naturally exist in the VLP surface. In a particular embodiment, the substance is small enough to pass through these pores and has a high affinity for the interior of the VLP.

[0098] EXAMPLES

[0099] Formation of Xylazine Conjugated Qβ VLPs

[0100] Qβ VLPs were purified after expression in E. coli. Xylazine-GLY4Cys was chemically conjugated to the surface of purified Qβ VLPs using the bifunctional crosslinker SMPH . SMPH was incubated for 2 hours at room temperature with Qβ VLPs at a 4:1 molar ratio. Excess SMPH was then removed using a 10K MW cutoff Amicon centrifugation filter. Xylazine-Gly4Cys was then added at a 10:1 molar ratio and incubated at 4degrees C overnight. Successful conjugation was confirmed by SDS-PAGE and Coomassie Blue staining, showing a ladder of additional higher molecular weight bands compared to control unconjugated Qβ coat protein. The results of this experiment are presented in enclosed FIGURE 1.

[0101] Immunization of Balb / C Mice and In Vitro Response

[0102] Balb / c mice (n=10, M / F) were immunized with Qβ-xylazine or unconjugated Qβ- control via intramuscular injection (20μg / 50μL) on day 0 and day 21. Blood samples were collected by retro-orbital bleed and sera isolated by centrifugation on days 3,7,14,21,28,35 at 42 post-first immunization. Serum antibody responses to xylazine were measured by enzyme linked immunosorbent assay (ELISA). Ninety-six well plates were coated for 2 hours at room temperature with 250 ng / well of xylazine-BSA in PBS. After blocking with 0.5% Milk in PBS overnight at 4°C, diluted mouse sera (using four-fold dilutions from 1:40 to 1:655360) were added to each well and incubated at room temperature for 2 hours. After five washes using PBS, HRP-conjugated goat anti-mouse IgG was added at a 1:5000 dilution in blocking buffer and incubated for 1 hour at room temperature. Following five washes, 50μl substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added to each well then plates were incubated for 10 min while shaking. The reaction was stopped by addition of 50μl 1% hydrochloric acid to each well. The absorbance of each well at 450 nm was measured using a microplate reader. Endpoint dilution IgG is reported as the final serum dilution that generated A450 greater than twice that of background. The results of this experiment are shown in enclosed FIGURE 2.

[0103] Assessment of Xylazine-induced Respiratory Depression

[0104] Xylazine-induced respiratory depression was assessed in naive mice (n=4, BALB / c M / F). Animals were assigned one of four subcutaneous xylazine doses (5mg / kg, 10mg / kg, 25 mg / kg or 50mg / kg). Prior to drug administration, mice were acclimated to respiratory chambers of a whole-body plethysmography (WBP) system for 30-min to collect baseline respiratory frequency (breaths per in minute; BPM). Once steady-state respiration was established, xylazine was administered at the assigned dosage. Respiratory frequency was then measured in response to the drug over a 90 min time-course. Data collection was conducted using a WBP AHR universal study with pull bias flow mode. These results are presented in enclosed FIGURE 3.

Claims

Claims:

1. A composition comprising: (a) a virus-like particle (VLP) comprising a bacteriophage coat protein; and (b) at least one conjugated xylazine determinant; wherein said xylazine determinant is displayed on said virus-like particle, and wherein said determinant comprises a conjugated xylazine derived from xylazine or a xylazine derivative compound.

2. The composition of claim 1, wherein said xylazine conjugate determinant is displayed at one or more lysine residues at the A-B loop, N-terminus or carboxy terminus of said bacteriophage coat protein.

3. The composition of claim 1 or 2, wherein said xylazine conjugate determinant is displayed at high density on the surface of said VLP.

4. The composition of any of claims 1-3 wherein the bacteriophage coat protein is a coat protein derived from Qbeta or AP205 bacteriophage.

5. The composition of claim 1, 3 or 4 wherein said bacteriophage coat protein is a coat protein derived from Qbeta bacteriophage.

6. The composition of claim 1, 3 or 43 wherein said bacteriophage coat protein is a coat protein derived from AP205 bacteriophage.

7. The composition according to any one of claims 1-6 wherein said xylazine conjugate determinant is displayed at one or more lysine residues on the surface of the bacteriophage.

8. The composition according to claim 2 or 7 wherein said xylazine conjugate is displayed on said bacteriophage at said lysine residues by covalently binding a xylazine or xylazine derivative molecule to said lysine residues through a linker group.

9. The composition according to claim 8 wherein said linker group comprises an oligopeptide covalently bonded to a crosslinker.

10. The composition according to claim 9 wherein said oligopeptide is covalently bonded to an electrophilic or nucleophilic group on the xylazine molecule and the crosslinker is bonded to said lysine residues on the surface of said bacteriophage.

11. The composition according to claim 10 wherein said nucleophilic group is the exocyclic amine group of xylazine.

12. The composition according to claims 8-11 wherein said oligopeptide is a 4 to 15 mer oligopeptide comprising neutral amino acid residues bonded to an amine group on said xylazine molecule.

13. The composition according to claim 12 wherein said neutral amino acid residues are selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, methione, proline, serine and mixtures thereof.

14. The composition according to claim 12 or 13 wherein said neutral amino acids are selected from the group consisting of glycine, serine and mixture thereof.

15. The composition according to any of claims 12-14 wherein said amino acid residues are glycine residues.

16. A composition according to any of claims 1, 3, 4, 5 and 7-15 as depicted in FIGURE 1B hereof.

17. A population of virus-like particles according to any of claims 1-16.

18. A pharmaceutical composition comprising a population of virus-like particles according to claim 18 in combination with a pharmaceutically acceptable carrier, additive and / or excipient.

19. The composition according to claim 18 which is formulated as a vaccine for administration to a subject or patient.

20. The composition according to claim 19 wherein said vaccine comprises an adjuvant.

21. A method for enhancing an immune response against xylazine or a xylazine derivative compound in a patient or subject in need comprising introducing the composition of claim 18 or 19 into said subject or patient, wherein an enhanced immune response against said xylazine or xylazine derivative compound is produced in said patient or subject.

22. The method of claim 25, wherein the composition is prophylactic for a xylazine induced disorder.

23. A method of inducing an immunogenic response in a patient or subject comprising administering a composition according to claim 18 or 19 to said patient or subject.

24. A method for treating or inhibiting xylazine use disorder, xylazine overdose or a symptom or side effect thereof in a patient or subject in need comprising administering to said patient a composition according to claim 18 or 19 to said patient or subject.

25. The method of claim 24 wherein said disorder is xylazine dependency.

26. The method of claim 24 wherein said symptom is high blood sugar, low blood pressure, low heart rate, blurry vision, coma, contracted pupils (miosis), disorientation, drowsiness, shallow breathing, central nervous system depression, staggering and disfiguring and potentially life-threating ulcers, including necrotic ulcers.

27. A method for treating or reducing the likelihood of a xylazine overdose or a symptom thereof in a patient or subject in need comprising administering to said patient a composition according to claim 18 or 19 to said patient or subject.

28. The method according to claim 31 wherein said symptom is high blood sugar, low blood pressure, low heart rate, blurry vision, coma, contracted pupils (miosis), disorientation, drowsiness, shallow breathing, central nervous system depression, staggering and disfiguring and potentially life-threating ulcers, including necrotic ulcers.

29. A method for treating or reducing the likelihood of drug-induced coma in a patient or subject in need comprising administering to said patient or subject a composition according to claim 18 or 19 to said patient or subject.

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