Opioid-Based Haptens: Development of Immunotherapy.

Over the past decades, extensive preclinical research has been conducted to develop vaccinations to protect against substance use disorder caused by opioids, nicotine, cocaine, and designer drugs. Morphine or fentanyl derivatives are small molecules, and these compounds are not immunogenic, but when conjugated as haptens to a carrier protein will elicit the production of antibodies capable of reacting specifically with the unconjugated hapten or its parent compound. The position of the attachment in opioid haptens to the carrier protein will influence the specificity of the antiserum produced in immunized animals with the hapten-carrier conjugate. Immunoassays for the determination of opioid drugs are based on the ability of drugs to inhibit the reaction between drug-specific antibodies and the corresponding drug-carrier conjugate or the corresponding labelled hapten. Pharmacological studies of the hapten-carrier conjugates resulted in the development of vaccines for treating opioid use disorders (OUDs). Immunotherapy for opioid addiction includes the induction of anti-drug vaccines which are composed of a hapten, a carrier protein, and adjuvants. In this review we survey the design of opioid haptens, the development of the opioid radioimmunoassay, and the results of immunotherapy for OUDs.

Cardiorespiratory anomalies and increased brainstem microglia in a rat model of neonatal opioid withdrawal syndrome.

Infants born with neonatal opioid withdrawal syndrome (NOWS) can display abnormal cardiorespiratory patterns including tachypnea, tachycardia, and impaired ventilatory responses to hypoxia (HVR) and hypercapnia (HCVR). Chronic morphine exposure is associated with increased midbrain microglial expression. Using a rat model of pre- and post-natal morphine exposure, we assessed cardiorespiratory features of NOWS (resting tachycardia and tachypnea) including the attenuated HVR and HCVR and whether they are associated with increased brainstem microglia expression. Pregnant rats (dams) received twice-daily subcutaneous injections of morphine (5 mg/kg) during the third (last) week of pregnancy to simulate 3rd trimester in utero opioid exposure. Offspring then received once-daily subcutaneous injections of morphine (0.5 mg/kg) until postnatal (P) day P10 days of age to simulate postnatal morphine therapy. Cardiorespiratory responses were assessed 24 h later (P11 days) following spontaneous withdrawal. Compared to saline-treated pups, morphine-exposed offspring exhibited tachycardia and tachypnea as well as an attenuated HVR and HCVR. Microglial cell counts were increased in the nucleus tractus solitarius (nTS), dorsal motor nucleus of the vagus (DMNV) and nucleus ambiguous (NAamb), but not the retrapezoid nucleus (RTN) or the non-cardiorespriatory region, the cuneate nucleus (CN). These data suggest that the cardiorespiratory features and autonomic dysregulation in NOWS infants may be associated with altered microglial function in specific brainstem cardiorespiratory control regions.

The morphine/heroin vaccine decreased the heroin-induced antinociceptive and reinforcing effects in three inbred strains mouse.

Clinical trials have indicated that a vaccine must be immunogenic in genetically diverse human populations and that immunogenicity and protective efficacy in animal models are two key indices required for the approval of a new vaccine. Additionally, the immune response (immunogenicity) and immunoprotection are dependent on the mouse strain. Therefore, the objective of the present study was to determine the immune response (immunogenicity) and the protective efficacy (behavioral response) in three inbred mouse strains immunized with the M6TT vaccine. Female BALB/c, C57Bl/6, and DBA/2 inbred mice were immunized with the M6-TT vaccine. A solid-phase antibody-capture ELISA was used to monitor antibody titer responses after each booster dose in vaccinated animals. The study used tail-flick testing to evaluate the antinociceptive effects induced by heroin. Additionally, heroin-induced locomotor activity and place preference were evaluated. The M6-TT vaccine was able to generate a specific antibody titer in the three inbred mouse strains evaluated. The antibodies reduced the antinociceptive effect of different doses of heroin. In addition, they decreased the heroin-induced locomotor activity and place preference. These findings suggest that the M6-TT vaccine generates a powerful immunogenic response capable of reducing the antinociceptive and reinforcing effects of heroin in different inbred mouse strains, which supports its possible future use in clinical trials in genetically diverse human populations.

Developing Translational Vaccines against Heroin and Fentanyl through Investigation of Adjuvants and Stability.

The nearly insurmountable adversity that accompanies opioid use disorder (OUD) creates life-altering complications for opioid users. To worsen matters, existing small-molecule drugs continue to inadequately address OUD due to their engagement of the opioid receptor, which can leave the user to deal with side effects and financial hardships from their repeated use. An alternative therapeutic approach utilizes endogenously generated antibodies through active vaccination to reduce the effect of opioids without modulating the opioid receptor. Here, we explore different adjuvants and storage conditions to improve opioid vaccine efficacy and shelf life. Our results revealed that inulin-based formulations (Advax) containing a CpG oligodeoxynucleotide (ODN) acted as effective adjuvants when combined with a heroin conjugate: immunized mice showed excellent recovery from heroin-induced antinociception accompanied by high titer, high opioid affinity serum antibodies similar to the immunopotentiating properties of traditional alum-based adjuvants. Moreover, nonhuman primates vaccinated with a heroin/fentanyl combination vaccine demonstrated potent antibody responses against opioids when formulated with both inulin and alum adjuvants. Finally, storing a freeze-dried opioid vaccine formulation maintained efficacy for up 1 year at room temperature. The results from our studies represent an advance toward a clinically feasible opioid vaccine.

A double-edged sword of using opioids and COVID-19: a toxicological view.

Today, COVID-19 is spreading around the world. Information about its mechanism, prognostic factors, and management is minimal. COVID-19, as a human disease, has several identifying phases. Physicians of patients with COVID-19 may be interested in knowing whether opioid use disorder may affect their patients' course or prognosis. This information may be crucial when considering the opioid epidemic in the US and other parts of the world. Opioid use at high doses and over several months duration can mitigate the immune system's function, which may complicate the course of COVID-19 disease. Potential suppression of parts of the immune response may be important in prevention, clinical support, and therapeutic use of medications in various phases of the COVID-19. Specifically, opioid use disorders via an inhalation route may enhance the "late hyper-inflammatory phase" or result in end-organ damage. It is well established that opioids decrease ventilation as their effect on the medullary respiratory centers increases the risk of pneumonia. This increased risk has been associated with immune-suppressive opioids. The ultimate role of opioids in COVID-19 is not clear. This paper endorses the need for clinical studies to decipher the role and impact of chronic opioid use on viral diseases such as COVID-19.

Effect of Opioid Use on Immune Activation and HIV Persistence on ART.

While there is an emerging consensus that engagement of the Mu opioid receptor by opioids may modulate various stages the HIV life cycle (e.g.: increasing cell susceptibility to infection, promoting viral transcription, and depressing immune responses to virally-infected cells), the overall effect on latency and viral reservoirs remains unclear. Importantly, the hypothesis that the increase in immune activation observed in chronic opioid users by direct or indirect mechanisms (i.e., microbial translocation) would lead to a larger HIV reservoir after ART-suppression has not been supported to date. The potential for a subsequent decrease in reservoirs after ART-suppression has been postulated and is supported by early reports of opioid users having lower latent HIV burden. Here, we review experimental data supporting the link between opioid use and HIV modulation, as well as the scientific premise for expecting differential changes in immune activation and HIV reservoir between different medications for opioid use disorder. A better understanding of potential changes in HIV reservoirs relative to the engagement of the Mu opioid receptor and ART-mediated immune reconstitution will help guide future cure-directed studies in persons living with HIV and opioid use disorder. Graphical Abstract Review. HIV replication, immune activation and dysbiosis: opioids may affect immune reconstitution outcomes despite viral suppression.

Opioid Use Disorder and COVID-19: Biological Plausibility for Worsened Outcomes.

BACKGROUND: Alarms have been raised that COVID-19 may disproportionately affect certain populations with substance use disorders, particularly Opioid Use Disorder (OUD), however warnings have largely focused on social risks such as reduced availability of services. Objectives: This commentary highlights three plausible biological mechanisms for potentially worsened outcomes in patients with OUD who contract COVID-19. Results: Opioid-related respiratory depression may amplify risks of hypoxemia from COVID-19 viral pneumonia. Complex opioid immune modulation may impact host response to COVID-19, though the effect direction and clinical significance are unclear. Drug-drug interactions may affect individuals with OUD who are co-administered medications for OUD and medications for COVID-19, particularly due to cardiac adverse effects. Conclusions/Importance: There are plausible biological mechanisms for potentially worsened outcomes in patients with OUD who contract COVID-19; these mechanisms require further study, and should be considered in individuals with OUD.

Single cell transcriptomics reveals opioid usage evokes widespread suppression of antiviral gene program.

Chronic opioid usage not only causes addiction behavior through the central nervous system, but also modulates the peripheral immune system. However, how opioid impacts the immune system is still barely characterized systematically. In order to understand the immune modulatory effect of opioids in an unbiased way, here we perform single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from opioid-dependent individuals and controls to show that chronic opioid usage evokes widespread suppression of antiviral gene program in naive monocytes, as well as in multiple immune cell types upon stimulation with the pathogen component lipopolysaccharide. Furthermore, scRNA-seq reveals the same phenomenon after a short in vitro morphine treatment. These findings indicate that both acute and chronic opioid exposure may be harmful to our immune system by suppressing the antiviral gene program. Our results suggest that further characterization of the immune modulatory effects of opioid is critical to ensure the safety of clinical opioids.

Preclinical Evaluation of Vaccines to Treat Opioid Use Disorders: How Close are We to a Clinically Viable Therapeutic?

The ongoing opioid crisis, now into its second decade, represents a global public health challenge. Moreover, the opioid crisis has manifested despite clinical access to three approved opioid use disorder medications: the full opioid agonist methadone, the partial opioid agonist buprenorphine, and the opioid antagonist naltrexone. Although current opioid use disorder medications are underutilized, the ongoing opioid crisis has also identified the need for basic research to develop both safer and more effective opioid use disorder medications. Emerging preclinical evidence suggests that opioid-targeted vaccines or immunopharmacotherapies may be promising opioid use disorder therapeutics. One premise for this article is to critically examine whether vaccine effectiveness evaluated using preclinical antinociceptive endpoints is predictive of vaccine effectiveness on abuse-related endpoints such as drug self-administration, drug discrimination, and conditioned place preference. A second premise is to apply decades of knowledge in the preclinical evaluation of candidate small-molecule therapeutics for opioid use disorder to the preclinical evaluation of candidate opioid use disorder immunopharmacotherapies. We conclude with preclinical experimental design attributes to enhance preclinical-to-clinical translatability and potential future directions for immunopharmacotherapies to address the dynamic illicit opioid environment.

Glial neuroimmune signaling in opioid reward.

The opioid epidemic is a growing public concern affecting millions of people worldwide. Opioid-induced reward is the initial and key process leading to opioid abuse and addiction. Therefore, a better understanding of opioid reward may be helpful in developing a treatment for opioid addiction. Emerging evidence suggests that glial cells, particularly microglia and astrocytes, play an essential role in modulating opioid reward. Indeed, glial cells and their associated immune signaling actively regulate neural activity and plasticity, and directly modulate opioid-induced rewarding behaviors. In this review, we describe the neuroimmune mechanisms of how glial cells affect synaptic transmission and plasticity as well as how opioids can activate glial cells affecting the glial-neuronal interaction. Last, we summarize current attempts of applying glial modulators in treating opioid reward.

Alum adjuvant is more effective than MF59 at prompting early germinal center formation in response to peptide-protein conjugates and enhancing efficacy of a vaccine against opioid use disorders.

Opioid use disorders (OUD) and fatal overdoses are a national emergency in the United States. Therapeutic vaccines offer a promising strategy to treat OUD and reduce the incidence of overdose. Immunization with opioid-based haptens conjugated to immunogenic carriers elicits opioid-specific antibodies that block opioid distribution to the brain and reduce opioid-induced behavior and toxicity in pre-clinical models. This study tested whether the efficacy of a lead oxycodone conjugate vaccine was improved by formulation in either aluminum hydroxide or the squalene-based oil-in-water emulsion MF59 adjuvant, which was recently FDA-approved for influenza vaccines in subjects 65+ years old. In adult BALB/c mice, alum formulation was more effective than MF59 at promoting the early expansion of hapten-specific B cells and the production of oxycodone-specific serum IgG antibodies, as well as blocking oxycodone distribution to the brain and oxycodone-induced motor activity. Alum was also more effective than MF59 at promoting early differentiation of peptide-specific MHCII-restricted CD4+ Tfh and GC-Tfh cells in adult C57Bl/6 mice immunized with a model peptide-protein conjugate. In contrast, alum and MF59 were equally effective in promoting hapten-specific B cells and peptide-specific MHCII-restricted CD4+ T cell differentiation in older C57Bl/6 mice. These data suggest that alum is a more effective adjuvant than MF59 for conjugate vaccines targeting synthetic small molecule haptens or peptide antigens in adult, but not aged, mice.

Neuroimmune mechanisms of psychostimulant and opioid use disorders.

Substance use disorders are global health problems with few effective treatment options. Unfortunately, most potential pharmacological treatments are hindered by abuse potential of their own, limited efficacy, or adverse side effects. As a consequence, there is a pressing need for the development of addiction treatments with limited abuse potential and fewer off target effects. Given the difficulties in developing new pharmacotherapies for substance use disorders, there has been growing interest in medications that act on non-traditional targets. Recent evidence suggests a role for dysregulated immune signaling in the pathophysiology of multiple psychiatric diseases. While there is evidence that immune responses in the periphery and the central nervous system are altered by exposure to drugs of abuse, the contributions of neuroimmune interactions to addictive behaviors are just beginning to be appreciated. In this review, we discuss the data on immunological changes seen in clinical populations with substance use disorders, as well as in translational animal models of addiction. Importantly, we highlight those mechanistic findings showing causal roles for central or peripheral immune mediators in substance use disorder and appropriate animal models. Based on the literature reviewed here, it is clear that brain-immune system interactions in substance use disorders are much more complex and important than previously understood. While much work remains to be done, there are tremendous potential therapeutic implications for immunomodulatory treatments in substance use disorders.

Preclinical Efficacy and Characterization of Candidate Vaccines for Treatment of Opioid Use Disorders Using Clinically Viable Carrier Proteins.

Vaccines may offer a new treatment strategy for opioid use disorders and opioid-related overdoses. To speed translation, this study evaluates opioid conjugate vaccines containing components suitable for pharmaceutical manufacturing and compares analytical assays for conjugate characterization. Three oxycodone-based haptens (OXY) containing either PEGylated or tetraglycine [(Gly)4] linkers were conjugated to a keyhole limpet hemocyanin (KLH) carrier protein via carbodiimide (EDAC) or maleimide chemistry. The EDAC-conjugated OXY(Gly)4-KLH was most effective in reducing oxycodone distribution to the brain in mice. Vaccine efficacy was T cell-dependent. The lead OXY hapten was conjugated to the KLH, tetanus toxoid, diphtheria cross-reactive material (CRM), as well as the E. coli-expressed CRM (EcoCRM) and nontoxic tetanus toxin heavy chain fragment C (rTTHc) carrier proteins. All vaccines induced early hapten-specific B cell expansion and showed equivalent efficacy against oxycodone in mice. However, some hapten-protein conjugates were easier to characterize for molecular weight and size. Finally, heroin vaccines formulated with either EcoCRM or KLH were equally effective in reducing heroin-induced antinociception and distribution to the brain of heroin and its metabolites in mice. This study identifies vaccine candidates and vaccine components for further development.

Blocking interleukin-4 enhances efficacy of vaccines for treatment of opioid abuse and prevention of opioid overdose.

Vaccines offer an option to treat heroin and prescription opioid abuse and prevent fatal overdoses. Opioid vaccines elicit antibodies that block opioid distribution to the brain and reduce opioid-induced behavioral effects and toxicity. The major limitation to the translation of addiction vaccines is that efficacy is observed only in subjects achieving optimal drug-specific serum antibody levels. This study tested whether efficacy of a vaccine against oxycodone is increased by immunomodulators targeting key cytokine signaling pathways involved in B and T cell lymphocyte activation. Blockage of IL-4 signaling increased vaccine efficacy in blocking oxycodone distribution to the brain and protection against opioid-induced behavior and toxicity in mice. This strategy generalized to a peptide-protein conjugate immunogen, and a tetanus-diphtheria-pertussis vaccine. These data demonstrate that cytokine-based immunomodulators increase efficacy of vaccines against small molecules, peptides and proteins, and identify IL-4 as a pharmacological target for improving efficacy of next-generation vaccines.

A Stable Heroin Analogue That Can Serve as a Vaccine Hapten to Induce Antibodies That Block the Effects of Heroin and Its Metabolites in Rodents and That Cross-React Immunologically with Related Drugs of Abuse.

An improved synthesis of a haptenic heroin surrogate 1 (6-AmHap) is reported. The intermediate needed for the preparation of 1 was described in the route in the synthesis of 2 (DiAmHap). A scalable procedure was developed to install the C-3 amido group. Using the Boc protectng group in 18 allowed preparation of 1 in an overall yield of 53% from 4 and eliminated the necessity of preparing the diamide 13. Hapten 1 was conjugated to tetanus toxoid and mixed with liposomes containing monophosphoryl lipid A as an adjuvant. The 1 vaccine induced high anti-1 IgG levels that reduced heroin-induced antinociception and locomotive behavioral changes following repeated subcutaneous and intravenous heroin challenges in mice and rats. Vaccinated mice had reduced heroin-induced hyperlocomotion following a 50 mg/kg heroin challenge. The 1 vaccine-induced antibodies bound to heroin and other abused opioids, including hydrocodone, oxycodone, hydromorphone, oxymorphone, and codeine.

More inflammation but less brain-derived neurotrophic factor in antisocial personality disorder.

Antisocial personality disorder (ASPD) is highly comorbid with substance use disorders (SUDs). We hypothesize that chronic neuroinflammation and the loss of neurotrophic factors prompts the pathogenesis of both disorders. We used ELISA to measure plasma levels of proinflammatory (tumor necrosis factor-α [TNF-α], C-reactive protein [CRP]) and anti-inflammatory factors (transforming growth factor-ß1 [TGF-ß1] and interleukin-10 [IL-10]), and brain-derived neurotrophic factor (BDNF) in male patients with ASPD (n=74), SUDs (n=168), ASPD comorbid with SUDs (ASPD+SUDs) (n=438), and Healthy Controls (HCs) (n=81). A multivariate analysis of covariance (MANCOVA) controlled for possible confounders was used to compare cytokines and BDNF levels between groups. The results of MANCOVA adjusted for age showed a significant (p<0.001) main effect of diagnosis on inflammatory factors and BDNF expression in these groups. ASPD, SUDs, and ASPD+SUDs patients had significantly (p<0.001) higher TNF-α levels but lower TGF-ß1 and BDNF levels. SUDs and ASPD+SUDs patients had higher IL-10 levels than did ASPD patients and HCs. There was no difference in IL-10 levels between HCs and ASPD. Moreover, subgrouping SUDs and ASPD±SUDs into opioid use disorder (OUD) and other SUDs groups showed that the IL-10 levels were specifically higher in OUD and ASPD±OUD groups than other SUDs (P≤0.001). We conclude that uncontrolled inflammation and losing neurotrophic factors, with or without comorbid SUDs, underlies ASPD. IL-10 expression might be more specifically associated with OUD.