Multivalent Vaccination Strategies Protect against Exposure to Polydrug Opioid and Stimulant Mixtures in Mice and Rats.

Illicit drug mixtures containing opioids and stimulants have been responsible for the majority of fatal drug overdoses among occasional users, and those with either opioid use disorder (OUD) or substance use disorder (SUD). As a complementary strategy to current pharmacotherapies, active immunization with conjugate vaccines has been proposed as a viable intervention to treat OUD as well as other SUD for which there are either limited or no treatment options. Vaccination against opioids and stimulants could help address the limitations of current medications (e.g., patient access, compliance, misuse liability, and safety) by providing an additional tool to prevent drug misuse and/or overdoses. However, more research is needed to fully understand the potential benefits and limitations of using vaccines to treat SUD and overdose and to inform us on how to deploy this strategy in the field. Previous reports have shown promise by combining two vaccines into bivalent vaccine formulations to concurrently target multiple drugs. Here, multiple individual candidate monovalent vaccines were incrementally combined in multivalent vaccine formulations to simultaneously target fentanyl, carfentanil, oxycodone, heroin, methamphetamine, and their analogs or metabolites. Bi-, tri-, and quadrivalent vaccine formulations induced the formation of independent serum antibody responses against their respective opioid targets and selectively attenuated the distribution of each individual drug to the brain in mice and rats. Results indicate that a single injection of an admixed multivalent vaccine formulation may be more effective than coinjecting multiple monovalent vaccines at multiple sites. Finally, adding a methamphetamine conjugate vaccine to an quadrivalent opioid vaccine in a pentavalent formulation did not interfere with the production of effective antiopioid IgG antibodies. Multivalent vaccines could provide multifaceted, yet selective, protection against polydrug use and exposure.

Self-Adjuvanting TLR7/8 Agonist and Fentanyl Hapten Co-Conjugate Achieves Enhanced Protection against Fentanyl Challenge.

Currently approved pharmacotherapies for opioid use disorders (OUDs) and overdose reversal agents are insufficient to slow the spread of OUDs due to the proliferation of fentanyl. This is evident in the 31% rise in drug overdose deaths from 2019 to 2022, with rates increasing from 21.6 to 28.3 overdoses per 100,000 deaths. Vaccines are a potential alternative or adjunct therapy for the treatment of several substance use disorders (nicotine, cocaine) but have shown limited clinical success due to suboptimal antibody titers. In this study, we demonstrate that coconjugation of a Toll-like receptor 7/8 (TLR7/8) agonist (UM-3006) alongside a fentanyl-based hapten (F1) on the surface of the carrier protein cross-reactive material 197 (CRM) significantly increased generation of high-affinity fentanyl-specific antibodies. This demonstrated enhanced protection against fentanyl challenges relative to an unconjugated (admix) adjuvant control in mice. Inclusion of aluminum hydroxide (alum) adjuvant further increased titers and enhanced protection, as determined by analysis of fentanyl concentration in serum and brain tissue. Collectively, our findings present a promising approach to enhance the efficacy of antiopioid vaccines, underscoring the need for extensive exploration of TLR7/8 agonist conjugates as a compelling strategy to combat opioid use disorders.

A TLR7/8 agonist increases efficacy of anti-fentanyl vaccines in rodent and porcine models.

Opioid use disorders (OUD) and overdose are public health threats worldwide. Widespread access to highly potent illicit synthetic opioids such as fentanyl is driving the recent rise in fatal overdoses. Vaccines containing fentanyl-based haptens conjugated to immunogenic carrier proteins offer a long-lasting, safe, and cost-effective strategy to protect individuals from overdose upon accidental or deliberate exposure to fentanyl and its analogs. Prophylactic or therapeutic active immunization with an anti-fentanyl vaccine induces the production of fentanyl-specific antibodies that bind the drug in the blood and prevent its distribution to the brain, which reduces its reinforcing effects and attenuates respiratory depression and bradycardia. To increase the efficacy of a lead anti-fentanyl vaccine, this study tested whether the incorporation of synthetic toll-like receptor (TLR) 4 and TLR7/8 agonists as vaccine adjuvants would increase vaccine efficacy against fentanyl challenge, overdose, and self-administration in either rats or Hanford miniature pigs. Formulation of the vaccine with a nucleolipid TLR7/8 agonist enhanced its immunogenicity and efficacy in preventing fentanyl-induced respiratory depression, analgesia, bradycardia, and self-administration in either rats or mini-pigs. These studies support the use of TLR7/8 adjuvants in vaccine formulations to improve their clinical efficacy against OUD and potentially other substance use disorders (SUD).

A lipidated TLR7/8 adjuvant enhances the efficacy of a vaccine against fentanyl in mice.

Opioid use disorders (OUD) and opioid-related fatal overdoses are a public health concern in the United States. Approximately 100,000 fatal opioid-related overdoses occurred annually from mid-2020 to the present, the majority of which involved fentanyl or fentanyl analogs. Vaccines have been proposed as a therapeutic and prophylactic strategy to offer selective and long-lasting protection against accidental or deliberate exposure to fentanyl and closely related analogs. To support the development of a clinically viable anti-opioid vaccine suitable for human use, the incorporation of adjuvants will be required to elicit high titers of high-affinity circulating antibodies specific to the target opioid. Here we demonstrate that the addition of a synthetic TLR7/8 agonist, INI-4001, but not a synthetic TLR4 agonist, INI-2002, to a candidate conjugate vaccine consisting of a fentanyl-based hapten, F1, conjugated to the diphtheria cross-reactive material (CRM), significantly increased generation of high-affinity F1-specific antibody concentrations, and reduced drug distribution to the brain after fentanyl administration in mice.

Evaluation of missed influenza vaccination opportunities in the emergency department.

BACKGROUND: Seasonal influenza is associated with significant healthcare resource utilization. An estimated 490,000 hospitalizations and 34,000 deaths were attributed to influenza during the 2018-2019 season. Despite robust influenza vaccination programs in both the inpatient and outpatient setting, the emergency department (ED) represents a missed opportunity to vaccinate patients at high risk for influenza who do not have access to routine preventive care. Feasibility and implementation of ED-based influenza vaccination programs have been previously described but have stopped short of describing the predicted health resource impact. The goal of our study was to describe the potential impact of an influenza vaccination program in an urban adult emergency department population using historic patient data. METHODS: This was a retrospective study of all encounters within a tertiary care hospital-based ED and three freestanding EDs during influenza season (defined as October 1 - April 30) over a two-years, 2018-2020. Data was obtained from the electronic medical record (EPIC®). All ED encounters during the study period were screened for inclusion using ICD 10 codes. Patients with a confirmed positive influenza test and no documented influenza vaccine for the current season were reviewed for any ED encounter at least 14 days prior to the influenza-positive encounter and during the concurrent influenza season. These ED visits were deemed a missed opportunity to provide vaccination and potentially prevent the influenza-positive encounter. Healthcare resource utilization, including subsequent ED encounters and inpatient admissions, were evaluated for patients with a missed vaccination opportunity. RESULTS: A total of 116,140 ED encounters occurred during the study and were screened for inclusion. Of these, 2115 were influenza-positive encounters, which represented 1963 unique patients. There were 418 patients (21.3%) that had a missed opportunity to be vaccinated during an ED encounter at least 14 days prior to the influenza-positive encounter. Of those with a missed vaccination opportunity, 60 patients (14.4%) had subsequent influenza-related encounters, including 69 ED visits and 7 inpatient admissions. CONCLUSION: Patients presenting to the ED with influenza frequently had opportunities to be vaccinated during prior ED encounters. An ED-based influenza vaccination program could potentially reduce influenza-related burden on healthcare resources by preventing future influenza-related ED encounters and hospitalizations.

IL-4 Predicts the Efficacy of a Candidate Antioxycodone Vaccine and Alters Vaccine-Specific Antibody-Secreting Cell Proliferation in Mice.

Opioid use disorders (OUDs) are a public health concern in the United States and worldwide. Current medications for OUDs may trigger side effects and are often heavily regulated. A novel treatment strategy to be used alone or in combination with existing medications is active immunization with antiopioid vaccines, which stimulate production of opioid-specific Abs that bind to the target drug and prevent its distribution to the brain. Although antiopioid vaccines have shown promising preclinical efficacy, prior clinical evaluations of vaccines targeting stimulants indicate that efficacy is limited to a subset of subjects who achieve optimal Ab responses. We have previously reported that depletion of IL-4 with a mAb increased opioid-specific IgG2a and total IgG, and it increased the number of germinal centers and germinal center T follicular helper cells in response to antiopioid vaccines via type I IL-4 signaling. The current study further investigates the mechanisms associated with IL-4-mediated increases in efficacy and whether IL-4 depletion affects specific processes involved in germinal center formation, including affinity maturation, class switching, and plasma cell differentiation in mice. Additionally, results demonstrate that preimmunization production of IL-4 after ex vivo whole blood stimulation predicted in vivo vaccine-induced Ab titers in outbred mice. Such mechanistic studies are critical for rational design of next-generation vaccine formulations, and they support the use of IL-4 as a predictive biomarker in ongoing OUD vaccine clinical studies.

Efficacy and Selectivity of Monovalent and Bivalent Vaccination Strategies to Protect against Exposure to Carfentanil, Fentanyl, and Their Mixtures in Rats.

Drug-related fatal overdoses have significantly increased in the past decade due to the widespread availability of illicit fentanyl and other potent synthetic opioids such as carfentanil. Deliberate or accidental consumption or exposure to carfentanil, fentanyl, and their mixture induces respiratory depression and bradycardia that can be difficult to reverse with the opioid receptor antagonist naloxone. Vaccines offer a promising strategy to reduce the incidence of fatalities associated with fentanyl-related substances, as well as treatment for opioid use disorder (OUD). This study reports monovalent and bivalent vaccination strategies that elicit polyclonal antibody responses effective in protecting against the pharmacological actions of carfentanil, fentanyl, or carfentanil/fentanyl mixtures. Rats were prophylactically immunized with individual conjugate vaccines containing either carfentanil- or fentanyl-based haptens, or their combination in bivalent vaccine formulations, and then challenged with carfentanil, fentanyl, or their mixture. First, these studies identified a lead vaccine protective against carfentanil-induced antinociception, respiratory depression, and bradycardia. Then, efficacy against both carfentanil and fentanyl was achieved through bivalent vaccination strategies that combined lead anti-carfentanil and anti-fentanyl vaccines via either heterologous prime/boost or co-administration immunization regimens. These preclinical data support the development of vaccines as a viable strategy to prevent toxicity from exposure to excessive doses of carfentanil, fentanyl, or their mixtures.

The glycerol-3-phosphate dehydrogenases GpsA and GlpD constitute the oxidoreductive metabolic linchpin for Lyme disease spirochete host infectivity and persistence in the tick.

We have identified GpsA, a predicted glycerol-3-phosphate dehydrogenase, as a virulence factor in the Lyme disease spirochete Borrelia (Borreliella) burgdorferi: GpsA is essential for murine infection and crucial for persistence of the spirochete in the tick. B. burgdorferi has a limited biosynthetic and metabolic capacity; the linchpin connecting central carbohydrate and lipid metabolism is at the interconversion of glycerol-3-phosphate and dihydroxyacetone phosphate, catalyzed by GpsA and another glycerol-3-phosphate dehydrogenase, GlpD. Using a broad metabolomics approach, we found that GpsA serves as a dominant regulator of NADH and glycerol-3-phosphate levels in vitro, metabolic intermediates that reflect the cellular redox potential and serve as a precursor for lipid and lipoprotein biosynthesis, respectively. Additionally, GpsA was required for survival under nutrient stress, regulated overall reductase activity and controlled B. burgdorferi morphology in vitro. Furthermore, during in vitro nutrient stress, both glycerol and N-acetylglucosamine were bactericidal to B. burgdorferi in a GlpD-dependent manner. This study is also the first to identify a suppressor mutation in B. burgdorferi: a glpD deletion restored the wild-type phenotype to the pleiotropic gpsA mutant, including murine infectivity by needle inoculation at high doses, survival under nutrient stress, morphological changes and the metabolic imbalance of NADH and glycerol-3-phosphate. These results illustrate how basic metabolic functions that are dispensable for in vitro growth can be essential for in vivo infectivity of B. burgdorferi and may serve as attractive therapeutic targets.

Housing conditions and microbial environment do not affect the efficacy of vaccines for treatment of opioid use disorders in mice and rats.

Vaccines offer a promising prophylactic and therapeutic intervention to counteract opioid use disorders (OUD) and fatal overdoses. Vaccines generate opioid-specific antibodies that bind the target opioid, reducing drug distribution to the brain and preventing drug-induced behavioral and pharmacological effects. Due to their selectivity, anti-opioid vaccines can be administered in combination with FDA-approved medications. Because patients with OUD or other substance use disorders may be affected by other multifactorial co-morbidities, such as infection or depression, it is important to test whether vaccine efficacy is modified by factors that may impact individual innate or adaptive immunity. To that end, this study tested whether housing conditions would affect the efficacy of two lead vaccine formulations targeting oxycodone and fentanyl in male mice and rats, and further analyzed whether differences in the gastrointestinal (GI) microbiome would be correlated with either vaccine efficacy or housing conditions. Results showed that housing mice and rats in either conventional (non-controlled) or specific pathogen-free (SPF, sterile barrier maintained) environment did not affect vaccine-induced antibody responses against oxycodone and fentanyl, nor their efficacy against oxycodone- and fentanyl-induced antinociception, respiratory depression, and bradycardia. Differences in the GI microbiome detected via 16S rRNA gene sequencing were related to the housing environment. This study supports use of anti-opioid vaccines in clinical populations that may display deficits in microbiome function.

Therapeutic and Prophylactic Vaccines to Counteract Fentanyl Use Disorders and Toxicity.

The incidence of fatal overdoses has increased worldwide due to the widespread access to illicit fentanyl and its potent analogues. Vaccines offer a promising strategy to reduce the prevalence of opioid use disorders (OUDs) and to prevent toxicity from accidental and deliberate exposure to fentanyl and its derivatives. This study describes the development and characterization of vaccine formulations consisting of novel fentanyl-based haptens conjugated to carrier proteins. Vaccine efficacy was tested against opioid-induced behavior and toxicity in mice and rats challenged with fentanyl and its analogues. Prophylactic vaccination reduced fentanyl- and sufentanil-induced antinociception, respiratory depression, and bradycardia in mice and rats. Therapeutic vaccination also reduced fentanyl intravenous self-administration in rats. Because of their selectivity, vaccines did not interfere with the pharmacological effects of commonly used anesthetics nor with methadone, naloxone, oxycodone, or heroin. These preclinical data support the translation of vaccines as a viable strategy to counteract fentanyl use disorders and toxicity.

Mechanisms of interleukin 4 mediated increase in efficacy of vaccines against opioid use disorders.

Opioid use disorders (OUD) affect over 27 million people worldwide. Anti-opioid vaccines offer a promising strategy to treat OUD and prevent overdose. Using immunomodulation of cytokine signaling to increase vaccine efficacy, this study found that blocking IL-4 improved the efficacy of vaccines targeting oxycodone and fentanyl in male and female mice. Genetic deletion of the IL-4 receptor, STAT6, or antibody-based depletion of IL-13, did not increase vaccine efficacy against opioids, suggesting the involvement of type I IL-4 receptors. Enhancement of vaccine efficacy with blockade of IL-4 was associated with improved germinal center formation in secondary lymphoid organs and selective transcriptome signatures in the activated CD4+ T cell population subset. These data suggest that IL-4 is both a pharmacological target and a potential biomarker of vaccine efficacy against OUD.