Amira Y. Moreno

Immunopharmacotherapy: Vaccination Strategies as a Treatment for Drug Abuse and Dependence

Despite intensive efforts for its eradication, addiction to both legal and illicit drugs continues to be a major worldwide medical and social problem. Drug addiction is defined as a disease state in which the body relies on a substance for normal functioning and develops physical dependence leading to compulsive and repetitive use despite negative consequences to the users health, mental state or social life. Psychoactive substances such as cocaine, nicotine, alcohol, and amphetamines are able to cross the blood-brain barrier once ingested and temporarily alter the chemical balance of the brain. Current medications used for the treatment of dependence are typically agonists or antagonists of the drugs of abuse. The complex interrelations of the neuronal circuits have made it difficult to accurately predict the actions of potential agonist/antagonist drugs and have led to undesirable side effects within the central nervous system. Nearly forty years ago, a handful of groups began to explore the possibility of utilizing an individuals own immune machinery to counteract the effects of drug exposure in an approach later termed by our laboratory, immunopharmacotherapy.Immunopharmacotherapy aims to use highly specific antibodies to sequester the drug of interest while the latter is still in the bloodstream. Thus, creation of the antibody-drug complex will blunt crossing of the blood brain barrier (BBB) not only counteracting the reinforcing effects of the drug but also preventing any detrimental side effects on the CNS. In the present mini-review we aim to present a focused summary, including relevant challenges and future directions, of the current state of cocaine and nicotine vaccines as these two programs have been the most successful to date.

A critical evaluation of a nicotine vaccine within a self-administration behavioral model.

(S)-Nicotine is a psychostimulant legal drug responsible for causing addiction to tobacco smoking. Tobacco smoking has been irrevocably linked to a number of serious diseases and at present is considered the leading cause of preventable death in the United States. Despite well-documented adverse medical consequences, nicotine addiction has historically been one of the hardest to break. Current therapies have offered limited success and show high rates of relapse, emphasizing the need to engineer alternative therapies to aid nicotine cessation. The current study presents a protein-based immunopharmacotherapy approach for the treatment of nicotine addiction. Immunopharmacotherapy aims to use highly specific antibodies to blunt passage of drug into the brain thus minimizing reinforcing effects on the reward pathways of the central nervous system. Generation of a successful vaccine heavily relies on appropriate optimization of hapten design, immunogenic carrier and adjuvant. Modification of a classical nicotine hapten in conjugation with three distinct carrier proteins allowed for priming of a nicotine vaccine able to elicit significant amounts of nicotine-specific antibodies. Increased self-administration with use of a high drug dose (0.03 mg/kg/infusion; approximately 2 cigarettes in human) was observed in the vaccinated versus control animals suggesting a compensatory pattern and possibly reduced passage of nicotine to the brain. These results support the hypothesis that proper optimization of vaccine formulations could lead to successful nicotine vaccines for human use.

Cocaine Analog Coupled to Disrupted Adenovirus: A Vaccine Strategy to Evoke High-titer Immunity Against Addictive Drugs

Based on the concept that anticocaine antibodies could prevent inhaled cocaine from reaching its target receptors in the brain, an effective anticocaine vaccine could help reverse cocaine addiction. Leveraging the knowledge that E1(-)E3(-) adenovirus (Ad) gene transfer vectors are potent immunogens, we have developed a novel vaccine platform for addictive drugs by covalently linking a cocaine analog to the capsid proteins of noninfectious, disrupted Ad vector. The Ad-based anticocaine vaccine evokes high-titer anticocaine antibodies in mice sufficient to completely reverse, on a persistent basis, the hyperlocomotor activity induced by intravenous administration of cocaine.

A Methamphetamine Vaccine Attenuates Methamphetamine-Induced Disruptions in Thermoregulation and Activity in Rats

BACKGROUND There are no approved pharmacotherapies for d-methamphetamine (METH) addiction and existing therapies have limited efficacy. Advances in using immunotherapeutic approaches for cocaine and nicotine addiction have stimulated interest in creating a similar approach for METH addiction. This study investigated whether active vaccination against METH could potentially attenuate responses to METH in vivo. METHODS Male Sprague Dawley rats (n = 32) received a four-boost series with one of three candidate anti-METH vaccines (MH2[R], MH6, and MH7) or a control keyhole limpet hemocyanin conjugate vaccine. Effects of METH on rectal temperature and wheel activity at 27°C ambient temperature were determined. The most efficacious vaccine, MH6, was then contrasted with keyhole limpet hemocyanin conjugate vaccine in a subsequent experiment (n = 16), wherein radiotelemetry determined home cage locomotor activity and body temperature at 23°C ambient temperature. RESULTS The MH6 vaccine produced high antibody titers with nanomolar affinity for METH and sequestered METH in the periphery of rats. In experiment 1, the thermoregulatory and psychomotor responses produced by METH at 27°C were blocked in the MH6 group. In experiment 2, METH-induced decreases in body temperature and locomotor activity at 23°C were also attenuated in the MH6 group. A pharmacokinetic study in experiment 2 showed that MH6-vaccinated rats had higher METH serum concentrations, yet lower brain METH concentrations, than control rats, and METH concentrations correlated with individual antibody titer. CONCLUSIONS These data demonstrate that active immunopharmacotherapy provides functional protection against physiological and behavioral disruptions induced by METH.

Impact of Distinct Chemical Structures for the Development of a Methamphetamine Vaccine

(+)-Methamphetamine (METH) use and addiction has grown at alarming rates over the past two decades, while no approved pharmacotherapy exists for its treatment. Immunopharmacotherapy has the potential to offer relief through producing highly specific antibodies that prevent drug penetration across the blood-brain barrier thus decreasing reinforcement of the behavior. Current immunotherapy efforts against methamphetamine have focused on a single hapten structure, namely linker attachment at the aromatic ring of the METH molecule. Hapten design is largely responsible for immune recognition, as it affects presentation of the target antigen and thus the quality of the response. In the current paper we report the systematic generation of a series of haptens designed to target the most stable conformations of methamphetamine as determined by molecular modeling. On the basis of our previous studies with nicotine, we show that introduction of strategic molecular constraint is able to maximize immune recognition of the target structure as evidenced by higher antibody affinity. Vaccination of GIX(+) mice with six unique METH immunoconjugates resulted in high antibody titers for three particularly promising formulations (45-108 μg/mL, after the second immunization) and high affinity (82, 130, and 169 nM for MH2, MH6, and MH7 hapten-based vaccines, respectively). These findings represent a unique approach to the design of new vaccines against methamphetamine abuse.

Novel Cocaine Vaccine Linked to a Disrupted Adenovirus Gene Transfer Vector Blocks Cocaine Psychostimulant and Reinforcing Effects

Immunotherapy is a promising treatment for drug addiction. However, insufficient immune responses to vaccines in most subjects pose a challenge. In this study, we tested the efficacy of a new cocaine vaccine (dAd5GNE) in antagonizing cocaine addiction-related behaviors in rats. This vaccine used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid). Three groups of rats were immunized with dAd5GNE. One group was injected with 3H-cocaine, and radioactivity in the blood and brain was determined. A second group was tested for cocaine-induced locomotor sensitization. A third group was examined for cocaine self-administration, extinction, and reinstatement of responding for cocaine. Antibody titers were determined at various time-points. In each experiment, we added a control group that was immunized with dAd5 without a hapten. The vaccination with dAd5GNE produced long-lasting high titers (>105) of anti-cocaine antibodies in all of the rats. The vaccination inhibited cocaine-induced hyperlocomotor activity and sensitization. Vaccinated rats acquired cocaine self-administration, but they showed less motivation to self-administer cocaine under a progressive-ratio schedule than control rats. When cocaine was not available in a session, control rats exhibited ‘extinction burst’ responding, whereas vaccinated rats did not. Moreover, when primed with cocaine, vaccinated rats did not reinstate responding, suggesting a blockade of cocaine-seeking behavior. These data strongly suggest that our dAd5GNE vector-based vaccine may be effective in treating cocaine abuse and addiction.

Disrupted Adenovirus-Based Vaccines Against Small Addictive Molecules Circumvent Anti-Adenovirus Immunity

Adenovirus (Ad) vaccine vectors have been used for many applications due to the capacity of the Ad capsid proteins to evoke potent immune responses, but these vectors are often ineffective in the context of pre-existing anti-Ad immunity. Leveraging the knowledge that E1(-)E3(-) Ad gene transfer vectors are potent immunogens, we have developed a vaccine platform against small molecules by covalently coupling analogs of small molecules to the capsid proteins of disrupted Ad (dAd5). We hypothesized that the dAd5 platform would maintain immunopotency even in the context of anti-Ad neutralizing antibodies. To test this hypothesis, we coupled cocaine and nicotine analogs, GNE and AM1, to dAd5 capsid proteins to generate dAd5GNE and dAd5AM1, respectively. Mice were pre-immunized with Ad5Null, resulting in high titer anti-Ad5 neutralizing antibodies comparable to those observed in the human population. The dAd5GNE and dAd5AM1 vaccines elicited high anti-cocaine and anti-nicotine antibody titers, respectively, in both naive and Ad5-immune mice, and both functioned to prevent cocaine or nicotine from reaching the brain of anti-Ad immune mice. Thus, disrupted Ad5 evokes potent humoral immunity that is effective in the context of pre-existing neutralizing anti-Ad immunity, overcoming a major limitation for current Ad-based vaccines.

Anti-Cocaine Vaccine Based on Coupling a Cocaine Analog to a Disrupted Adenovirus

The challenge in developing an anti-cocaine vaccine is that cocaine is a small molecule, invisible to the immune system. Leveraging the knowledge that adenovirus (Ad) capsid proteins are highly immunogenic in humans, we hypothesized that linking a cocaine hapten to Ad capsid proteins would elicit high-affinity, high-titer antibodies against cocaine, sufficient to sequester systemically administered cocaine and prevent access to the brain, thus suppressing cocaine-induced behaviors. Based on these concepts, we developed dAd5GNE, a disrupted E1-E3- serotype 5 Ad with GNE, a stable cocaine analog, covalently linked to the Ad capsid proteins. In pre-clinical studies, dAd5GNE evoked persistent, high titer, high affinity IgG anti-cocaine antibodies, and was highly effective in blocking cocaine-induced hyperactivity and cocaine self-administration behavior in rats. Future studies will be designed to expand the efficacy studies, carry out relevant toxicology studies, and test dAd5GNE in human cocaine addicts.

Onchocerca volvulus-neurotransmitter tyramine is a biomarker for river blindness

Onchocerciasis, also known as “river blindness”, is a neglected tropical disease infecting millions of people mainly in Africa and the Middle East but also in South America and Central America. Disease infectivity initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious third-stage larvae. Ivermectin has controlled transmission of microfilariae, with an African Program elimination target date of 2025. However, there is currently no point-of-care diagnostic that can distinguish the burden of infection—including active and/or past infection—and enable the elimination program to be effectively monitored. Here, we describe how liquid chromatography-MS–based urine metabolome analysis can be exploited for the identification of a unique biomarker, N-acetyltyramine-O,β-glucuronide (NATOG), a neurotransmitter-derived secretion metabolite from O. volvulus. The regulation of this tyramine neurotransmitter was found to be linked to patient disease infection, including the controversial antibiotic doxycycline treatment that has been shown to both sterilize and kill adult female worms. Further clues to its regulation have been elucidated through biosynthetic pathway determination within the nematode and its human host. Our results demonstrate that NATOG tracks O. volvulus metabolism in both worms and humans, and thus can be considered a host-specific biomarker for onchocerciasis progression. Liquid chromatography-MS–based urine metabolome analysis discovery of NATOG not only has broad implications for a noninvasive host-specific onchocerciasis diagnostic but provides a basis for the metabolome mining of other neglected tropical diseases for the discovery of distinct biomarkers and monitoring of disease progression.

Probing the Effects of Hapten Stability on Cocaine Vaccine Immunogenicity

Judicious hapten design has been shown to be of importance when trying to generate a viable vaccine against a drug of abuse. Hapten design has typically been predicated upon faithfully emulating the unique chemical architecture that each drug presents. However, the need for drug-hapten congruency may also compromise vaccine immunogenicity if the drug-hapten conjugate possesses chemical epitope instability. There has been no systematic study on the impact of hapten stability as it relates to vaccine immunogenicity. As a starting point, we have probed the stability of a series of cocaine haptens through varying several of its structural elements, including functionality at the C2-position, the nature of the linker, and its site of attachment. Accordingly, a hydrolytic stability profile of four cocaine haptens (GNNA, GNNS, GNE, and GNC) was produced, and these results were compared through each haptens immunological properties, which were generated via active vaccination. From this group of four, three of the haptens, GNE, GNNA, and GNC, were further examined in an animal behavioral model, and findings here were again measured in relationship to hapten stability. We demonstrate a corresponding relationship between the half-life of the hapten and its immunogenicity, wherein haptens presenting a fully representative cocaine framework elicited higher concentrations of cocaine-specific IgG in sera and also conferred better protection against cocaine-induced locomotor activity. Our results indicate that hapten half-life plays an important role in vaccine immunogenicity and this in turn can impact animal behavioral effects when challenged with a drug of abuse.