Viral delivery of a peptide-based immunomodulator enhances T cell priming during vaccination.

Modern, subunit-based vaccines have so far failed to induce significant T cell responses, contributing to ineffective vaccination against many pathogens. Importantly, while today's adjuvants are designed to trigger innate and non-specific immune responses, they fail to directly stimulate the adaptive immune compartment. Programmed cell death 1 (PD-1) partly regulates naïve-to-antigen-specific effector T cell transition and differentiation by suppressing the magnitude of activation. Indeed, we previously reported on a microbial-derived, peptide-based PD-1 checkpoint inhibitor, LD01, which showed potent T cell-stimulating activity when combined with a vaccine. Here we sought to improve the potency of LD01 by designing and testing new LD01 derivatives. Accordingly, we found that a modified version of an 18-amino acid metabolite of LD01, LD10da, improved T cell activation capability in a malaria vaccine model. Specifically, LD10da demonstrates improved antigen-specific CD8+ T cell expansion when combined prophylactically with an adenovirus-based malaria vaccine. A single dose of LD10da at the time of vaccination is sufficient to increase antigen-specific CD8+ T cell expansion in wild-type mice. Further, we show that LD10 can be encoded and delivered by a Modified Vaccinia Ankara viral vector and can enhance antigen-specific CD8+ T cell expansion comparable to that of synthetic peptide administration. Therefore, LD10da represents a promising biologic-based immunomodulator that can be genetically encoded and delivered, along with the antigen, by viral or other nucleic acid vectors to improve the efficacy and delivery of vaccines for ineradicable and emerging infectious diseases.

A single immunization with a modified vaccinia Ankara vectored vaccine producing Sudan virus-like particles protects from lethal infection.

A new vectored vaccine MVA-VLP-SUDV was generated against Sudan ebolavirus (SUDV) combining the advantages of the immunogenicity of a live attenuated vaccine vector (Modified Vaccinia Ankara, MVA) with the authentic conformation of virus-like particles (VLPs). The vaccine expresses minimal components to generate self-assembling VLPs in the vaccinee: the envelope glycoprotein GP and the matrix protein VP40. Guinea pigs vaccinated with one dose of MVA-VLP-SUDV generated SUDV-specific binding and neutralizing antibody responses as well as Fc-mediated protective effects. These responses were boosted by a second vaccine dose. All vaccinated animals which received either one or two vaccine doses were protected from death and disease symptoms following challenge with a lethal dose of SUDV. These data demonstrate single dose protection and potency of the MVA-VLP platform for use in emergency situations to contain outbreaks.

Modified vaccinia Ankara vaccine expressing Marburg virus-like particles protects guinea pigs from lethal Marburg virus infection.

We introduce a new vaccine platform against Marburg virus (MARV) combining the advantages of the immunogenicity of a highly attenuated vaccine vector (Modified Vaccinia Ankara, MVA) with the authentic conformation of virus-like particles (VLPs). Our vaccine, MVA-MARV-VLP, expresses the minimal components of MARV VLPs: the envelope glycoprotein GP and the matrix protein VP40. Electron microscopy confirmed self-assembly and budding of VLPs from infected cells. Prime/boost vaccination of guinea pigs with MVA-MARV-VLP-elicited MARV-specific binding and neutralizing antibody responses. Vaccination also induced Fc-mediated innate immune effector functions including activation of NK cells and antibody-dependent phagocytosis by neutrophils and monocytes. Inoculation of vaccinated animals with guinea pig-adapted MARV demonstrated 100% protection against death and disease with no viremia. Therefore, our vaccine platform, expressing two antigens resulting in assembly of VLPs in the native conformation in vaccinated hosts, can be used as a potent vaccine against MARV.

In Search of Reward Deficiency Syndrome (RDS)-free Controls: The "Holy Grail" in Genetic Addiction Risk Testing.

BACKGROUND: The search for an accurate, gene-based test to identify heritable risk factors for Reward Deficiency Syndrome (RDS) was conducted based on hundreds of published studies about the role of dopamine in addictive behaviors, including risk for drug dependence and compulsive/impulsive behavior disorders. The term RDS was first coined by Blum's group in 1995 to identify a group of behaviors with a common neurobiological mechanism associated with a polymorphic allelic propensity for hypodopaminergia. OBJECTIVES: To outline the process used to select risk alleles of reward genes for the Genetic Addiction Risk Score (GARS) test. Consequently, to address the limitations caused by inconsistent results that occur in many case-control behavioral association studies. These limitations are perhaps due to the failure of investigators to adequately screen controls for drug and alcohol use disorder, and any of the many RDS behaviors, including nicotine dependence, obesity, pathological gambling, and internet gaming addiction. METHODS: Review of the literature related to the function of risk alleles of reward genes associated with hypodopaminergia relevant case-control association studies for the selection of alleles to be measured by the Genetic Addiction Risk Score (GARS) test. RESULTS: The prevalence of the DRD2 A1 allele in unscreened controls (33.3%), compared to "Super-Controls" [highly screened RDS controls (3.3%) in proband and family] is used to exemplify a possible solution. CONCLUSION: Unlike one gene-one disease (OGOD), RDS is polygenetic, and very complex. In addition, any RDS-related behaviors must be eliminated from the control group in order to obtain the best possible statistical analysis instead of comparing the phenotype with disease-ridden controls.

Buprenorphine Treatment for Opioid Use Disorder in Community-Based Settings: Outcome Related to Intensity of Services and Urine Drug Test Results.

BACKGROUND AND OBJECTIVES: Variables contributing to the outcome of buprenorphine treatment for opiate use disorder have been studied, including patient characteristics and the treatment approach applied. It is also valuable to study the types of clinical facilities that can affect outcome. METHODS: We evaluated patients (N = 20 993) in 573 facilities where buprenorphine was prescribed. Urine drug test results were analyzed for those (N = 13 281) who had buprenorphine prescribed at least twice in the period January 2015 through June 2017. Facilities were divided into three categories: medication management (MM) only, limited psychosocial (LP) therapy, and recovery-oriented (with more extensive counseling and a 12-step orientation) (RO). RESULTS: Urine drug tests negative for other opioids at the time of the second buprenorphine prescription were 34% for MM, 56% for LP, and 62% for RO (P < .001). A comparison was made between the most recent and the established patients at the facilities. The decrement in urinalyses positive for other opioids in this latter comparison was 3% for MM, 7% for LP, and 23% for RO (P < .001). DISCUSSION AND CONCLUSIONS: In a large sample of community settings, buprenorphine patients' urinalyses positive for opioids can vary considerably across treatment facilities, and more intensive recovery orientation may yield a better outcome in terms of secondary opioid use. SCIENTIFIC SIGNIFICANCE: The majority of buprenorphine patients are treated in community facilities. It is important that research be done by facility type in such settings in order to plan for optimal treatment. (© 2020 The Authors. The American Journal on Addictions published by Wiley Periodicals, Inc.;00:00-00).

A Single Dose of Modified Vaccinia Ankara Expressing Lassa Virus-like Particles Protects Mice from Lethal Intra-cerebral Virus Challenge.

Lassa fever surpasses Ebola, Marburg, and all other hemorrhagic fevers except Dengue in its public health impact. Caused by Lassa virus (LASV), the disease is a scourge on populations in endemic areas of West Africa, where reported incidence is higher. Here, we report construction, characterization, and preclinical efficacy of a novel recombinant vaccine candidate GEO-LM01. Constructed in the Modified Vaccinia Ankara (MVA) vector, GEO-LM01 expresses the glycoprotein precursor (GPC) and zinc-binding matrix protein (Z) from the prototype Josiah strain lineage IV. When expressed together, GP and Z form Virus-Like Particles (VLPs) in cell culture. Immunogenicity and efficacy of GEO-LM01 was tested in a mouse challenge model. A single intramuscular dose of GEO-LM01 protected 100% of CBA/J mice challenged with a lethal dose of ML29, a Mopeia/Lassa reassortant virus, delivered directly into the brain. In contrast, all control animals died within one week. The vaccine induced low levels of antibodies but Lassa-specific CD4+ and CD8+ T cell responses. This is the first report showing that a single dose of a replication-deficient MVA vector can confer full protection against a lethal challenge with ML29 virus.

Cryo-EM Reveals Architectural Diversity in Active Rotavirus Particles.

Rotavirus is a well-studied RNA virus that causes severe gastroenteritis in children. During viral entry, the outer layer of the virion is shed, creating a double-layered particle (DLP) that is competent to perform viral transcription (i.e., mRNA synthesis) and launch infection. While inactive forms of rotavirus DLPs have been structurally characterized in detail, information about the transcriptionally-active DLP remains limited. Here, we used cryo-Electron Microscopy (cryo-EM) and 3D image reconstructions to compare the structures of internal protein components in transcriptionally-active versus inactive DLPs. Our findings showed that transcriptionally-active DLPs gained internal order as mRNA synthesis unfolded, while inactive DLPs remained dynamically disordered. Regions of viral protein/RNA constituents were analyzed across two different axes of symmetry to provide a more comprehensive view of moving components. Taken together, our results bring forth a new view of active DLPs, which may enable future pharmacological strategies aimed at obliterating rotavirus transcription as a therapeutic approach.

Promoting Precision Addiction Management (PAM) to Combat the Global Opioid Crisis.

It is universally agreed that dopamine is a major neurotransmitter in terms of reward dependence, however, there remains controversy regarding how to modulate its role clinically to treat and prevent relapse for both substance and non-substance-related addictive behaviors. It is also agreed by most that there is a need to provide early genetic identification possibly through a novel researched technology referred to Genetic Addiction Risk Score(GARS).™ The existing FDA-approved medications promote blocking dopamine, however, we argue that a more prudent paradigm shift should be biphasic-short-term blockade and long-term upregulation, enhancing functional connectivity of brain reward. It is critical to understand that the real phenotype is not any specific drug or non -drug addictive behavior, but instead is Reward Deficiency Syndrome (RDS). Thus the true phenotype of all addictive behaviors is indeed RDS. Finally, we are suggesting that one way to combat the current out of control Opioid /Alcohol crisis worldwide is to seriously reconsider treating RDS by simply supplying powerful narcotic agents (e.g. Buprenorphine). This type of treatment will only keep people addicted. A more reasonable solution involving genetic testing, urine drug screens using Comprehensive Analysis of Reported Drugs (CARD) and dopamine homeostasis we call " Precision Addiction Management" ™ seems parsonomiuos.

The Benefits of Genetic Addiction Risk Score (GARS™) Testing in Substance Use Disorder (SUD).

Following 25 years of extensive research by many scientists worldwide, a panel of ten reward gene risk variants, called the Genetic Addiction Risk Score (GARS), has been developed. In unpublished work, when GARS was compared to the Addiction Severity Index (ASI), which has been used in many clinical settings, GARS significantly predicted the severity of both alcohol and drug dependency. In support of early testing for addiction and other RDS subtypes, parents caught up in the current demographic of 127 people, both young and old, dying daily from opiate/opioid overdose, need help. In the past, families would have never guessed that their loved ones would die or could be in real danger due to opiate addiction. Author, Bill Moyers, in Parade Magazine, reported that as he traveled around the United States, he found many children with ADHD and other spectrum disorders like Autism, and noted that many of these children had related conditions like substance abuse. He called for better ways to identify these children and treat them with approaches other than addictive pharmaceuticals. To our knowledge, GARS is the only panel of genes with established polymorphisms reflecting the Brain Reward Cascade (BRC), which has been correlated with the ASI-MV alcohol and drug risk severity score. While other studies are required to confirm and extend the GARS test to include other genes and polymorphisms that associate with an hypodopaminergic trait, these results provide clinicians with a non-invasive genetic test. Genomic testing, such as GARS, can improve clinical interactions and decision-making. Knowledge of precise polymorphic associations can help in the attenuation of guilt and denial, corroboration of family gene-o-grams; assistance in risk-severity-based decisions about appropriate therapies, including pain medications and risk for addiction; choice of the appropriate level of care placement (i.e., inpatient, outpatient, intensive outpatient, residential); determination of the length of stay in treatment; determination of genetic severity-based relapse and recovery liability and vulnerability; determination of pharmacogenetic medical monitoring for better clinical outcomes (e.g., the A1 allele of the DRD2 gene reduces the binding to opioid delta receptors in the brain, thus, reducing Naltrexone's clinical effectiveness); and supporting medical necessity for insurance scrutiny.

A Systematic, Intensive Statistical Investigation of Data from the Comprehensive Analysis of Reported Drugs (CARD) for Compliance and Illicit Opioid Abstinence in Substance Addiction Treatment with Buprenorphine/naloxone.

BACKGROUND: Buprenorphine and naloxone (bup/nal), a combination partial mu receptor agonist and low-dose delta mu antagonist, is presently recommended and used to treat opioid-use disorder. However, a literature review revealed a paucity of research involving data from urine drug tests that looked at compliance and abstinence in one sample. METHOD: Statistical analysis of data from the Comprehensive Analysis of Reported Drugs (CARD) was used to assess compliance and abstinence during treatment in a large cohort of bup/nal patients attending chemical-dependency programs from eastern USA in 2010 and 2011. RESULTS: Part 1: Bup/nal was present in 93.4% of first (n = 1,282; p <.0001) and 92.4% of last (n = 1,268; p <.0001) urine samples. Concomitantly, unreported illicit drugs were present in 47.7% (n = 655, p =.0261) of samples. Patients who were compliant to the bup/nal prescription were more likely than noncompliant patients to be abstinent during treatment (p =.0012; odds ratio = 1.69 with 95% confidence interval (1.210, 2.354). Part 2: An analysis of all samples collected in 2011 revealed a significant improvement in both compliance (p < 2.2 × 10-16) and abstinence (p < 2.2 × 10-16) during treatment. Conclusion/Importance: While significant use of illicit opioids during treatment with bup/nal is present, improvements in abstinence and high compliance during maintenance-assisted therapy programs may ameliorate fears of diversion in comprehensive programs. Expanded clinical datasets, the treatment modality, location, and year of sampling are important covariates, for further studies. The potential for long-term antireward effects from bup/nal use requires consideration in future investigations.

"Dopamine homeostasis" requires balanced polypharmacy: Issue with destructive, powerful dopamine agents to combat America's drug epidemic.

The well-researched pro-dopamine regulator KB220 and variants result in increased functional connectivity in both animal and human brains, and prolonged neuroplasticity (brain cell repair) having been observed in rodents. Moreover, in addition to increased functional connectivity, recent studies show that KB220Z increases overall brain connectivity volume, enhances neuronal dopamine firing, and eliminates lucid dreams in humans over a prolonged period. An unprecedented number of clinical studies validating this patented nutrigenomic technology in re-balancing brain chemistry and optimizing dopamine sensitivity and function have been published. On another note, it is sad that unsuspecting consumers could be deceived and endangered by false promises of knock-off marketers with look- and- sound-alike products. Products containing ingredients having potential dangers (i.e., combinations of potent D2 agonists including L-Dopa and L-Theanine) threaten the credibility and reputation of validated, authentic, and ethical products. We encourage clinicians and neuroscientists to continue to embrace the concept of "dopamine homeostasis" and search for safe, effective, validated and authentic means to achieve a lifetime of recovery, instead of reverting to anti-dopaminergic agents doomed to fail in the war against the devastating drug epidemic, or promoting powerful D2 agonists that compromise needed balance.

GLOBAL OPIOID EPIDEMIC: DOOMED TO FAIL WITHOUT GENETICALLY BASED PRECISION ADDICTION MEDICINE (PAM™): LESSONS LEARNED FROM AMERICA.

It is a reality that globally opioid deaths have soared for men and women of all social, economic status and age from heroin and fentanyl overdoses. Specifically, in the United States, deaths from narcotic overdoses have reached alarming metrics since 2010. In fact, the Fentanyl rise is driven by drug dealers who sell it as heroin or who use it to lace cocaine or to make illegal counterfeit prescription opioids. The President's Commission on the crisis has linked the death toll as equivalent to "September 11th every three weeks." In fact, The U.S. Centre for Disease Control (CDC) released data showing that opioid-related overdoses were up 15% in the first three quarters of 2016 compared to 2015. Various governmental organizations including NIDA, are actively seeking solutions. However, we argue that unless the scientific community embraces genetic addiction risk coupled with potential precision or personalized medicine to induce "dopamine homeostasis" it will fail. We now have evidence that a ten-gene and eleven single nucleotide polymorphism (SNP) panel predicts Addiction Severity Index (ASI) for both alcohol and drugs of abuse (e.g., Opioids). In a large multi-addiction centre study involving seven diverse treatment programs, the genetic addiction risk score (GARS™) was shown to have a predictive relationship with ASI-MV derived alcohol (≥ seven alleles), and other drugs (≥ 4 alleles) severity risk scores. In a number of neuroimaging studies, we also display that in both animal (bench) and abstinent Chinese severe heroin-dependent patients (bedside), BOLD dopamine activation across the brain reward circuitry revealed increases in resting state functional connectivity as well volume connectivity. It is also known that published nutrigenomic (coupling gene polymorphisms with altered KB220z) studies reveal improved clinical outcomes related to obesity.

Molecular Genetic Testing in Pain and Addiction: Facts, Fiction and Clinical Utility.

The Brain Reward Cascade (BRC) is an interaction of neurotransmitters and their respective genes to control the amount of dopamine released within the brain. Any variations within this pathway, whether genetic or environmental (epigenetic), may result in addictive behaviors as well as altered pain tolerance. While there are many studies claiming a genetic association with addiction and other behavioral infractions, defined as Reward Deficiency Syndrome (RDS), not all are scientifically accurate and in some case just wrong. Albeit our bias, we discuss herein the facts and fictions behind molecular genetic testing in RDS (including pain and addiction) and the significance behind the development of the Genetic Addiction Risk Score (GARSPREDX™), the first test to accurately predict one's genetic risk for RDS.

Withdrawal from Buprenorphine/Naloxone and Maintenance with a Natural Dopaminergic Agonist: A Cautionary Note.

BACKGROUND: While numerous studies support the efficacy of methadone and buprenorphine for the stabilization and maintenance of opioid dependence, clinically significant opioid withdrawal symptoms occur upon tapering and cessation of dosage. METHODS: We present a case study of a 35 year old Caucasian female (Krissie) who was prescribed increasing dosages of prescription opioids after carpel tunnel surgery secondary to chronic pain from reflex sympathetic dystrophy and fibromyalgia. Over the next 5 years, daily dosage requirements increased to over 80 mg of Methadone and 300 ug/hr Fentanyl transdermal patches, along with combinations of 12-14 1600 mcg Actig lollipop and oral 100 mg Morphine and 30 mg oxycodone 1-2 tabs q4-6hr PRN for breakthrough pain. Total monthly prescription costs including supplemental benzodiazepines, hypnotics and stimulants exceeded $50,000. The patient was subsequently transferred to Suboxone® in 2008, and the dosage was gradually tapered until her admission for inpatient detoxification with KB220Z a natural dopaminergic agonist. We carefully documented her withdrawal symptoms when she precipitously stopped taking buprenorphine/naloxone and during follow-up while taking KB220Z daily. We also genotyped the patient using a reward gene panel including (9 genes 18 alleles): DRD 2,3,4; MOA-A; COMT; DAT1; 5HTTLLR; OPRM1; and GABRA3. FINDINGS: At 432 days post Suboxone® withdrawal the patient is being maintained on KB220Z, has been urine tested and is opioid free. Genotyping data revealed a moderate genetic risk for addiction showing a hypodopaminergic trait. This preliminary case data suggest that the daily use of KB220Z could provide a cost effective alternative substitution adjunctive modality for Suboxone®. We encourage double-blind randomized -placebo controlled studies to test the proposition that KB220Z may act as a putative natural opioid substitution maintenance adjunct.

Antiviral responses by Swine primary bronchoepithelial cells are limited compared to human bronchoepithelial cells following influenza virus infection.

Swine generate reassortant influenza viruses because they can be simultaneously infected with avian and human influenza; however, the features that restrict influenza reassortment in swine and human hosts are not fully understood. Type I and III interferons (IFNs) act as the first line of defense against influenza virus infection of respiratory epithelium. To determine if human and swine have different capacities to mount an antiviral response the expression of IFN and IFN-stimulated genes (ISG) in normal human bronchial epithelial (NHBE) cells and normal swine bronchial epithelial (NSBE) cells was evaluated following infection with human (H3N2), swine (H1N1), and avian (H5N3, H5N2, H5N1) influenza A viruses. Expression of IFNλ and ISGs were substantially higher in NHBE cells compared to NSBE cells following H5 avian influenza virus infection compared to human or swine influenza virus infection. This effect was associated with reduced H5 avian influenza virus replication in human cells at late times post infection. Further, RIG-I expression was lower in NSBE cells compared to NHBE cells suggesting reduced virus sensing. Together, these studies identify key differences in the antiviral response between human and swine respiratory epithelium alluding to differences that may govern influenza reassortment.