Inundative, Dry-Powder, Inhaled Measles Vaccination to Prevent Deaths of Young Children in War-torn Regions.

Children living in war-torn and geographically remote regions often die from measles due to undervaccination. Protective community immunity could be safely improved through the comprehensive use of small, inexpensive, easy-to-use, dry-powder aerosolized measles vaccination inhalers. Influential local community members could be engaged to provide risk counseling and inform their peers of measles risks to inspire vaccine uptake. Vaccination by inhaled live attenuated measles vaccine has been shown to be safe and protective among several million research subjects and omits (1) needles, syringes, glass vials, and specialized disposal systems; (2) deadly vaccine reconstitution errors; (3) cold chain technology to protect temperature-sensitive vaccine; (4) vaccine wastage associated with suboptimal use of multidose vials; (5) trained vaccinators; (6) food, housing, and transportation costs associated with centralized vaccination campaigns; and (7) risk of violence to vaccinators and associated staff. All elements for inhaler-based measles vaccination are readily available. Dry-powder measles vaccine inhalers can be assembled and distributed to save lives.

Incentivized self-vaccination for global measles eradication.

Measles-we've become inured to its cruel, insidious impact as it kills over 100,000 children yearly because of suboptimal vaccination coverage. It does not have to be this way. A familiar, safe, exceptionally effective measles vaccine saves lives and permanent, global measles eradication is within reach. But now we need to be clever and courageously explore new strategies to save lives. Firstly, let us enable people to vaccinate themselves, not with a needle and syringe, but with a quick inhaled puff of dry powder vaccine. Secondly, let us provide micro-payments using digital currency to incentivize those who vaccinate themselves. Thirdly, let us leverage learnings from how our social networks guide our behaviors to further encourage self-vaccination. Fourthly, let us inspire friendly regional competition among communities vying for the highest proportion of citizens who show measles neutralizing antibodies in spot saliva samples. With global cooperation and relentless determination, we eradicated smallpox. Next up? Measles.

Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines.

The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines. CBD treatment leads to alterations in the abundance of metabolites, mRNA transcripts, and proteins associated with activation of cholesterol biosynthesis, transport, and storage. We found that CBD rapidly incorporates into cellular membranes, alters cholesterol accessibility, and disrupts cholesterol-dependent membrane properties. Sustained treatment with high concentrations of CBD induces apoptosis in a dose-dependent manner. CBD-induced apoptosis is rescued by inhibition of cholesterol synthesis and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.

A durable protective immune response to wild-type measles virus infection of macaques is due to viral replication and spread in lymphoid tissues.

Infection with wild-type (WT) measles virus (MeV) is an important cause of childhood mortality that leads to lifelong protective immunity in survivors. WT MeV and the live-attenuated MeV used in the measles vaccine (LAMV) are antigenically similar, but the determinants of attenuation are unknown, and protective immunity induced by LAMV is less robust than that induced by WT MeV. To identify factors that contribute to these differences, we compared virologic and immunologic responses after respiratory infection of rhesus macaques with WT MeV or LAMV. In infected macaques, WT MeV replicated efficiently in B and T lymphocytes with spreading throughout lymphoid tissues resulting in prolonged persistence of viral RNA. In contrast, LAMV replicated efficiently in the respiratory tract but displayed limited spread to lymphoid tissue or peripheral blood mononuclear cells. In vitro, WT MeV and LAMV replicated similarly in macaque primary respiratory epithelial cells and human lymphocytes, but LAMV-infected lymphocytes produced little virus. Plasma concentrations of interleukin-1ß (IL-1ß), IL-12, interferon-γ (IFN-γ), CCL2, CCL11, CXCL9, and CXCL11 increased in macaques after WT MeV but not LAMV infection. WT MeV infection induced more protective neutralizing, hemagglutinin-specific antibodies and bone marrow plasma cells than did LAMV infection, although numbers of MeV-specific IFN-γ- and IL-4-producing T cells were comparable. Therefore, MeV attenuation may involve altered viral replication in lymphoid tissue that limited spread and decreased the host antibody response, suggesting a link between lifelong protective immunity and the ability of WT MeV, but not LAMV, to spread in lymphocytes.

Safety and immunogenicity of dry powder measles vaccine administered by inhalation: a randomized controlled Phase I clinical trial.

BACKGROUND: Measles is a highly infectious respiratory disease which causes 122,000 deaths annually. Although measles vaccine is extremely safe and effective, vaccine coverage could be improved by a vaccine that is more easily administered and transported. We developed an inhalable dry powder measles vaccine (MVDP) and two delivery devices, and demonstrated safety, immunogenicity, and efficacy of the vaccine in preclinical studies. Here we report the first clinical trial of MVDP delivered by inhalation. METHODOLOGY: Sixty adult males aged 18 to 45 years, seropositive for measles antibody, were enrolled in this controlled Phase I clinical study. Subjects were randomly assigned in 1:1:1 ratio to receive either MVDP by Puffhaler(®) or by Solovent™ devices or the licensed subcutaneous measles vaccine. Adverse events (AEs) were recorded with diary cards until day 28 post-vaccination and subjects were followed for 180 days post-vaccination to assess potential serious long term adverse events. Measles antibody was measured 7 days before vaccination and at days 21 and 77 after vaccination by ELISA and a plaque reduction neutralization test. RESULTS: All subjects completed the study according to protocol. Most subjects had high levels of baseline measles antibody. No adverse events were reported. MVDP produced serologic responses similar to subcutaneous vaccination. CONCLUSIONS: MVDP was well tolerated in all subjects. Most subjects had high baseline measles antibody titer which limited ability to measure the serologic responses, and may have limited the adverse events following vaccination. Additional studies in subjects without pre-existing measles antibody are needed to further elucidate the safety and immunogenicity of MVDP.

Successful respiratory immunization with dry powder live-attenuated measles virus vaccine in rhesus macaques.

Measles remains an important cause of childhood mortality worldwide. Sustained high vaccination coverage is the key to preventing measles deaths. Because measles vaccine is delivered by injection, hurdles to high coverage include the need for trained medical personnel and a cold chain, waste of vaccine in multidose vials and risks associated with needle use and disposal. Respiratory vaccine delivery could lower these barriers and facilitate sustained high coverage. We developed a novel single unit dose, dry powder live-attenuated measles vaccine (MVDP) for respiratory delivery without reconstitution. We tested the immunogenicity and protective efficacy in rhesus macaques of one dose of MVDP delivered either with a mask or directly intranasal with two dry powder inhalers, PuffHaler and BD Solovent. MVDP induced robust measles virus (MeV)-specific humoral and T-cell responses, without adverse effects, which completely protected the macaques from infection with wild-type MeV more than one year later. Respiratory delivery of MVDP was safe and effective and could aid in measles control.

Dry powder measles vaccine: particle deposition, virus replication, and immune response in cotton rats following inhalation.

A stable and high potency dry powder measles vaccine with a particle size distribution suitable for inhalation was manufactured by CO(2)-Assisted Nebulization with a Bubble Dryer(®) (CAN-BD) process from bulk liquid Edmonston-Zagreb live attenuated measles virus vaccine supplied by the Serum Institute of India. A novel dry powder inhaler, the PuffHaler(®) was adapted for use in evaluating the utility of cotton rats to study the vaccine deposition, vaccine virus replication, and immune response following inhalation of the dry powder measles vaccine. Vaccine deposition in the lungs of cotton rats and subsequent viral replication was detected by measles-specific RT-PCR, and viral replication was confined to the lungs. Inhalation delivery resulted in an immune response comparable to that following injection. The cotton rat model is useful for evaluating new measles vaccine formulations and delivery devices.

Preparation of active proteins, vaccines and pharmaceuticals as fine powders using supercritical or near-critical fluids.

Supercritical or near-critical fluid processes for generating microparticles have enjoyed considerable attention in the past decade or so, with good success for substances soluble in supercritical fluids or organic solvents. In this review, we survey their application to the production of protein particles. A recently developed process known as CO2-assisted nebulization with a Bubble Dryer (CAN-BD) has been demonstrated to have broad applicability to small-molecule as well as macromolecule substances (including therapeutic proteins). The principles of CAN-BD are discussed as well as the stabilization, micronization and drying of a wide variety of materials. More detailed case studies are presented for three proteins, two of which are of therapeutic interest: anti-CD4 antibody (rheumatoid arthritis), alpha1-antitrypsin (cystic fibrosis and emphysema), and trypsinogen (a model enzyme). Dry powders were formed in which stability and activity are maintained and which are fine enough to be inhaled and reach the deep lung. Enhancement of apparent activity after CAN-BD processing was also observed in some formulation and processing conditions.

Stabilizing formulations for inhalable powders of live-attenuated measles virus vaccine.

Carbon dioxide Assisted Nebulization with a Bubble Dryer((R)) (CAN-BD) processing allows particles to be made in the 3-5 mum size range, which is desirable for lung delivery, without destroying biological activity. In response to the Grand Challenge in Global Health Initiative #3, we have been developing an inhalable needle-free live-attenuated measles virus vaccine for use in developing countries. Measles was chosen because it is the number one vaccine preventable killer of children worldwide. Powders were processed by CAN-BD, where a solution containing excipients and live-attenuated measles virus in water was mixed intimately with supercritical or near superctitical carbon dioxide to form an emulsion. The emulsion was expanded to atmospheric pressure through a flow restrictor. The resulting plume was dried by heated nitrogen and the powders collected on a filter at the bottom of the drying chamber. Powders were analyzed using varying techniques including X-ray diffraction, scanning electron microscopy, Andersen cascade impaction, differential scanning calorimetery, Karl Fischer titration, and viral plaque assay. CAN-BD has been used to produce powders of live-attenuated measles virus vaccine with characteristics desirable for lung delivery. The powders retain viral activity through forming and drying the microparticles by CAN-BD, and have passed the WHO stability test for 1 week at 37 degrees C. The powders have an amorphous character and a glass transition temperature of around 60 degrees C. Lyophilization, the present standard commercial method of processing measles vaccine makes solids with a water content of less than 1%. By substituting myo-inositol for sorbitol and using the CAN-BD drying technique the water content can be lowered to 0.5%. The most successful formulations to date have been based conceptually on the current lyophilized formulation, but with modifications to the type and amounts of sugar. Of current interest are formulations containing myo-inositol, as they retain high viral activity and have low initial water content.

Stabilization of measles virus for vaccine formulation.

An attenuated live measles virus (MV) was characterized by several biophysical methods as a function of temperature and pH. Following a method developed previously, the resultant light scattering and spectroscopic data were synthesized into an empirical phase diagram that visually and simultaneously represents the entire data set. Using this empirically-based phase diagram, screening assays were developed to identify potential vaccine stabilizers. Various compounds are shown by these assays to inhibit the temperature-induced aggregation of viral particles, and also to protect the integrity of the viral envelope. Accelerated stability assays show that, upon thermal challenge, MV formulated with these excipients retains its infectivity to a significant extent. Thus, the enhanced physical stability produced by this method is shown to protect the biological activity of this important but labile vaccine.