Multivalent DNA Vaccines as A Strategy to Combat Multiple Concurrent Epidemics: Mosquito-Borne and Hemorrhagic Fever Viruses.

The emergence of multiple concurrent infectious diseases localized in the world creates a complex burden on global public health systems. Outbreaks of Ebola, Lassa, and Marburg viruses in overlapping regions of central and West Africa and the co-circulation of Zika, Dengue, and Chikungunya viruses in areas with A. aegypti mosquitos highlight the need for a rapidly deployable, safe, and versatile vaccine platform readily available to respond.

Protection against infection mediated by a synthetically engineered DNA vaccine.

Lyme disease is the most common vector-borne disease in North America. The etiological agent is the spirochete burgdorferi, transmitted to mammalian hosts by the Ixodes tick. In recent years there has been an increase in the number of cases of Lyme disease.

Active immunoprophylaxis with a synthetic DNA-encoded monoclonal anti-respiratory syncytial virus scFv-Fc fusion protein confers protection against infection and durable activity.

Respiratory Syncytial virus (RSV) is a major threat to many vulnerable populations. There are currently no approved vaccines, and RSV remains a high unmet global medical need. Here we describe the employment of a novel synthetic DNA-encoded antibody technology platform to develop and deliver an engineered human DNA-encoded monoclonal antibody (dMAb) targeting the fusion protein (F) of RSV as a new approach to prevention or therapy of at risk populations.

Immunogenicity of a protective intradermal DNA vaccine against lassa virus in cynomolgus macaques.

Lassa virus (LASV) is a hemorrhagic fever virus of the Arenaviridae family with high rates of mortality and co-morbidities, including chronic seizures and permanent bilateral or unilateral deafness. LASV is endemic in West Africa and Lassa fever accounts for 10-16% of hospitalizations annually in parts of Sierra Leone and Liberia according to the CDC. An ongoing outbreak in Nigeria has resulted in 144 deaths in 568 cases confirmed as LASV as of November 2018, with many more suspected, highlighting the urgent need for a vaccine to prevent this severe disease.

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses.

Novel IFN-γ ELISpot reveals robust T cell responses elicited after influenza nucleoprotein DNA vaccination in New Zealand White rabbits.

The New Zealand White rabbit is a highly accessible animal model which is regularly employed in biomedical research. However, the paucity of rabbit-specific reagents available limits its use in certain fields. Specifically, the lack of a reliable T cell assay has limited its employment in immune prophylactic and therapeutic studies.

Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer.

Chimeric antigen receptor T (CAR-T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR-T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody-based switch. Herein, we apply this approach to Her2-expressing breast cancers by engineering switch molecules through site-specific incorporation of FITC or grafting of a peptide neo-epitope (PNE) into the anti-Her2 antibody trastuzumab (clone 4D5).

Versatile strategy for controlling the specificity and activity of engineered T cells.

The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR-T--cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell.

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure.

Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL).

Multiformat T-cell-engaging bispecific antibodies targeting human breast cancers.

Four different formats of bispecific antibodies (bsAbs) were generated that consist of anti-Her2 IgG or Fab site-specifically conjugated to anti-CD3 Fab using the genetically encoded noncanonical amino acid. These bsAbs varied in valency or in the presence or absence of an Fc domain. Different valencies did not significantly affect antitumor efficacy, whereas the presence of an Fc domain enhanced cytotoxic activity, but triggered antigen-independent T-cell activation.

When is it okay to cry? An end-of-life simulation experience.

This article details how a small college of nursing affiliated with a faith-based health care corporation integrated the education of end-of-life care into a megacode simulation. Students participated in a high-fidelity simulated megacode scenario in which the simulator died. Following de-briefing, student groups participated in an additional scenario in which faculty coached them through postmortem care and interaction with a family member and a hospital chaplain.

A CXCR4-targeted site-specific antibody-drug conjugate.

A chemically defined anti-CXCR4-auristatin antibody-drug conjugate (ADC) was synthesized that selectively eliminates tumor cells overexpressing the CXCR4 receptor. The unnatural amino acid p-acetylphenylalanine (pAcF) was site-specifically incorporated into an anti-CXCR4 immunoglobulin G (IgG) and conjugated to an auristatin through a stable, non-cleavable oxime linkage to afford a chemically homogeneous ADC. The full-length anti-CXCR4 ADC was selectively cytotoxic to CXCR4(+) cancer cells in vitro (half maximal effective concentration (EC50 )≈80-100 pM).

Discovery and optimization of piperidyl-1,2,3-triazole ureas as potent, selective, and in vivo-active inhibitors of α/β-hydrolase domain containing 6 (ABHD6).

α/β-Hydrolase domain containing 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) to regulate certain forms of cannabinoid receptor-dependent signaling in the nervous system. The full spectrum of ABHD6 metabolic activities and functions is currently unknown and would benefit from selective, in vivo-active inhibitors. Here, we report the development and characterization of an advanced series of irreversible (2-substituted)-piperidyl-1,2,3-triazole urea inhibitors of ABHD6, including compounds KT182 and KT203, which show exceptional potency and selectivity in cells (<5 nM) and, at equivalent doses in mice (1 mg kg(-1)), act as systemic and peripherally restricted ABHD6 inhibitors, respectively.

Development and optimization of piperidyl-1,2,3-triazole ureas as selective chemical probes of endocannabinoid biosynthesis.

We have previously shown that 1,2,3-triazole ureas (1,2,3-TUs) act as versatile class of irreversible serine hydrolase inhibitors that can be tuned to create selective probes for diverse members of this large enzyme class, including diacylglycerol lipase-β (DAGLβ), a principal biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). Here, we provide a detailed account of the discovery, synthesis, and structure-activity relationship (SAR) of (2-substituted)-piperidyl-1,2,3-TUs that selectively inactivate DAGLβ in living systems. Key to success was the use of activity-based protein profiling (ABPP) with broad-spectrum and tailored activity-based probes to guide our medicinal chemistry efforts.

DAGLβ inhibition perturbs a lipid network involved in macrophage inflammatory responses.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLβ. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLβ in living systems.