A computationally designed H5 antigen shows immunological breadth of coverage and protects against drifting avian strains.

Since the first identification of the H5N1 Goose/Guangdong lineage in 1996, this highly pathogenic avian influenza virus has spread worldwide, becoming endemic in domestic poultry. Sporadic transmission to humans has raised concerns of a potential pandemic and underscores the need for a broad cross-protective influenza vaccine. Here, we tested our previously described methodology, termed Computationally Optimized Broadly Reactive Antigen (COBRA), to generate a novel hemagglutinin (HA) gene, termed COBRA-2, that was based on H5 HA sequences from 2005 to 2006.

Chimeric yellow fever 17D-Zika virus (ChimeriVax-Zika) as a live-attenuated Zika virus vaccine.

Zika virus (ZIKV) is an emerging mosquito-borne pathogen representing a global health concern. It has been linked to fetal microcephaly and other birth defects and neurological disorders in adults. Sanofi Pasteur has engaged in the development of an inactivated ZIKV vaccine, as well as a live chimeric vaccine candidate ChimeriVax-Zika (CYZ) that could become a preferred vaccine depending on future ZIKV epidemiology.

Application of replication-defective West Nile virus vector to non-flavivirus vaccine targets.

The RepliVax vaccine platform(RV) is based on flavivirus genomes that are rationally attenuated by deletion. The self-limiting infection provided by RV has been demonstrated to be safe, highly immunogenic and efficacious for several vaccine candidates against flaviviruses. Here respiratory syncytial virus (RSV) F, influenza virus HA, and simian immunodeficiency virus (SIV) Env proteins were expressed in place of either prM-E or C-prM-E gene deletions of the West Nile (WN) virus genome.

A novel approach to a rabies vaccine based on a recombinant single-cycle flavivirus vector.

The RepliVax® vaccine (RV) platform is based on flavivirus genomes that are rationally attenuated by deletion. These single-cycle RV vaccine candidates targeting flavivirus pathogens have been demonstrated to be safe, highly immunogenic, and efficacious in animal models, including non-human primates. Here we show utility of the technology for delivery of a non-flavivirus immunogen by engineering several West Nile-based RV vectors to express full-length rabies virus G protein.

Preparation of pure, high titer, pseudoinfectious Flavivirus particles by hollow fiber tangential flow filtration and anion exchange chromatography.

Purification of enveloped viruses such as live flavivirus vaccine candidates poses a challenge as one must retain viral infectivity to preserve immunogenicity. Here we describe a laboratory-scale purification procedure for two replication defective (single-cycle) flavivirus variants for use in a pre-clinical setting. The two step purification scheme based on hollow fiber tangential flow filtration (TFF) followed by anion exchange chromatography using convective interaction media (CIM(®)) monoliths results in a ∼60% recovery of infectious virus titer and can be used to prepare nearly homogenous, highly purified vaccine viruses with titers as high as 1×10(9) focus forming units per mL.

Distinct cellular origins for serotonin-expressing and enterochromaffin-like cells in the gastric corpus.

Background & Aims: The alimentary tract contains a diffuse endocrine system comprising enteroendocrine cells that secrete peptides or biogenic amines to regulate digestion, insulin secretion, food intake, and energy homeostasis. Lineage analysis in the stomach revealed that a significant fraction of endocrine cells in the gastric corpus did not arise from Neurogenin3 (Neurog3)-expressing cells, unlike enteroendocrine cells elsewhere in the digestive tract. We aimed to isolate enriched serotonin-secreting and enterochromaffin-like (ECL) cells from the stomach and to clarify their cellular origin.

Single-dose vaccine against tick-borne encephalitis.

Tick-borne encephalitis (TBE) virus is the most important human pathogen transmitted by ticks in Eurasia. Inactivated vaccines are available but require multiple doses and frequent boosters to induce and maintain immunity. Thus far, the goal of developing a safe, live attenuated vaccine effective after a single dose has remained elusive.

Notch signaling differentially regulates the cell fate of early endocrine precursor cells and their maturing descendants in the mouse pancreas and intestine.

Notch signaling inhibits differentiation of endocrine cells in the pancreas and intestine. In a number of cases, the observed inhibition occurred with Notch activation in multipotential cells, prior to the initiation of endocrine differentiation. It has not been established how direct activation of Notch in endocrine precursor cells affects their subsequent cell fate.

Progenitor cell capacity of NeuroD1-expressing globose basal cells in the mouse olfactory epithelium.

The basic helix-loop-helix transcription factor NeuroD1 is expressed in embryonic and adult mouse olfactory epithelium (OE), as well as during epithelial regeneration, suggesting that it plays an important role in olfactory neurogenesis. We characterized NEUROD1-expressing progenitors, determined their progeny in the adult OE, and identified a subtle phenotype in ΔNeuroD1-knockout mice. All olfactory sensory neurons (OSNs) derive from a NeuroD1-expressing progenitor as shown by recombination-mediated lineage tracing, as do other sensory receptors of the nose, including vomeronasal, nasal septal, and Grunenberg ganglion neurons.

Characterization of the RepliVax platform for replication-defective flavivirus vaccines.

RepliVax, a novel replication-defective vaccine platform has recently been described as a suitable means of generating potent vaccines targeting flaviviruses. In this study, we directly compared attenuation, immunogenicity and efficacy of several prototype RepliVax constructs to available, well characterized live attenuated (LAV) and inactivated (INV) flavivirus vaccine controls in mice and hamsters. Other important aspects of general mechanisms and properties of RepliVax vaccines were also studied.

The Lbc Rho guanine nucleotide exchange factor α-catulin axis functions in serotonin-induced vascular smooth muscle cell mitogenesis and RhoA/ROCK activation.

Serotonin (5-hydroxytryptamine, 5-HT) is mitogenic for several cell types including pulmonary arterial smooth muscle cells (PASMC), and is associated with the abnormal vascular smooth muscle remodeling that occurs in pulmonary arterial hypertension. RhoA/Rho kinase (ROCK) function is required for 5-HT-induced PASMC mitogenesis, and 5-HT activates RhoA; however, the signaling steps are poorly defined. Rho guanine nucleotide exchange factors (Rho GEFs) transduce extracellular signals to Rho, and we found that 5-HT treatment of PASMC led to increased membrane-associated Lbc Rho GEF, suggesting modulation by 5-HT.

Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry: comparison of transgenic strains and relevance for fetal cell microchimerism.

Animal models are increasingly being used for the assessment of fetal cell microchimerism in maternal tissue. We wished to determine the optimal transgenic mouse strain and analytic technique to facilitate the detection of rare transgenic microchimeric fetal cells amongst a large number of maternal wild-type cells. We evaluated two strains of mice transgenic for the enhanced green fluorescent protein (EGFP): a commercially available, commonly used strain (C57BL/6-Tg(ACTB-EGFP)10sb/J) (CAG) and a newly created strain (ROSA26-EGFP) using three different techniques: in vivo and ex vivo fluorescent imaging (for whole body and dissected organs, respectively), PCR amplification of gfp, and flow cytometry (FCM).

Enteroendocrine precursors differentiate independently of Wnt and form serotonin expressing adenomas in response to active beta-catenin.

Wnt signaling is required for the maintenance of intestinal stem cells and self-renewal of the intestinal epithelium. Intestinal cancers are frequently associated with mutations that activate the Wnt pathway. The role of Wnt signaling on differentiation of lineage-specific precursors in the intestine is not well characterized.

Ubiquitous and uniform in vivo fluorescence in ROSA26-EGFP BAC transgenic mice.

Transplantation studies and cell lineage analyses require the ability to explicitly distinguish morphologically identical cells that have an identifiable marker indicating their origin in vivo. Several reporter mouse strains have been generated for such studies, but pancellular detection of the marker in all tissues has not been achieved. In this report, we describe the generation of transgenic mice that express enhanced green fluorescent protein (EGFP) under control of a 187 kb bacterial artificial chromosome (BAC) containing the murine ROSA26 locus, and show several advantages over existing EGFP reporter lines.

Functional proteomic screen identifies a modulating role for CD44 in death receptor-mediated apoptosis.

Apoptotic evasion is a hallmark of cancer and its resistance to chemotherapeutic drugs. Identification of cellular proteins that mediate apoptotic programs is a critical step toward the development of therapeutics aimed at overcoming apoptosis resistance. We developed an innovative high-throughput screen to identify proteins that modulate Fas ligand-mediated apoptosis using fluorophore-assisted light inactivation (HTS-FALIpop).

Neurogenin 3-expressing progenitor cells in the gastrointestinal tract differentiate into both endocrine and non-endocrine cell types.

Mice deficient for the transcription factor neurogenin 3 (ngn3) fail to develop endocrine cells in the intestine and pancreas and show partial endocrine differentiation in the stomach. We expressed Cre recombinase under control of a ngn3 BAC to achieve high fidelity cell lineage tracing in vivo to determine whether endocrine cells in these organs differentiate from NGN3+ precursor cells. Our results indicate that all small intestinal enteroendocrine cells arise from ngn3-expressing cells and confirm that NGN3+ cells give rise to all pancreatic endocrine cells as noted previously.

Minireview: Development and differentiation of gut endocrine cells.

For over 30 yr, it has been known that enteroendocrine cells derive from common precursor cells in the intestinal crypts. Until recently, relatively little was understood about the events that result in commitment to endocrine differentiation or the segregation of over 10 different hormone-expressing cell types in the gastrointestinal tract. The earliest cell fate decisions appear to be regulated by the Notch signaling pathway.