Receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential

The host adaptation of influenza virus is partly dependent on the sialic acid (SA) isoform bound by the viral hemagglutinin (HA). Avian influenza viruses preferentially bind the α-2,3 SA and human influenza viruses the α-2,6 isoform. Each isoform is predominantly associated with different surface epithelial cell types of the human upper airway. Using recombinant HAs and human tracheal airway epithelial cells in vitro and ex vivo, we show that many avian HA subtypes do not adhere to this canonical view of SA specificity. The propensity of avian viruses to adapt to human receptors may thus be more widespread than previously supposed.

Large is fast, small is tight: determinants of speed and affinity in subunit capture by a periplasmic chaperone

The chaperone/usher pathway assembles surface virulence organelles of Gram-negative bacteria, consisting of fibers of linearly polymerized protein subunits. Fiber subunits are connected through 'donor strand complementation': each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh β-strand in trans. Whereas the folding of Ig domains is a fast first-order process, folding of Ig modules into the fiber conformation is a slow second-order process. Periplasmic chaperones separate this process in two parts by forming transient complexes with subunits. Interactions between chaperones and subunits are also based on the principle of donor strand complementation. In this study, we have performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 capsular subunit from Yersinia pestis and analyzed the effect of the mutations on the structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. The results suggest that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enables Caf1M to rapidly bind an early folding intermediate of Caf1 and direct its partial folding. The switch from the Caf1M-Caf1 contact to the less hydrophobic, but considerably tighter and less dynamic Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M was engineered to bind Caf1 faster, tighter, or both faster and tighter. To our knowledge, this is the first successful attempt to rationally design an assembly chaperone with improved chaperone function.

The p85 subunit of phosphatidylinositol 3-kinase associates with the Fc receptor gamma-chain and linker for activitor of T cells (LAT) in platelets stimulated by collagen and convulxin.

There is extensive evidence to show that phosphatidylinositol 3-kinase plays an important role in signaling by the immune family of receptors, which has recently been extended to include the platelet collagen receptor, glycoprotein VI. In this report we present two potential mechanisms for the regulation of this enzyme on stimulation of platelets by collagen. We show that on stimulation with collagen, the regulatory subunit of phosphatidylinositol 3-kinase associates with the tyrosine-phosphorylated form of the adapter protein linker for activator of T Cells (LAT) and the tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif of the Fc receptor gamma-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor gamma-chain and LAT with p85 are rapid and supported by the Src-homology 2 domains of the regulatory subunit. We did not obtain evidence to support previous observations that the regulatory subunit of phosphatidylinositol 3-kinase is regulated through association with the tyrosine kinase Syk. The present results provide a molecular basis for the regulation of the p85/110 form of phosphatidylinositol 3-kinase by GPVI, the collagen receptor that underlies activation.

Possible role of aerosol transmission in a hospital outbreak of influenza.

BACKGROUND: We examined the role of aerosol transmission of influenza in an acute ward setting. METHODS: We investigated a seasonal influenza A outbreak that occurred in our general medical ward (with open bay ward layout) in 2008. Clinical and epidemiological information was collected in real time during the outbreak. Spatiotemporal analysis was performed to estimate the infection risk among patients. Airflow measurements were conducted, and concentrations of hypothetical virus-laden aerosols at different ward locations were estimated using computational fluid dynamics modeling. RESULTS: Nine inpatients were infected with an identical strain of influenza A/H3N2 virus. With reference to the index patient's location, the attack rate was 20.0% and 22.2% in the "same" and "adjacent" bays, respectively, but 0% in the "distant" bay (P = .04). Temporally, the risk of being infected was highest on the day when noninvasive ventilation was used in the index patient; multivariate logistic regression revealed an odds ratio of 14.9 (95% confidence interval, 1.7-131.3; P = .015). A simultaneous, directional indoor airflow blown from the "same" bay toward the "adjacent" bay was found; it was inadvertently created by an unopposed air jet from a separate air purifier placed next to the index patient's bed. Computational fluid dynamics modeling revealed that the dispersal pattern of aerosols originated from the index patient coincided with the bed locations of affected patients. CONCLUSIONS: Our findings suggest a possible role of aerosol transmission of influenza in an acute ward setting. Source and engineering controls, such as avoiding aerosol generation and improving ventilation design, may warrant consideration to prevent nosocomial outbreaks.

Localization and function of the Kv3.1b subunit in the rat medulla oblongata: focus on the nucleus tractus solitarii

The voltage-gated potassium channel subunit Kv3.1 confers fast firing characteristics to neurones. Kv3.1b subunit immunoreactivity (Kv3.1b-IR) was widespread throughout the medulla oblongata, with labelled neurones in the gracile, cuneate and spinal trigeminal nuclei. In the nucleus of the solitary tract (NTS), Kv3.1b-IR neurones were predominantly located close to the tractus solitarius (TS) and could be GABAergic or glutamatergic. Ultrastructurally, Kv3.1b-IR was detected in NTS terminals, some of which were vagal afferents. Whole-cell current-clamp recordings from neurones near the TS revealed electrophysiological characteristics consistent with the presence of Kv3.1b subunits: short duration action potentials (4.2 +/- 1.4 ms) and high firing frequencies (68.9 +/- 5.3 Hz), both sensitive to application of TEA (0.5 mm) and 4-aminopyridine (4-AP; 30 mum). Intracellular dialysis of an anti-Kv3.1b antibody mimicked and occluded the effects of TEA and 4-AP in NTS and dorsal column nuclei neurones, but not in dorsal vagal nucleus or cerebellar Purkinje cells (which express other Kv3 subunits, but not Kv3.1b). Voltage-clamp recordings from outside-out patches from NTS neurones revealed an outward K(+) current with the basic characteristics of that carried by Kv3 channels. In NTS neurones, electrical stimulation of the TS evoked EPSPs and IPSPs, and TEA and 4-AP increased the average amplitude and decreased the paired pulse ratio, consistent with a presynaptic site of action. Synaptic inputs evoked by stimulation of a region lacking Kv3.1b-IR neurones were not affected, correlating the presence of Kv3.1b in the TS with the pharmacological effects.

What have dendritic cells ever done for adjuvant design? Cellular and molecular methods for the rational development of vaccine adjuvants

Our new molecular understanding of immune priming states that dendritic cell activation is absolutely pivotal for expansion and differentiation of naïve T lymphocytes, and it follows that understanding DC activation is essential to understand and design vaccine adjuvants. This chapter describes how dendritic cells can be used as a core tool to provide detailed quantitative and predictive immunomics information about how adjuvants function. The role of distinct antigen, costimulation, and differentiation signals from activated DC in priming is explained. Four categories of input signals which control DC activation – direct pathogen detection, sensing of injury or cell death, indirect activation via endogenous proinflammatory mediators, and feedback from activated T cells – are compared and contrasted. Practical methods for studying adjuvants using DC are summarised and the importance of DC subset choice, simulating T cell feedback, and use of knockout cells is highlighted. Finally, five case studies are examined that illustrate the benefit of DC activation analysis for understanding vaccine adjuvant function.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Prostate cancer: important steps and considerations in the design of therapeutic vaccines

Vaccine-based immunotherapy can increase the overall survival of patients with advanced prostate cancer. However, the efficacy of vaccine-elicited anticancer immune responses is heavily influenced by the physical, nutritional, and psychological status of the patient. Given their importance, these parameters should be carefully considered for the design of future clinical trials testing this immunotherapeutic paradigm in prostate cancer patients.

Evaluating and optimizing oral formulations of live bacterial vaccines using a gastro-small intestine model

Gastrointestinal (GI) models that mimic physiological conditions in vitro are important tools for developing and optimizing biopharmaceutical formulations. Oral administration of live attenuated bacterial vaccines (LBV) can safely and effectively promote mucosal immunity but new formulations are required that provide controlled release of optimal numbers of viable bacterial cells, which must survive gastrointestinal transit overcoming various antimicrobial barriers. Here, we use a gastro-small intestine gut model of human GI conditions to study the survival and release kinetics of two oral LBV formulations: the licensed typhoid fever vaccine Vivotif comprising enteric coated capsules; and an experimental formulation of the model vaccine Salmonella Typhimurium SL3261 dried directly onto cast enteric polymer films and laminated to form a polymer film laminate (PFL). Neither formulation released significant numbers of viable cells when tested in the complete gastro-small intestine model. The poor performance in delivering viable cells could be attributed to a combination of acid and bile toxicity plus incomplete release of cells for Vivotif capsules, and to bile toxicity alone for PFL. To achieve effective protection from intestinal bile in addition to effective acid resistance, bile adsorbent resins were incorporated into the PFL to produce a new formulation, termed BR-PFL. Efficient and complete release of 4.4x107 live cells per dose was achieved from BR-PFL at distal intestinal pH, with release kinetics controlled by the composition of the enteric polymer film, and no loss in viability observed in any stage of the GI model. Use of this in vitro GI model thereby allowed rational design of an oral LBV formulation to maximize viable cell release.