Predictive markers of safety and immunogenicity of adjuvanted vaccines

Vaccination represents one of the greatest public health triumphs; in part due to the effect of adjuvants that have been included in vaccine preparations to boost the immune responses through different mechanisms. Although a variety of novel adjuvants have been under development, only a limited number have been approved by regulatory authorities for human vaccines. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the current state of the art in the adjuvant field. Held at the U.S. Pharmacopeial Convention (USP) in Rockville, Maryland, USA, from 18 to 19 April 2013 and organized by the International Association for Biologicals (IABS), the conference focused particularly on the future development of effective adjuvants and adjuvanted vaccines and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. More information on the conference output can be found on the IABS website, http://www.iabs.org/.     

ATP inhibits hydroxyl radical formation and the inflammatory response of stimulated whole blood even under circumstances of severe oxidative stress

We recently reported that Adenosine-5′-triphosphate (ATP) is able to inhibit the inflammatory reaction in stimulated whole blood. Many diseases, in which inflammatory reactions are involved, are associated with oxidative stress. In the present study, we therefore, investigated the effect of ATP on cytokine release in stimulated whole blood under conditions of oxidative stress, as simulated by pre-incubation of blood with hydrogen peroxide (H₂O₂). In the presence of H₂O₂, ATP at concentrations of 100 and 300 μM inhibited Tumour Necrosis factor-alpha (TNF-α) release and stimulated IL-10 release in LPS-PHA stimulated whole blood. Moreover, electron spin resonance (ESR) measurements showed that ATP and its breakdown product Adenosine-5′-diphosphate (ADP) attenuated spin trap-hydroxyl radical adduct formation in the Fenton reaction. Our results demonstrate that even in circumstances of severe oxidative stress, ATP has marked anti-inflammatory properties in stimulated whole blood. Moreover, the inhibition of the hydroxyl radical ESR signal indicates a direct attenuation of oxidative stress by ATP.     

A Novel Method for the Preparation of Substrate-Free Cytochrome P-45011β

A new method for the removal of the stabilizing substrate, deoxycorticosterone, from adrenal cytochrome P-450_11β, has been developed. Dextran coated charcoal is used for the adsorption of the steroid and the adsorbed steroid is separated from the cytochrome P-450-preparation by low speed centrifugation. The substrate-free enzyme, obtained in this manner, has all the characteristic spectral properties of low-spin cytochrome P-450_11β, and may be converted to the high-spin form by the addition of deoxycorticosterone.

The dextran coated charcoal method has the following advantages over the previously used method of substrate removal. It does not require the addition of the cofactors for cytochrome P-450-dependant hydroxyla-tion of deoxycorticosterone, small amounts of enzyme may be prepared in a short time and the enzyme preparation is not diluted to any great extent during the process.     

Plant response to climate change along the forest‐tundra ecotone in northeastern Siberia

Russia's boreal (taiga) biome will likely contract sharply and shift northward in response to 21st century climatic change, yet few studies have examined plant response to climatic variability along the northern margin. We quantified climate dynamics, trends in plant growth, and growth–climate relationships across the tundra shrublands and Cajander larch (Larix cajanderi Mayr.) woodlands of the Kolyma river basin (657 000 km²) in northeastern Siberia using satellite‐derived normalized difference vegetation indices (NDVI), tree ring‐width measurements, and climate data. Mean summer temperatures (T_s) increased 1.0 °C from 1938 to 2009, though there was no trend (P > 0.05) in growing year precipitation or climate moisture index (CMI_gy). Mean summer NDVI (NDVI_s) increased significantly from 1982 to 2010 across 20% of the watershed, primarily in cold, shrub‐dominated areas. NDVI_s positively correlated (P < 0.05) with T_s across 56% of the watershed (r = 0.52 ± 0.09, mean ± SD), principally in cold areas, and with CMI_gy across 9% of the watershed (r = 0.45 ± 0.06), largely in warm areas. Larch ring‐width measurements from nine sites revealed that year‐to‐year (i.e., high‐frequency) variation in growth positively correlated (P < 0.05) with June temperature (r = 0.40) and prior summer CMI (r = 0.40) from 1938 to 2007. An unexplained multi‐decadal (i.e., low‐frequency) decline in annual basal area increment (BAI) occurred following the mid‐20th century, but over the NDVI record there was no trend in mean BAI (P > 0.05), which significantly correlated with NDVI_s (r = 0.44, P < 0.05, 1982–2007). Both satellite and tree‐ring analyses indicated that plant growth was constrained by both low temperatures and limited moisture availability and, furthermore, that warming enhanced growth. Impacts of future climatic change on forests near treeline in Arctic Russia will likely be influenced by shifts in both temperature and moisture, which implies that projections of future forest distribution and productivity in this area should take into account the interactions of energy and moisture limitations.     

A Novel Assay for Measurement of Membrane‐Protein Surface Expression using a β‐lactamase Reporter

The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Therefore, it is important to have sensitive techniques that allow measurement of surface expression. The current assays for such measurement are time consuming and low throughput. Here, we describe a quantitative, one‐step and potentially high‐throughput assay, using the β‐lactamase enzyme (βlac) as a reporter, for measurement of surface expression of proteins. In this assay, the βlac is fused to the extracellular portion of the plasma membrane protein of interest. To selectively measure surface expression, a cell‐impermeable substrate of βlac, nitrocefin, is used. We demonstrate the utility of the βlac assay using well‐established paradigms of internalization and molecular chaperoning, applied to two G‐protein‐coupled receptors and a monoamine transporter. Considering its simplicity and low cost, this assay could become a standard technique in the measurement of protein surface expression .      

Antibodies to Heteromeric Glycolipid Complexes in Guillain-Barré Syndrome

Autoantibodies are infrequently detected in the sera of patients with the demyelinating form of Guillain-Barré syndrome most commonly encountered in the Western world, despite abundant circumstantial evidence suggesting their existence. We hypothesised that antibody specificities reliant on the cis interactions of neighbouring membrane glycolipids could explain this discrepancy, and would not have been detected by traditional serological assays using highly purified preparations of single gangliosides. To assess the frequency of glycolipid complex antibodies in a Western European cohort of patients GBS we used a newly developed combinatorial glycoarray methodology to screen against large range of antigens (11 gangliosides, 8 other single glycolipids and 162 heterodimeric glycolipid complexes). Serum samples of 181 patients from a geographically defined, Western European cohort of GBS cases were analysed, along with 161 control sera. Serum IgG binding to single gangliosides was observed in 80.0% of axonal GBS cases, but in only 11.8% of cases with demyelinating electrophysiology. The inclusion of glycolipid complexes increased the positivity rate in demyelinating disease to 62.4%. There were 40 antigens with statistically significantly increased binding intensities in GBS as compared to healthy control sera. Of these, 7 complex antigens and 1 single ganglioside also produced statistically significantly increased binding intensities in GBS versus neurological disease controls. The detection of antibodies against specific complexes was associated with particular clinical features including disease severity, requirement for mechanical ventilation, and axonal electrophysiology. This study demonstrates that while antibodies against single gangliosides are often found in cases with axonal-type electrophysiology, antibodies against glycolipid complexes predominate in cases with demyelinating electrophysiology, providing a more robust serum biomarker than has ever been previously available for such cases. This work confirms the activation of the humoral immune system in the dysimmune disease process in GBS, and correlates patterns of antigen recognition with different clinical features.