An Opsonophagocytic Killing Assay for the Evaluation of Group A Streptococcus Vaccine Antisera

Group A Streptococcus (GAS) is a major cause of global mortality, yet there are no licensed GAS vaccines. Vaccine progress has been hampered, in part, by a lack of standardized assays able to quantify antibody function in test antisera. The most widely used assay was developed over 50 years ago by Rebecca Lancefield and relies on human whole blood as a source of complement and neutrophils. Recently, an opsonophagocytic killing (OPK) assay has been developed for GAS by adapting the OPK methods utilized in Streptococcus pneumoniae vaccine testing. This assay uses dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60 cells) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in the Lancefield assays. This protocol outlines methods for performing a GAS OPK assay including titering test sera to generate an opsonic index. This in vitro assay could aid in selecting vaccine candidates by demonstrating whether candidate-induced antibodies lead to complement deposition and opsonophagocytic killing

The Role of Tungsten Chemical State and Boron on Ammonia Formation Using N₂-H₂ Radiofrequency Discharges

This work aims at investigating the role of tungsten and boron surfaces on ammonia production with N2textendash H2 radiofrequency plasmas at 3 Pa. The experiments combine the analysis of the reaction products and surface chemical environment using mass spectrometry and x-ray photoelectron spectroscopy (XPS). We show that NH3 is formed upon discharges of N2 or H2 after having exposed a tungsten (W) foil to H2 or N2, respectively. A higher amount of ammonia is formed for the N2-then-H2 case, which we explain by the larger number of Eleytextendash Rideal reaction channels for the formation of NH x (s) and the lower surface diffusion barrier for adsorbed hydrogen, calculated using the density functional theory (DFT). As a result, H(s) combines with N(s) or NH x (s) through Langmuirtextendash Hinshelwood at a faster rate than N(s) combines with another N(s). The amount of NH3 formed with N2textendash H2 discharges after conditioning the tungsten foil with H2, N2 or O2 was also investigated. We observed that this pre-conditioning plays no major role on the amount of NH3 detected with the residual gas analyser, albeit a small decrease was observed after H2 contamination. With DFT, the adsorption energies of H on WO3 and W are found to be similar, while the adsorption of N on WO3 is significantly weaker. The similar NH3 concentrations obtained with a clean and oxidized tungsten surface thus suggest that the adsorption of N does not limit the formation rate of ammonia. The production of NH3 on boron was evaluated as well. The boron surface reduced the amount of detected ammonia almost by half. On the one side, a significant amount of H2 was removed from the surface during the Ar cleaning that followed, which suggests a strong retention of hydrogen. On the other side, the XPS data reveals that nitrogen forms strong bonds with boron and impurities on the surface, regardless on whether hydrogen is previously present on the surface or in the plasma volume. The presence of hydrogen in the plasma volume, simultaneously with nitrogen or after nitrogen exposure, is nevertheless necessary for the formation of NH(s) and NH2(s). No NH3(s) was however detected with XPS. The increased retention of both hydrogen and nitrogen on the boron surface may thus hinder the formation of NH3

New Antibody Weapons against an Old Foe

Antibodies have been used in a diagnostic capacity for many diseases and for identifying serotypes within single species of pathogens, notably between the multiple capsular polysaccharide serotypes of Streptococcus pneumoniae. For many years, the functions of antibodies in infection were thought to be limited to the opsonization of microorganisms followed by phagocytosis and to the fixing of complement. The thought that antibodies could have other functions has emerged only recently. The study by Yano and coworkers from the laboratory of Liise-anne Pirofski published in mBio [M. Yano, S. Gohil, J. R. Coleman, C. Manix, and L.-A. Pirofski, mBio 2(5):e00176-11, 2011] identifies one mechanism whereby nonopsonic antibodies enhance the transformation competence of two S. pneumoniae serotypes, which leads to an increase in genetic exchange and bacterial variability with a resulting population reduction through fratricide. These new and revealing antibody functions will add another chapter to the burgeoning story of the diversity and versatility of the immune response to bacteria

The effect of age on the response to the pneumococcal polysaccharide vaccine

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>is a leading cause of morbidity and mortality in the elderly. To prevent invasive pneumococcal diseases, the 23-valent pneumococcal polysaccharide vaccine (PPV) is recommended in subjects over 65 years of age. Although it has been reported to provide approximately 50-80% protection against invasive disease in the general elderly population, there is still controversy as to the effectiveness of the PPV in the elderly.</p> <p>Methods</p> <p>To evaluate the immune response to the pneumococcal polysaccharide vaccine in the elderly, samples from young adults and elderly were obtained before and one month after vaccination. The quantitative and qualitative response to the vaccine were measured by the ELISA and opsonophagocytic killing assay for eight vaccine type serotypes (4, 6B, 9V, 14, 18C, 19A, 19F, 23F) and one vaccine-related serotype (6A).</p> <p>Results</p> <p>The response to the pneumococcal polysaccharide vaccine showed a similar response between adults and elderly when evaluated by the ELISA, however the functional activity of the antibodies elicited after vaccination were lower in the elderly group for more than half of the serotypes evaluated. In comparison of the antibody needed for 1:8 opsonic titer, more antibodies were needed in the elderly for serotypes Pn 4, 19F, 23F and 6A, suggesting the functional activity of antibody detected by the ELISA was lower in the elderly compared with the adult group for these serotypes. As for subjects with an opsonic titer <8 after vaccination, only one subject each for serotypes Pn 4, 9V and 6A were found in the adult group. However, up to 10 (30.3%) of the subjects did not show opsonic activity after vaccination in the elderly group for serotypes Pn 4, 9V, 14, 19A and 6A.</p> <p>Conclusions</p> <p>Although the amount of antibodies elicited were similar between the two age groups, distinct differences in function were noted. This report highlights the importance of a quantitative and qualitative evaluation of the immunogenic response to the PPV in the elderly age group.</p> <p>Trial registration</p> <p>This trial is registered with Clinical trials.gov. Registration number NCT00964769</p

Immune response to 19A serotype after immunization of 19F containing pneumococcal conjugate vaccine in Korean children aged 12-23 months

PurposeThe purpose of this study was to evaluate the immune response to serotype 19A in children aged 12-23 months after immunization of the 19F containing 7-valent pneumococcal conjugate vaccine (PCV7).MethodsBlood samples from a total of 45 subjects (age 12-23 months) were included in the study. Subjects were categorized according to immunization status into three groups as follows: 18 subjects with 3 primary doses and 1 booster dose of PCV7 (booster group), 21 subjects with 3 primary doses before 12 months of age (primary group), and 6 subjects with no vaccination history of PCV7 (control group). An ELISA and opsonophagocytic killing assay (OPKA) was done to evaluate the immune responses against serotypes 19F and 19A.ResultsAccording to the ELISA, all subjects had antibody titers ≥0.35 µg/mL for serotypes 19F and 19A in the booster and primary group and 83.0% and 66.7% in the control group, respectively. According to the OPKA, subjects with opsonic activity (≥20) against serotypes 19F and 19A were 100% and 61.1% of the subjects in the booster group and 66.7% and 19.0% in the primary group, respectively. No subjects in the control group had opsonic antibodies against both serotypes.ConclusionIn conclusion, in children 12-23 months age who were previously vaccinated with PCV7, a cross-reactive immune response is elicited against serotype 19A after a primary series of 3 doses in a small proportion of subjects, and this response is amplified after booster vaccination

Immune response to pneumococcal polysaccharides 4 and 14 in elderly and young adults. I Antibody concentrations, avidity and functional activity

Streptococcus pneumoniae is a serious worldwide pathogen and the focus of numerous vaccine development projects. Currently the most widely accepted surrogate marker for evaluating the efficacy of a given vaccine is to utilize ELISA. Measurement of antibody concentration by ELISA without reduction in cross-reactive antibodies causes an overestimation of antibody concentration and therefore protection, this is most notable in the aged, an at risk group for this infection. We compared the immune response to the pneumococcal polysaccharides (PPS) 4 and 14 of 20 young to 20 elderly adults. Pre-and post-vaccination IgG antibody concentrations and antibody avidity against PPS4 and PPS14 were measured using two different enzyme-linked immunosorbant assay (ELISA) absorption protocols. All sera were pre-absorbed with either cell-wall polysaccharide (CPS), or CPS and serotype 22F polysaccharide. Pre- and post-vaccination IgG antibody concentrations for serotype 4, but not 14, were significantly lowered with the additional absorption with serotype 22F polysaccharide in both age groups. Young and elderly demonstrated a significant increase from pre- to post-immunization antibody concentration, using either absorption method; and opsonophagocytic antibody titers in response to both PPS4 and PPS14. The correlation coefficients between ELISA and opsonophagocytic assays were improved by additional absorption with serotype 22F in response to serotype 4, but not serotype 14 in all age groups. Opsonophagocytic antibody titers in a sub-group of elderly (>77 years of age) were significantly lower than the opsonophagocytic antibody concentrations in young adults. These results suggest the importance of eliminating cross-reactive antibodies from ELISA measurements by absorption of serum and an age-related impairment in the antibody response to pneumococcal polysaccharides

N-Crotylphthalimide

In the title compound {systematic name: 2-[(E)-but-2-en-1-yl]isoindoline-1,3-dione}, C12H11NO2, the phthalimide ring system is essentially planar, with a maximum deviation of 0.008 (1) Å, while the plane of the N-crotyl substituent is orthogonal to the phthalimide ring system, making a dihedral angle of 87.5 (1)°

The persistent cosmic web and its filamentary structure I: Theory and implementation

We present DisPerSE, a novel approach to the coherent multi-scale identification of all types of astrophysical structures, and in particular the filaments, in the large scale distribution of matter in the Universe. This method and corresponding piece of software allows a genuinely scale free and parameter free identification of the voids, walls, filaments, clusters and their configuration within the cosmic web, directly from the discrete distribution of particles in N-body simulations or galaxies in sparse observational catalogues. To achieve that goal, the method works directly over the Delaunay tessellation of the discrete sample and uses the DTFE density computed at each tracer particle; no further sampling, smoothing or processing of the density field is required. The idea is based on recent advances in distinct sub-domains of computational topology, which allows a rigorous application of topological principles to astrophysical data sets, taking into account uncertainties and Poisson noise. Practically, the user can define a given persistence level in terms of robustness with respect to noise (defined as a "number of sigmas") and the algorithm returns the structures with the corresponding significance as sets of critical points, lines, surfaces and volumes corresponding to the clusters, filaments, walls and voids; filaments, connected at cluster nodes, crawling along the edges of walls bounding the voids. The method is also interesting as it allows for a robust quantification of the topological properties of a discrete distribution in terms of Betti numbers or Euler characteristics, without having to resort to smoothing or having to define a particular scale. In this paper, we introduce the necessary mathematical background and describe the method and implementation, while we address the application to 3D simulated and observed data sets to the companion paper.Comment: A higher resolution version is available at http://www.iap.fr/users/sousbie together with complementary material. Submitted to MNRA

Surface Modification of ITER-like Mirrors after One Hundred Cleaning Cycles Using Radio-Frequency Plasma

In ITER, the metallic first mirrors (FMs) will undergo erosion due to their proximity to the fusion plasma and deposition of materials originated from the first walls (mainly beryllium). In-situ plasma cleaning is a promising technique to conserve the FMs optical properties by means of ion sputtering. In this work, the evolution of the optical properties of single-crystal (Sc) and nanocrystalline (Nc) molybdenum (Mo) and rhodium (Rh) mirrors were investigated up to 100 cycles of consecutive contamination and cleaning. Aluminum oxide (AlO) was used as contaminant to replace the toxic beryllium. The plasma cleaning was carried out using a capacitively coupled argon (Ar) plasma excited by a 60 MHz radio-frequency generator resulting in the formation of a self-bias applied on the mirrors of -280 V. The plasma potential being around 30 V, the Ar ion energy was about 310 eV. The optical properties of the mirrors were assessed using ex-situ reflectivity measurements. Moreover, the surface topography was characterized by means of scanning electron microscopy (SEM), focused ion beam (FIB) and roughness measurements using atomic force microscopy (AFM). ScMo and ScRh mirrors formerly exposed to 80 successful cleaning cycles using aluminum/tungsten (Al/W) deposits and air storage exhibit drastic changes in their optical properties after being subject to cleaning cycles using AlO as contaminant. Additionally, freshly polished ScRh were exposed to identical cleaning cycles. All Sc mirrors exhibited pits induced by the polishing procedure using diamond paste in addition of mounds/wavy patterns. The carbon incorporated during the polishing process was demonstrated to be responsible for the pitting of the surface. The Nc mirrors preserved their initial reflectivities after up to 100 cycles. The surface topography was systematically characterized and an average erosion rate for NcRh mirrors of about 59 nm per cycle has been estimated from FIB cross-sections. The optical properties of the Nc mirrors showed a superiority in the present study in comparison to the Sc materials due to the influence of their polishin