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ORIGINAL RESEARCH article
Sec. Vaccines and Molecular Therapeutics
Volume 11 - 2020 | https://doi.org/10.3389/fimmu.2020.628971
Disease Severity, Fever, Age, and Sex Correlate With SARS-CoV-2 Neutralizing Antibody Responses
- 1Institute of Medical Immunology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
- 2Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany
- 3Institute of Virology, Charité-Universitätsmedizin Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, and German Centre for Infection Research (DZIF), Partner Site Charité, Berlin, Germany
- 4Institute of Transfusion Medicine, Charité - Universitätsmedizin Berlin, Berlin, Germany
- 5Department of Infectious Diseases and Respiratory Medicine, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
- 6Department of Tropical Medicine, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
- 7German Center for Lung Research (DZL), Partner Site Charité, Berlin, Germany
- 8Berlin Center for Advanced Therapies (BeCAT), Charité – Universitätsmedizin Berlin, Berlin, Germany
Clinical trials on the use of COVID-19 convalescent plasma remain inconclusive. While data on safety is increasingly available, evidence for efficacy is still sparse. Subgroup analyses hint to a dose-response relationship between convalescent plasma neutralizing antibody levels and mortality. In particular, patients with primary and secondary antibody deficiency might benefit from this approach. However, testing of neutralizing antibodies is limited to specialized biosafety level 3 laboratories and is a time- and labor-intense procedure. In this single center study of 206 COVID-19 convalescent patients, clinical data, results of commercially available ELISA testing of SARS-CoV-2 spike-IgG and –IgA, and levels of neutralizing antibodies, determined by plaque reduction neutralization testing (PRNT), were analyzed. At a medium time point of 58 days after symptom onset, only 12.6% of potential plasma donors showed high levels of neutralizing antibodies (PRNT50 ≥ 1:320). Multivariable proportional odds logistic regression analysis revealed need for hospitalization due to COVID-19 (odds ratio 6.87; p-value 0.0004) and fever (odds ratio 3.00; p-value 0.0001) as leading factors affecting levels of SARS-CoV-2 neutralizing antibody titers in convalescent plasma donors. Using penalized estimation, a predictive proportional odds logistic regression model including the most important variables hospitalization, fever, age, sex, and anosmia or dysgeusia was developed. The predictive discrimination for PRNT50 ≥ 1:320 was reasonably good with AUC: 0.86 (with 95% CI: 0.79–0.92). Combining clinical and ELISA-based pre-screening, assessment of neutralizing antibodies could be spared in 75% of potential donors with a maximal loss of 10% of true positives (PRNT50 ≥ 1:320).
Convalescent plasma therapy has been advocated since the beginning of the global spread of SARS-CoV-2 (1). Historically, convalescent plasma was applied in different diseases, including influenza, SARS, and MERS (2–4). Rapid availability and low costs of convalescent plasma therapy have spurred researchers worldwide to focus on this treatment approach. Alone in the US, convalescent plasma was used in >40,000 COVID-19 patients generating strong safety data (5). While observational findings in cases or smaller cohorts appeared promising, reporting improved survival, radiological resolution and viral load (6–8), data from two randomized controlled trials in China and the EU, were terminated prematurely, underpowered and unable to prove clinical benefit (9, 10). In a recently completed randomized controlled trial from India the majority of patients (~70%) received plasma with low levels of SARS-CoV-2 neutralizing antibodies (<1:80) (11). However, subgroup analyses report a dose-response relationship between convalescent plasma neutralizing antibody level and mortality, suggesting that treatment with high levels of neutralizing antibodies is likely to be more beneficial and advocating the administration of accordingly selected plasmas in clinical trials (12). Based on pathophysiological considerations and first case reports, it is expected that in particular patients with deficient antibody production may be more likely to benefit from convalescent plasma treatment (13, 14).
Identifying adequate donors is of critical importance for any ongoing trials and centers actively recruiting convalescent plasma donors. Collection of convalescent plasma is highly regulated by national authorities requiring, e.g., different periods for quarantine before convalescent plasma donation and for storage before approval for use in COVID-19 patients. Levels of neutralizing antibodies required for prevention or treatment in COVID-19 remain to be determined. However, based on general pathophysiological considerations it seems plausible to use higher levels of antibodies and FDA and EMA advocate levels of at least 1:80 to 1:160 and preferably higher titers (15). Unfortunately, evaluating neutralizing antibody capacities requires highly specialized expertise, availability of a biosafety level 3 laboratory and is time- and labor-intense. Consequently, and despite significant variability between different commercially available ELISA tests, these tests are applied frequently as a surrogate marker for the levels of neutralizing antibodies (16).
In order to assess convalescent plasma therapy in COVID-19, characterization of convalescent plasma by neutralizing antibody capacities is indispensable for interpretation of safety and efficacy. Preference should be given to donors of COVID-19 convalescent plasma with the higher neutralizing antibody response. Multiple assays for the detection of anti-SARS-CoV-2 antibodies are available, including several systems for high-throughput testing (17–20). These assays, although not of major importance in standard care settings, but can support clinicians to assess immune response in patients and are an important tool in epidemiological studies (21). The gold-standard to assess the neutralizing capacity of serum antibodies is the plaque-reduction neutralization test by using wild type virus isolates. However, this test needs access to wild-type isolates and BSL3 capacity, including trained personnel. Recently, PRNT surrogate assays, such as pseudotype-based neutralization assays (22) and surrogate virus neutralization test (sVNT) were established showing promising performance (23, 24). However, algorithms without multiple and complex test systems may streamline the identification process of adequate plasmas donors.
In this single center study, we characterized potential COVID-19 convalescent plasma donors for presence of IgG- and IgA-antibodies to the S1 domain of the SARS-CoV-2 spike (S) protein by ELISA and for neutralizing antibody capacity determined by plaque reduction neutralization test (PRNT). Furthermore, by correlating these results with clinical data we aimed to identify a clinical vignette that would help to improve the donor selection process. We prefer donors with a PRNT50 of ≥1:320 enabling to achieve a neutralizing antibody titer (PRNT50) of >1:40 with the administration of 440 ml of convalescent plasma in recipients with <75 kg body weight or 660 ml of convalescent plasma in recipients with a body weight of 75-110 kg (calculation example assuming 40 ml plasma volume per kg body weight: 70 kg × 40 ml = 2800 ml; 440 ml of convalescent plasma/(2800 + 440 ml) = 0.14; PRNT50 of 1:320 × 0.14 = 1:43 in the recipient).
Human Subjects and Serum Samples
In April 2020 we started screening patients who recovered from mild to moderate COVID-19 for convalescent plasma donation. Potential plasma donors were selected in agreement with German national plasma donation guidelines. Required age range is 18–60 years in first time donors and 18–68 in experienced plasma donors, individual exceptions due to extraordinary fitness and in absence of relevant comorbidities can apply. According to national guidelines, convalescent subjects required also to be clinically asymptomatic after COVID-19 disease for at least 4 weeks prior to plasma donation. The use of blood from healthy human subjects and from COVID-19-convalescent subjects was approved by the Institutional Review Board at Charité - Universitätsmedizin Berlin (EA2/092/20 and EA2/066/20) (25). All patients enrolled gave written informed consent in person. Convalescent subjects had a history of SARS-CoV-2 infection confirmed by a positive RT-PCR from pharyngeal swab. Eligibility of potential donors was checked according to national regulatory guidelines for plasma donors (Richtlinie Hämotherapie) (26). Clinical data was collected using a questionnaire for clinical symptoms including fever, dyspnea, cough and anosmia/dysgeusia as well as for requirement for hospitalization due to COVID-19. For serologic and neutralization testing, 9-ml blood from each donor were collected in serum tubes. Serum tubes were centrifuged at 1,500 g and 20°C for 15 min and aliquoted into 500- to 1,000-μl aliquots and stored at −20°C until further processing.
Enzyme-Linked Immunosorbent Assay for Anti-SARS-CoV-2 S1-IgG and -IgA
For the detection of IgG and IgA to the S1 domain of the SARS-COV-2 spike (S) protein, anti-SARS-CoV-2 assay was used according to the manufacturer´s instructions (Euroimmun, Lübeck, Germany). Serum samples were tested at a 1:101 dilution using the fully EUROIMMUN Analyzer. Optical density (OD) ratios were calculated by dividing the OD at 450 nm by the OD of the calibrator included in the kit. The calculated OD ratios can be used as a relative measure for the concentration of antibodies in the serum. For IgG and IgA response, an OD ratio < 1.1 was considered to be non-reactive.
Plaque Reduction Neutralization Test for SARS-CoV-2
PRNTs for SARS-CoV-2 were performed as previously described (27, 28). Briefly, Vero E6 cells were seeded in a 24-well plate format. Sera were diluted and mixed with 100 plaque forming units of SARS-CoV-2 (strain: SARS-CoV-2/human/DEU/BavPat2-ChVir984, NCBI GenBank Acc. No. MT270112.1). Each 24-well was incubated with serum-virus solution for 1 h at 37°C. After 1 h, supernatants were discarded, cells were washed once with PBS, and 1.2% Avicel solution in DMEM was added to the wells. After 3 days at 37°C, all supernatants were discarded and cells were fixed using a 6% formaldehyde/PBS solution and stained with crystal violet. Serum dilutions with a plaque reduction of 50% (PRNT50) are referred to as titers.
All analyses were conducted in R (version 4.0.2.) using the Hmisc (version 4.4-0) and rms packages (version 6.0-0). For proportional odds (PO) logistic regression on categories of neutralizing SARS-CoV-2 antibody titers PRNT50 values for 1:320 (10 donors), 1:640 (7 donors), and 1:1,280 (9 donors) were pooled into a single category (≥1:320) to gain sufficient per-category sample sizes and 3 of 206 clinical records were excluded due to missing entries in several variables. The full model was prespecified to include variables sex, age in years, days after positive SARS-CoV-2 RT-PCR testing, fever, dyspnea, cough, anosmia/dysgeusia, and requirement for hospitalization. For developing a predictive model, shrunken coefficients of the full model were first re-estimated by L2-penalized regression and the linear predictors approximated by ordinary linear regression and step-down backward selection of the top 4 important predictors with only minute loss of precision (R2 > 0.99 compared to full model). Correct estimates of the variance of the coefficients of the reduced model were calculated using equation 5.2 in (29).
A second PO logistic regression model was used to calculate L2-penalized regression coefficients of anti-SARS-CoV-2 S1-IgG OD ratios for higher SARS-CoV-2 neutralizing antibody titers. Coefficients and covariance matrices of both models were used to create spreadsheets (Supplementary Data Sheet 2 and 3) in large adaptation of an example given by (30). Receiver-operator characteristics were analyzed using the pROC package (version 1.16.2). Specificities at 0.95 target sensitivity and 95% confidence intervals (CI) for all performance measures were computed with 2,000 stratified bootstrap replicates. The true positive number after two consecutively performed tests A and B can be estimated by TPAB = T × (SeA × SeB − covSeAB), with T the number of real cases and the final serial-test sensitivity which is given by the product of the individual tests’ sensitivities SeA and SeB, adjusted by their conditional covariance covSeAB. The false positive number calculates as FPAB = N × ((1 − SpA) × (1 − SpB) + covSpAB), with N the number of real negative cases, and SpA, SpB the individual tests’ specificities and their covariance covSpAB. The final specificity of two consecutive tests can be estimated by SpAB = SpA + SpB − SpA × SpB − covSpAB (31).
Potential plasma donors contacted our clinic after being diagnosed with COVID-19. General suitability for plasma donation was evaluated before presentation at our clinic by telephone interview. In total, 206 potential convalescent plasma donors were assessed. In agreement with national recommendations, all convalescent patients had a symptomatic confirmed SARS-CoV-2 infection (mandatory positive SARS-CoV-2 test by RT-PCR testing in pharyngeal swab). In addition, patients required to be clinically asymptomatic after COVID-19 disease for at least 4 weeks.
All 206 donors were tested by ELISA for IgG and IgA against SARS-CoV-2 S1 spike and by plaque reduction neutralization test (PRNT50). In total, 54% of donors were female, and median age of donors was 37.4 years. Median time point of antibody testing was 53 days after positive SARS-CoV-2 PCR testing and 58 days after symptom onset. Patients were interviewed for clinical symptoms including fever, dyspnea, cough, and anosmia/dysgeusia. In addition, patients were asked to report requirement for hospitalization due to COVID-19.
Fever was reported by 109 donors (53%), dyspnea by 70 donors (34%), cough by 130 (63%), and anosmia/dysgeusia by 111 donors (54%). Hospitalization due to COVID-19 was reported by 19 donors (9%) (Table 1).
Detection of Anti-SARS-CoV-2 S1-IgG and IgA by ELISA
Despite positive SARS-CoV-2 RT-PCR result, IgG-and IgA-antibodies against S1 spike protein were only detectable in 89% of donors. Using the manufactures cut-off for SARS-CoV-2 spike ELISA with a ratio < 1.1, a total of 23 donors showed seronegativity for both (IgG(−)/IgA(−)), 42 donors were IgG(+)/IgA(−), six donors were IgG(−)/IgA(+).
Detection of Neutralizing Antibodies by Plaque Reduction Neutralization Test (PRNT50)
No detectable neutralizing SARS-CoV-2 antibodies (PRNT50 < 1:20) were found in 33 donors (16%). A PRNT50 of ≥1:20–<1:40 was detectable in 39 donors (19%), a value of ≥1:40–<1:80 was found in 38 donors (18%), PRNT50 levels of ≥1:80–<1:160 were seen in 40 donors (19%), levels of ≥1:160–<1:320 in 30 donors (15%) and PRNT50 levels ≥ 1:320 were detected in 26 (13%) (Table 1).
Correlation of Anti-SARS-CoV-2 S1-IgG and IgA With Levels of Neutralizing Antibodies (PRNT50)
SARS-CoV-2 neutralizing antibody titers significantly correlated both with anti-S1 IgG and anti-S1 IgA antibodies (Figure 1). Receiver-operator characteristics (ROC curves) for prediction of a PRNT50 titer of at least 1:320 revealed only moderate discrimination when using anti-S1 IgA as predictor (AUC: 0.76 with 95% CI: 0.67–0.84, Figure 1A) whereas anti-S1 IgG performed very well (AUC: 0.92 with 95% CI: 0.87–0.96, Figure 1B).
Figure 1 SARS-CoV-2 neutralizing antibody titers correlate with anti-S1 IgG and to a lesser extend with IgA antibodies. Plaque reduction neutralization test (PRNT50) titers of 206 plasma donors were correlated with anti-S1 IgA (A) and anti-S1 IgG (B) ELISA measurements (ρ, Spearman’s rank correlation coefficients). Values for 1:320, 1:640 (squares), and 1:1,280 (triangles) titers were pooled into a single category (≥1:320) to gain sufficient per-category sample sizes and to represent the recommended minimum titer for convalescent plasma therapy (dashed vertical line). Dashed horizontal lines indicate ELISA positivity cutoffs provided by the manufacturer EUROIMMUN). Right panels show receiver-operator characteristics (ROC curves) for prediction of a PRNT50 titer of at least 1:320 (AUC, area under the curve).
Bivariate analyses using non-parametric rank correlation coefficients further revealed strong association of PRNT50 titers with presence of fever and hospitalization status, the latter of which positively correlated with other variables indicative of disease severity, such as fever, dyspnea, age, and time since positive PCR testing. A summarizing correlation matrix between all variables analyzed is shown by heat plot representation in Figure 2A.
Figure 2 Hospitalization, fever and age are main predictors of high SARS-CoV-2 neutralizing antibody titers in convalescent plasma. (A) Heat plot representation summarizing bivariate rank correlation coefficients between all variables analyzed. Shown is only the upper triangle of the correlation matrix where correlation coefficients are given as Spearman’s ρ for continuous variables (four leftmost columns) and Somers’ D for bivariate correlations including at least one binary variable. Positive and negative correlations are colored in red and blue, respectively. Numbers within circles represent correlation coefficients expressed as percent while circle sizes are proportional to the absolute correlation coefficient values. (B) Adjusted odds ratios (OR) estimated by multivariable proportional odds (PO) logistic regression analysis are shown for each predictor of PRNT50 titer as response variable. OR of continuous variables are calculated for 4 weeks increase in time since PCR+ and 10 years increase in age. Blue of different transparencies indicates 90, 95, and 99% confidence levels. (C) Predicted probabilities for PRNT50 titers of at least 1:320 for varying predictor settings with 95% confidence bands. Shown are predicted probabilities for age ranging from 18 to 75 years in females (top row) and males (bottom row) by all possible combinations of hospitalization status with presence of anosmia or dysgeusia (columns) and fever (blue and red lines). Effects were estimated by a L2-penalized PO logistic regression model including all shown, most important variables age, sex, fever, anosmia or dysgeusia, and hospitalization.
Multivariable Analysis of Clinical Features With Levels of Neutralizing Antibodies (PRNT50)
Multivariable PO logistic regression analysis revealed need for hospitalization for COVID-19 (odds ratio 6.87; p-value 0.0004) and fever (odds ratio 3.00; p-value 0.0001) as leading factors affecting levels of SARS-CoV-2 neutralizing antibody titers in convalescent plasma donors (Table 2 and Figure 2B). Also increasing age (odds ratio 1.32; p-value 0.0165) and male sex (odds ratio 0.47; p-value 0.006) influenced neutralizing antibody titers. There was a trend toward significance for anosmia/dysgeusia (odds ratio 1.58; p-value 0.0847), while coefficients for time point since RT-PCR positivity, dyspnea and coughing were not significant in the model (Table 2 and Figure 2B).
Table 2 Multivariable proportional odds logistic regression analysis of factors affecting SARS-CoV-2 neutralizing antibody titers in convalescent plasma.
Model for Neutralizing Anti-SARS-CoV-2 Antibody Levels to Tailor an Individual Screening Strategy
Using penalized estimation, a predictive PO logistic regression model including the most important variables age, sex, fever, hospitalization, and anosmia or dysgeusia was developed (see Supplementary Data Sheet 3). The predictive discrimination for PRNT50 ≥ 1:320 was reasonably good with AUC: 0.86 (CI: 0.79–0.92). Probabilities for PRNT50 ≥ 1:320 were modelled to demonstrate effects of the individual predictors (Figure 2C). Both male and female donors showed an increase of predicted probability with increasing age. Female donors and fever during COVID-19 resulted in a higher predicted probability for PRNT50 ≥ 1:320. Predicted probability increased further when donors were hospitalized for COVID-19 and additionally developed anosmia or dysgeusia. The highest probabilities for PRNT50 ≥1:320 can therefore be expected in male and female patients aged 60 years or older with fever, previous hospitalization for COVID-19 and accompanying anosmia or dysgeusia.
In order to reduce labor and cost in performing plaque reduction neutralization testing, a clinical prediction model and anti-S1 IgG testing may be utilized with an appropriately defined sensitivity for preselection with minute loss of donors having potentially high neutralizing anti-SARS-CoV-2 antibody titers (see Supplementary Material section).
Using the clinical vignette with a classification cutoff at 0.95 target sensitivity, approximately, one in four positively screened donors is expected to have a titer ≥ 1:320, corresponding to a positive predictive value (PPV) of 0.27 (CI: 0.19–0.34) and a specificity of 0.62 (CI: 0.41–0.73). At 0.95 sensitivity, the anti-S1 IgG ELISA has 0.71 specificity (CI: 0.62–0.88) and a PPV of 0.32 (CI: 0.26–0.54), i.e., one in three donors tested above the corresponding cutoff IgG OD ratio of 3.83 will have a PRNT50 titer of 1:320 or higher.
Aiming on gain in pre-screening specificity, i.e., to further reduce numbers of false-positives that may enter PRNT, a serial testing strategy can be devised where anti-S1 IgG ELISA is performed only in donors that were positively classified in the clinical vignette. Accepting 10% loss of donors with PRNT50 ≥ 1:320 (i.e., 0.95 × 0.95 target sensitivity), conditional pre-selection would result in a sensitivity of 0.89 (CI: 0.79–0.98), a specificity of 0.85 (CI: 0.79–0.95) and a PPV of 0.46 (CI: 0.38–0.72) corresponding to 23 true positive (CI: 21–25) and 27 false positive (CI: 9–38) out of the 203 available donors in our study. Thus, if anti-S1 IgG ELISA testing was performed in donors pre-identified via the clinical vignette, analyzing PRNT could have been spared in 75% of the donors of this cohort with the given prevalence of 12.6% for PRNT50 titers ≥ 1:320.
With the ongoing SARS-CoV-2 pandemic, convalescent plasma continues to be a plausible treatment option, in particular for patients with impaired specific SARS-CoV-2 antibody response (e.g., primary and secondary immunodeficiency). Although data showing clear efficacy is still missing, there is an increasing body of evidence, that clinical benefit is linked to the neutralizing capacity of convalescent plasma (12, 32–34). Selecting plasma donors with high probability for higher levels of neutralizing antibodies is therefore of importance for any ongoing or future convalescent plasma trial.
While levels of neutralizing antibodies to MERS-CoV were reported to remain stable for > 2 years (35), for SARS-CoV-2, it is now recognized, that antibody levels are already declining after 3 months (36, 37). Therefore, identifying potential COVID-19 plasma donors is underlying additional time constraints.
In line with the highly variable disease course in COVID-19, also humoral immune response expresses a great heterogeneity. In this study higher levels of neutralizing antibodies (≥1:320 in PRNT50) were only detectable in 12.6% of donors. Despite positive RT-PCR testing for SARS-CoV-2 in nasopharyngeal swab, 11% of donors did not develop any detectable SARS-CoV-2 S1-IgG or -IgA antibodies and 16% no detectable neutralizing antibodies, respectively.
The availability of prescreening algorithms in order to increase the probability of selecting plasma donors with higher neutralizing antibody titers could support convalescent plasma collection.
The assessment of clinical convalescent donor characteristics is readily available and could serve as a “pre-test” filter. Our study showed that in COVID-19 convalescent plasma donors, clinical parameters of disease severity (i.e., need for hospitalization, fever and anosmia or dysgeusia) as well as sex and age correlate with levels of neutralizing antibodies against SARS-CoV-2. Using additional ELISA tests, donors with a given level of neutralizing antibodies, i.e., ≥1:320, may reliably be identified with appropriate cutoffs.
Depending on the available local resources for testing neutralizing antibody titers and depending on the availability of convalescent plasma donors, i.e., high or low prevalence regions, different sensitivities and specificities may be preferred. Prediction models may help to develop an individual screening strategy for potential convalescent plasma donors. In our cohort, combining clinical vignette and IgG-ELISA, both with a cutoff at target sensitivity of 95%, PRNT assays could be spared in 75% of initial donors with the accepted loss of 10% of true positives. Our observation of 13% high neutralizing antibodies (PRNT50 of ≥1:320) is in line with recent reports from smaller cohorts in the US and Europe, reporting 13%–14.5% with PRNT50 of ≥ 1:500 (38, 39). In contrast, a recent publication from Brazil showed a PRNT50 > 1:320 in approximately 38% of donors, however donors were tested earlier. Wendel et al, also highlighted that 36.2% of convalescent plasma donors remained SARS-CoV-2 RT-PCR positive within 28-48 days of recovery (40). Klein et al. detected neutralizing antibodies (>1:20) in 80% but did not report specifically on different titer dilutions (41).
Our results confirm that clinical severity (i.e., need for hospitalization) and age predicts levels of neutralizing antibody responses (41). In addition, we found that fever and anosmia/dysgeusia correlates with neutralizing antibody titers which is also supported by other studies (42–44). It remains inconclusive, if and how sex correlates with neutralizing antibody responses in COVID-19 patients. In general, females develop more profound adaptive immune responses against viral infections and vaccines than males which might translate into the observed sex-differences in SARS-CoV-2 pathogenesis and that may underly reduced disease vulnerability in women (45–48). In patients with moderate COVID-19, Takahashi et al. report (49) significantly higher T cell activation and a trend for higher SARS-CoV-2-specific antibody titers in female patients. While we and others found that female donors had a higher probability of high neutralizing antibodies (18, 50) or SARS-CoV-2 IgG antibodies (49, 51, 52), other reports had the opposite finding (38, 41, 53–56) or did not find a significance at all (39, 57, 58). These seemingly contradictory findings might be due to sampling or selection bias in individual studies but also to apparently different dynamics in the SARS-CoV-2 neutralizing antibody response in male and female donors (50, 59). Despite our finding of higher antibody responses in female donors, it is important to stress that female donors after pregnancy carry an elevated risk of HLA- and HNA-antibodies. These antibodies are associated with increased transfusion reactions resulting in the exclusion for plasma donation. Generally excluding female donors would therefore spare resource for these additional tests.
Our study has limitations. It is of cross-sectional nature, and the narrow time window of sample collection with regard to COVID-19 disease onset does not allow to analyze possible kinetics of antibody responses. Results of our study are limited to a specific ELISA test system recognizing antibodies against spike protein, so we cannot apply our observations as a general rule to other ELISA systems. Our analysis is not representative of the humoral immune response to SARS-CoV-2 in all patients but focuses on a rather homogeneous cohort of convalescent patients that would qualify as plasma donors. Although application of our prediction tool on a publicly available dataset (18) demonstrated acceptable accuracy (Supplementary Data Sheet 4), predictive performance may be worse in another clinical setting, e.g., due to a different case-mix, thus requiring external validation and potential recalibration of the prescreening model. In addition, no standardization of the different neutralizing antibody assays has been conducted. However, type of cell line, real virus or pseudovirus, count of viral particles, culture volumes and other factors are all likely to affect test results. It is therefore difficult to compare levels of (neutralizing) antibodies to SARS-CoV-2 from different studies.
The wide spectrum of antibody responses in convalescent plasma donors with mild COVID-19 disease remains an important issue. Our data support a clinical evaluation of COVID-19 symptoms as a pre-filter in order to identify convalescent plasma donors with potentially higher neutralizing antibody levels. The availability of prescreening algorithms in order to increase the probability of selecting plasma donors with higher neutralizing antibody titers could support convalescent plasma collection by sparing resources for labor-intense testing of neutralizing antibodies.
Data Availability Statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
The studies involving human participants were reviewed and approved by Institutional Review Board at Charité Universitaetsmedizin Berlin (EA2/092/20 and EA2/066/20). The patients/participants provided their written informed consent to participate in this study.
SSc and LH made substantial contributions to conception and design. LH, KW, FK, and TZ made patient samples available. SSc, TS, VC, MM, and LH performed acquisition and analysis of data. SSc, TS, VC, and LH performed interpretation of data. TS, MM, CD, and VC performed analysis of neutralizing IgG and ELISA data. SSc, TS, VC, and LH wrote the article. SSt, KW, NAB, OM, FK, TZ, MW, LS, MM, CS, H-DV, and CD reviewed the manuscript critically for important intellectual content. All authors contributed to the article and approved the submitted version.
Parts of this work were funded by the German Ministry of Health (Konsiliarlabor für Coronaviren) to CD and VC. The other researchers did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. We acknowledge support from the Open Access Publication Fund of Charité – Universitätsmedizin Berlin.
Conflict of Interest
MM and VC are named together with Euroimmun GmbH on a patent application filed recently regarding the diagnostic of SARS-CoV-2 by antibody testing.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Y. Vural, M. Antelmann, S. Bauer, M. L. Schmidt, A. Richter, and F. Walper for expert technical assistance.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.628971/full#supplementary-material
Supplementary Data Sheet 1 | ROC analysis for PRNT50 ≥1:320. This Excel file contains two spreadsheets with receiver-operator characteristics for using anti-SARS-CoV-2 S1-IgG OD ratio as predictor of PRNT50 titers ≥1:320, and for using the PO logistic regression model with a four-variable clinical vignette (sex, age, fever, anosmia/dysgeusia, and requirement for hospitalization) to predict PRNT50 titers ≥1:320, respectively. The spreadsheets list several performance measures for a range of possible thresholds as given by each donor’s predicted value. These may be used to identify classification cutoffs at a certain target value of a performance measure, for example a sensitivity of 95%.
Supplementary Data Sheet 2 | Predictive PO model for ELISA IgG OD ratio. This Excel file contains spreadsheets to calculate probabilities of high PRNT50 titers using IgG OD ratio values as input. The first sheet provides a detailed description of how this may be used. In short, IgG OD ratio values as determined using Euroimmun anti-SARS-CoV-2 S1-IgG ELISA can be entered to calculate probabilities of higher PRNT50 titers in sheets for either of the categories “PRNT50>=320”, “PRNT50>=160”, “PRNT50>=80”, “PRNT50>=40”, or “PRNT50>=20”, respectively. For example, in “PRNT50>=320” an estimated probability higher than 0.06 (corresponding to IgG OD 3.83 for 95% target sensitivity) would qualify a sample for inclusion into final plaque reduction neutralization testing. For demonstration purposes, sheets are prefilled with data from this study.
Supplementary Data Sheet 3 | Predictive PO model for clinical data. This Excel file contains spreadsheets to calculate probabilities of high PRNT50 titers using a clinical vignette with four important variables as input. The first sheet provides a detailed description of how this may be used. In short, patient characteristics (sex, age, fever, anosmia/dysgeusia, and requirement for hospitalization) can be entered to calculate probabilities of higher PRNT50 titers in sheets for either of the categories “PRNT50>=320”, “PRNT50>=160”, “PRNT50>=80”, “PRNT50>=40”, or “PRNT50>=20”, respectively. For example, in “PRNT50>=320” an estimated probability higher than 0.1046 (for 95% target sensitivity) would qualify a sample for inclusion into further anti-SARS-CoV-2 S1-IgG ELISA screening or final plaque reduction neutralization testing. For demonstration purposes, sheets are prefilled with data from this study.
Supplementary Data Sheet 4 | Validation of Predictive PO using data from Padoan et al. Padoan et al. (18) have assessed the performance of different immunoassays for the detection of SARS-CoV-2 antibodies in comparison with neutralization activity. For 68 plasma donors an anti-spike IgG assay as well as PRNT testing was performed and results are publicly available in a dataset (60) which is used here to demonstrate the utility of the Excel prediction-tool. Data acquired at least 25 days after disease onset of 6 non-hospitalized and 22 hospitalized (12 moderate, 10 severe) recovered patients is used to calculate performance measures for the prediction of having a PRNT50 titer of ≥1:320. Accuracy (F-measure) was 0.79 for using the clinical vignette alone and 0.83 in combination with anti-Spike IgG.
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Keywords: COVID-19, SARS-CoV-2, immunodeficiency, antibody deficiency, coronavirus disease 2019, convalescent plasma, SARS-CoV-2 neutralizing antibody, plaque reduction neutralization test
Citation: Schlickeiser S, Schwarz T, Steiner S, Wittke K, Al Besher N, Meyer O, Kalus U, Pruß A, Kurth F, Zoller T, Witzenrath M, Sander LE, Müller MA, Scheibenbogen C, Volk H-D, Drosten C, Corman VM and Hanitsch LG (2021) Disease Severity, Fever, Age, and Sex Correlate With SARS-CoV-2 Neutralizing Antibody Responses. Front. Immunol. 11:628971. doi: 10.3389/fimmu.2020.628971
Received: 16 November 2020; Accepted: 29 December 2020;
Published: 29 January 2021.
Edited by:Karl Ljungberg, Eurocine Vaccines AB, Sweden
Reviewed by:Frauke Muecksch, The Rockefeller University, United States
Katelyn Ann Bruno, Mayo Clinic Florida, United States
Copyright © 2021 Schlickeiser, Schwarz, Steiner, Wittke, Al Besher, Meyer, Kalus, Pruß, Kurth, Zoller, Witzenrath, Sander, Müller, Scheibenbogen, Volk, Drosten, Corman and Hanitsch. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Leif G. Hanitsch, email@example.com
†These authors have contributed equally to this work
‡These authors share senior authorship

domain: medical


text:
Mar 19, 2013 (CIDRAP News) – The World Health Organization (WHO) yesterday updated its surveillance recommendations for the novel coronavirus (NCoV), advising clinicians to be wary of the virus even if patients with a severe respiratory illness have been confirmed to have another disease.
The agency also offered specific surveillance advice to nations in which NCoV has been detected, as well as investigations and studies to conduct where cases are detected. The WHO previously updated its guidance on Nov 28, 2012.
NCoV has infected at least 15 people worldwide, 9 of them fatally. Most cases have involved travel to or residence in the Middle East.
This year the third cluster of NCoV cases, in the United Kingdom, was confirmed, providing "clear evidence of limited, non-sustained human-to-human transmission," according to the guidance.
"A number of unanswered questions remain," the WHO added, "including the virus reservoir, the means by which seemingly sporadic infections are being acquired, the mode of transmission between infected persons, the clinical spectrum of infection, and the incubation period."
The UK cluster included a patient who originally tested positive for influenza A and was not thought to be infected with NCoV until tests came back positive, the agency said.
In part because of that experience, the WHO now recommends testing anyone who has a severe respiratory infection and develops unexpectedly severe disease despite appropriate treatment, even if another cause has been identified, if that other disease does not fully explain the patient's illness. Such a person should be tested regardless of place of residence or history of travel.
The guidance also recommends testing for patients who have a severe respiratory infection—without regard to residence or travel history and unless another cause has been identified—in these situations:
- The case is part of a cluster
- The case is in a healthcare worker who had contact with patients with severe acute respiratory infections
Additionally the WHO recommends testing of people with any acute respiratory disease who had contact, within 10 days before symptom onset, with a confirmed or probable NCoV case-patient while the case-patient was ill.
The agency strongly encourages countries in which a case has been detected to consider adding NCoV testing as part of routine sentinel respiratory disease surveillance. It also recommends additional testing of milder, unexplained community-acquired pneumonia that requires hospital admission if resources can support the testing.
The WHO "does not advise special screening at points of entry with regard to this event nor does it recommend that any travel or trade restrictions be applied," the guidance says.
The agency recommends several investigative steps, such as complete data collection during history taking, investigation of potential exposures, and contact tracing. It also details recommended epidemiologic studies, such as serologic surveys in healthcare workers and retrospective testing of stored specimens from patients who had respiratory disease.
Mar 18 WHO guidance
Nov 29, 2012, WHO guidance
Mar 14 CIDRAP News story "UK officials detail novel coronavirus cluster findings"
Mar 12 CIDRAP News story "Saudi Arabian death marks 15th novel coronavirus case"

domain: health


text:
Various points hold the attention of various people. We are all acquainted with songs, drama, dance, and storytelling. As enjoyment has actually developed throughout history, it has adjusted to a selection of various ranges, from receptions for two individuals to efficiencies intended for an audience of thousands. In today’s globe, amusement contains a wide variety of products, including prerecorded sound and video clip recordings. Most of these items are conveniently versatile to different occasions and also places.
Several types of amusement exist, varying from the passive kinds of home entertainment found in opera to the much more active activities of video games. Whatever form the entertainment takes, it is implied to be satisfying. Depending upon the medium used, this enjoyment can be life-enhancing, or it can be simply a kind of diversion. Home entertainment is a kind of excitement for a wide variety of people, and it assists us expand as a person as well as a family.
The lead single from the cd Bankrupt!, Amusement premiered on BBC Radio 1’s Zane Lowe show on 18 February 2013, and also was sent to alternate radio, influencing the United States throughout the week of 26 February. It has a striking cover including a mai-ti against a gray background and also a staccato guitar line. The album’s main remix attributes vocals from Mutya Keisha Siobhan and production by Dev Hynes.
Entertainment aids us get away from stress and anxiety and also advertises a culture of area. It additionally launches endorphins, which soothe discomfort and also stress and anxiety. We really feel happier and healthier when we are bordered by tasks that allow us to express our creative thinking and have fun. Usually, amusement activities are the best way to bond with enjoyed ones, friends, and also associates. Home entertainment likewise assists us unwind after a lengthy day at work, and develops social bonds.
One of the earliest forms of enjoyment is storytelling. It has actually influenced nearly every type of entertainment given that ancient times. The objective of narration is not just to captivate but to assess human problems. Stories are not just made use of as entertainment yet additionally as components of several forms of narrative art, such as poetry as well as drama. While composed tales have been adjusted to high-art standards, they are still one of the most usual means to entertain a crowd when they’re taking a trip together.
Lots of sporting activities have a strong home entertainment worth, depending upon the climate of the nation. While browsing is greatly connected with the Hawaiian Islands, snow snowboarding is most likely to have its beginnings in Scandinavia. While these sporting activities have actually spread around the globe, their native countries remain to be renowned for their prowess. Canada’s ice hockey team, for example, is a prominent form of home entertainment. In a similar way, the singular sports of hockey, boxing, and ice skating develop an entertaining blend of sport as well as society.
The business of home entertainment is very regulated, as well as attorneys who practice around are vital to ensure that lawful needs are satisfied. The Federal Communications Commission, for example, has produced many guidelines targeted at shielding the rights of artists. FCC guidelines aim to stop radio and also tv stations from owning too much control over a certain geographical market. Additionally, FCC regulations call for game program manufacturers to give equal time to various other prospects. The good news is, enjoyment legal representatives are equipped to help their clients comprehend as well as abide by these regulations and also laws.
Computer game have ended up being a significantly preferred resource of home entertainment. They have the prospective to eliminate tension and also supply interesting entertainment for an entire family members. They are likewise commonly easily accessible, and are a great way to spend time with the household. Streaming flicks on Netflix or watching a game on your tv is a remarkable way to spend quality time together. Ensure to purchase snacks for the ideal motion picture night! It will undoubtedly make your time rewarding. However what about the singular player?
The legislation of entertainment includes all sorts of kinds of regulation. The Sydney Music Hall, for instance, is an iconic example of home entertainment. In London, the O2 entertainment precinct includes an interior field, music club, movie theater, and also exhibition room. The Bayreuth Festspielhaus is a special musical composition – a theater devoted completely to a solitary musical piece. So whether you’re intending to arrange a performance or an impromptu celebration, an attorney can help you navigate the subtleties of home entertainment legislation.
For those interested in post-punk music, Amusement is a worthwhile listen. The band’s tunes are filled with snarling guitars, riffing over a drum beat, as well as split with split vocals. While it’s tough to explain just how this movie happened, it definitely supplies several hours of satisfaction. It’s additionally worth looking into the video clip for the song “Amusement”.
Storytelling is an ancient form of home entertainment, affecting virtually all other types of art. Stories offer a means of analyzing conflict, and also usually serve as a motivation for art. Written tales have usually been enriched with images, typically to high creative standards. Along with storytelling, stories are often utilized as a way to captivate a group traveling via a wild or on a long journey. On top of that, tales are an essential part of the innovative procedure that started with ancient mythology. 오피
The entertainment industry is an umbrella term for several sub-industries dedicated to producing and also distributing entertainment. Along with film, tv, and radio, the entertainment industry consists of cinema, art, dancing, opera, as well as other types of art. It likewise includes social media such as blogs, forums, and socials media. Amusement is not restricted to flicks, yet consists of social media, museums, as well as exhibits. Enjoyment includes anything that entails producing worth.

domain: artistic


This is a big one folks, 5 DAYS to go. Not only Microsoft’s decision regarding their consoles affect millions of console fans, but it also deeply affects PC Gaming. With a renewed focus on combining both Xbox and Windows 10 ecosystem under one wing, we are already seeing many Xbox One games coming to PC. And if Kotaku rumors are to be trusted, we might see a huge change in their plans these years.
With their Xbox brand success previously, they had bid adieu to PC Gaming only to re-enter with GFWL which was throughly rejected by PC fans as it should. Now that Xbox One is seeing some really harsh years while PS4 makes record sales, they are trying to look back at the lost opportunity in PC gaming and following Windows 10 very closely to what Apple did to Mac OS, rendering it into a walled garden.
Microsoft not only shows first and second party games on their conferences, but also purchased marketing deals with third party developers and possibly any new hardware. Last year they showcased Hololens, which wasn’t primarily build for gaming but it still left an impressive mark on us. This year we are going to see a whole lot of hardware…possibly more than any E3 before this.
Microsoft E3 2016 – New Upcoming Games
So lets get this thing started. Microsofts conference from E3 will be LIVE on 13th June at 10:00 PM (IST – New Delhi) on Youtube/Twitch.
XBOX ONE – MINI and XBOX ONE – “SCORPIO”
Nobody expected Sony, who are leading this generation, to jump the gun with PS4K “Neo”. Though not a generational leap, it is marked to be a mid-gen console with capabilities including 4K output (not to be confused with 4K Gaming) and with that a stronger CPU and GPU compared to original PS4. Microsoft couldn’t possibly let Sony do whatever they want to do on their own, this is not how competition works. So recent strong rumor suggests many new products under Xbox name is going to come. Among them will be Xbox One – Mini, which will be a slim version of Xbox
One with all the same capabilities original Xbox One has. And in 2017, another faster and stronger Xbox codenamed “Scorpio” will be released, and it will be able to support Oculus Rift. That sentence alone means it has to be far stronger than current XB1, maybe stronger than even PS4K, which is rumored to release this year.
Xbox One “Scorpio” is said to be a 6 TeraFlop machine with in-built VR solution. For comparison, original Xbox One was 1.32 TeraFlops. Sony’s PS4 was a little stronger with 1.84 TeraFlops. That should give you a slight idea how 0.5 TeraFlop difference made PS4 run games at 1080p while XB1 dabbled in and out on 900p. According to estimations, new PS4K “Neo” will have 4.14 TeraFlops, which still is weaker than Xbox One “Scorpio”, which begs the point how much costly would this new Xbox machine in 2017 will be.
This E3, XB1-mini is coming. Whether this will have the 4K output capabilities is difficult to say. The rumoured 2017 Xbox Scorpio announcement is anyones guess. Would Microsoft risk revealing a far stronger machine this early which will stop XB1 sales much more than they already had become. We will be sitting on the edge of our seats thinking the same thing this E3.
Also, Xbox brand is going really diverse. Expect a Xbox Stick (similar to Chromecast), Xbox TV (similar to AppleTV), Xbox One Mini model and maybe a hint of Xbox One “Scorpio”.
GEARS OF WAR 4
Microsoft needs this. Halo 5 was great and showed good sales but there is no denying the popularity of that ultra huge franchise has fallen miles below where it once started. With Fable being a third pillar of X360 days, it’s latest installment is cancelled and the studio shut down.
Forza are seeing relatively good sales but racing market is not what it was once. Microsoft needs this win. And nobody can deny that Gears of War certainly has the potential to be that ultra huge game.
With the reveal at 2015, a small section of gameplay and recently multiplayer was shown. With the release date already out, we expect Microsoft to turn the wheels on the marketing and will make us more excited than we already are. A lengthy gameplay, this time maybe a little something bright, (the previous E3 gameplay was so dark many people live couldn’t see things properly) and with a little more details about the story setting.
We know the main character is the son of the legend Marcus Fenix, whose statues can be clearly seen in the new Gears 4 trailer. And we know the entire campaign happens in the duration of one day – or more appropriately one night, like a Batman game.
It would be interesting to see the campaign dwell into gore and scary settings and how Coalition (the developer of Gears game forseeable future) will present a thrilling start to a trilogy of sorts. Just like Halo 4 was shifted to a new developer, Gears 4 is also being developed by a new team. The expectations are high, the fans, the developers and Microsoft are all pinning huge hopes from this entry. As your humble writer’s most anticipated game, I wish this game be good. And lets hope the inevitable PC port of Gears of War 4 will be announced shortly too because…it will come eventually.
Gears of War 4 is scheduled to release on 11th October on XB1.
Microsoft E3 2016 – New Upcoming Games – Literally nothing is known about this game. Revealed at E3 2015 with a CGI trailer, which did gave hints about its setting and the gameplay, but very little which we will go through here. The trailer featured a female main character with a pet-robot whose consciousness can be transferred to any new robot with the help of a glowing orb. You can increase you party to many robot pets who helps you in combat.
The female protagonist looks to be a teenager and sports a grappling hook and a gun. We don’t even know whether the game is a first person shooter or third person, but it does involves shooting.
The team behind this game have some impressive ranks. The creator of Mega Man games is involved in this and the director is Mark Pacini, who was director of Metroid Prime series. Seeing the roster, we believe it will involve exploration and puzzles to be a strong focus.
It has an estimation release date of 2016, but as we have not even seen a single gameplay footage, not in-door or out-door, which makes us believe that this game could be delayed to 2017. Ofcourse, Microsoft can pull a surprise release this E3 which might be the best thing for us gamers.
The game is confirmed to be on PC and XB1. But given the low coverage it has got, and MS still sticking to 2016 release date, it might be not a AAA game we all hoped it will be. Still, a new IP is always welcome, and we expect at least a gameplay footage this E3 if Recore is to be released this year or delayed to Q1-2017.
Who doesn’t want a game where you can level an entire city through explosions. This is exactly what Crackdown 3 multiplayer promises to give us. The Single Player will also have impressive destruction physics, as long as it doesn’t breaks the games mission structure completely.
Last Crackdown game was released in 2010 on Xbox 360. So even if a crackdown game wasn’t in development from the get go, the sequel must be very close to finish. Not much is know about the plot, but there are indications that it will be set in an alternate timeline than CrackDown 2 was set in.
Microsoft had hinted towards a Crackdown 3 – Multiplayer only Beta to hit in 2016. We are eager to get our hands upon all the destruction physics made possible by Cloud Computing. A official in-game trailer hasn’t been showed yet, and as this game has some hype situated to it, we highly expect atleast a gameplay trailer to be dropped on E3 or at Gamescom. The multiplayer needs to have some huge hooks besides destruction-physics, because in holiday 2016 we’re getting the likes of Battlefield 1, Call of Duty and Titanfall 2 among other MP games.
It would be hard to crack that bubble even when you’re just releasing a free Multiplayer Beta. More reason to tune up the game and release it in top notch conditions. Microsoft doesn’t want another Halo MCC fiasco in their hands. Crackdown 3 is expected to release in 2017, with multiplayer beta in 2016. It would be interesting to see how gamers will react to this game, and it may very well become a pillar in Xbox gaming roster.
Watch Crack Down 3 Trailer
SEA OF THIEVES, CUPHEAD and others
Microsoft E3 2016 – New Upcoming Games – Not every game needs to be a high budget explosion. Sometimes a genuine idea with the right presentation is all it takes to become gamers darling. CUPHEAD was one of those game. It is entirely hand drawn tough as nails platformer which reminds you of old cartoons. Just one look of this game will make almost anyone stun on their place and take notice. With an incredible artstyle and addictive platforming mechanics, this one is a sure shot success.
Sea of Thieves is a game in development from Rare Studios. You might remember this studio name because last year Microsoft released a remastered experience of their best 30 games in one collection. Though many amongst the team who made up their golden years had left, Sea Of Thieves looks like it can bring back Rare Studios back to glory days. The game will generate its content much like how Minecraft community works and will depend on user-generated content to give it a replay value.
Microsoft’s other game which had user-generated content concept “Spark”, didn’t really set the world on fire, but as far as trailers go, Sea of Thieves is something we expect to roam the world. No release window is announced, so we expect a reminder with a 2017 release date this E3. It has been confirmed for PC.
Undead Labs previously made State of Decay for XB1. From then there has been no information on their next project. We know they ramped up production in 2014 and hired a bunch of people. So lets see if we see some zombie madness from this studio this E3.
The developer of Gone Home showed us their new survival horror game TACOMA last E3. We expect the release to be late 2017, so its appearance here is hard to predict.
Watch Sea of Thieves Trailer
Microsoft’s new AAA IP’s are not doing well. Sunset Overdrive’s sun set just few weeks after the release, Quantum Break didn’t break into success and previous launch lineup like Ryse and Dead Rising 3 all ended up on PC to make up their losses, even still when Microsoft was not willing to let go of these IP’s. Scalebound’s future is of grave concern to Microsoft.
But still, you really can’t go wrong with an upbeat protagonist riding on a dragon, hack and slashing its way to the end, dawning his magical armor and super agile sword skills. Made by Platinum Games, ( maker of Bayonetta and MGS:Revengeance ) Hideki Kamiya is personally directing the game, (previous director of Devil May Cry) and has promised an advanced AI in your companion Dragon with a good combination of Swords and Ranged gameplay.
The game sport gigantic areas to fight huge monsters and small human enemies. It has 4-way co-op in special dungeons. And in terms of customization, the game doesn’t have a crafting system but special kinds of weapons will be dropped throughout the game, and you can even customize your dragon as you fit.
We should not forget that Microsoft has an only limited amount of time at E3, and with so many games and so many other conferences like Gamescom, they may spread out the gameplay details of many games into two. Still, we expect some details to come about it here. The release date is 2017, so we expect a firm release date to be announced alongside more gameplay.
HALO WARS 2
Microsoft E3 2016 – New Upcoming Games – Nothing warms my heart more than seeing an AAA RTS being made. Halo Wars 1 came back in 2009 on both Xbox 360 and PC and now 7 years later its sequel is coming, this time helmed by Creative Assembly ( Total War Series ). After putting out an explosive CGI trailer last E3 and pulling of a big surprise on all PC fans, we all are expecting a gameplay. Now RTS demos are a little confusing to people who aren’t really accustomed to it.
We hope Microsoft will take time out of their conference to showcase it in detail among the console crowd, but personally, I wouldn’t be too shocked if they left it out to reveal it at another time or save it for a PC centric conference like last years AMD PC Gaming show.
Halo as a franchise has climbed down to a believable level, and this might be a great effort to branch out the lore and get a hold of some new fans. With Microsoft combining Xbox and Windows10 ecosystem, many are acknowledging that the next mainlined title Halo could come to PC side by side with the console. A reintroduction of Halo Wars 2 on consoles and PC might be a great way for that.
Also, Halo 5 Forge mode is already on its way to PC. Last year Halo standalone multiplayer was released exclusively on PC in only Russia. So with Halo Wars 2, we could see more Halo games on PC. The release date is late 2016, so we should learn about it more in some coming days.
Watch Halo Wars 2 Trailer
FORZA HORIZON 3
Forza franchise is an annual series. Last year Forza Motorsport 6 released on XB1, and a Forza Motorsport 6 : Apex free version on PC this year. So Forza Horizon 3 is imminent. Racing genre has generally been in a slump with only handful of titles and not much great sales attached to them.
The announcement of Gran Turismo Sports very much lifted out the spirit, only to bring them down with a subpar graphics we weren’t expecting. Turn10 studios (Forza) has been consistent in delivering great Forza games, with additions like Drivatar and 3D water puddles. This year they’re ramping up the heat even more by announcing a “Special Forza Motorsport Event” at E3.
With great reception of Horizon 2, looks like Forza Horizon 3 is in a great place to counter any other racing game this holiday, and certainly, Microsoft doesn’t want Gran Turismo to steal all the spotlight with their FIA approved online races. It is almost guaranteed that a new Forza game will be announced and released in Q4-2016.
MULTIPLATFORM TITLES AND TIMED EXCLUSIVES
Many games we know are coming would certainly see their reveal at Microsoft’s or Sony’s booth.
Xbox has Battlefield 1 marketing deal, so expect a gameplay trailer in Xbox Conference. Titanfall 1 also had Xbox marketing, so it could be that EA went with Microsoft again with Titanfall 2 marketing, and thus we can see more details about it in Microsoft’s place too.
It would be interesting to see whether Overwatch would debut on Microsofts booth or Sony’s.
If Read Dead Redemption is having a sequel this E3, Microsoft showcasing it first would be a big win for them perception wise. Many backward compatible games will be name dropped for Xb1. All bets are off but expect many games whose publisher aren’t doing a conference to end up on anyone’s conference, team Green or Team Blue. It doesn’t really affect you much, other than how the DLC and season pass will be distributed among PS/XB users. But it makes up for an eye-grabbing show, and E3 is all about that.
Written By – Shubhendu Singh.
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Hi! This is Joyce (jakrn ST member) here to show you Karen Diamond Designs’ kit, “All Natural.” If you are looking for an out-doors type of kit, this is the kit for you! It contains a variety of natural textures such as burlap, wood. This kit is versatile for camping, hunting, any of those outdoor photos that you have to scrap.
I created this layout using this unique kit:
See gallery image for additional credits
Thanks for looking! Happy Scrapping!

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Jonathan Kuminga, the No. 1 2021 recruit, will be heading to the NBA G League next year. Instead of a final year of high school, the top recruit in next year’s class has reclassified.
— Shams Charania (@ShamsCharania) July 16, 2020
He is joining Isaiah Todd and other top recruits in heading to the G-league.

domain: news


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Neck Pain Florissant, MO
Neck Pain Florissant, MO. It not only causes great discomfort, but also limits and affects the movements and postures that you typically have no problem with. If you are like 75 percent of us, neck pain has affected you at one time or another. Though the figurative “pain in the neck” is typically not more than a minor annoyance that you can easily disregard, real neck pain is a serious issue. It is a pervasive problem that, when not promtly and properly cared for, can result in immense pain and limitations. Ranking as the number four cause of disability, neck pain accounts for a significant part of the 88 billion dollars in annual healthcare costs for neck and back pain in the US.
Neck pain should not be ignored. If your neck pain is taking a toll on your life, seek the right care as soon as possible. As the top Florissant, MO physical therapy and occupational therapy facility, we have what it takes to provide the individualized and proven Florissant, MO neck pain treatment that will reduce your pain and return you to what you love. To discover what we can do to address your Florissant, MO neck pain, contact us today.
Florissant, MO Neck Pain: Causes and Risk Factors
It is a fact that the large majority of us suffer from neck pain at some point in our lives. We depend upon the intricate and complex structures that make up the cervical spine to provide protection to our nervous system, offer support to our head, and give us a unique combination of flexibility range of motion that make it possible to execute many of the motions and tasks we do every day. As such a complex region of the body, the neck is vulnerable to pain and impairment that can be caused by overuse, injury and trauma, illnesses, and other conditions.
Common Florissant, MO Neck Pain Causes
- Sprains and strains
- Direct trauma
- Joint degeneration
- Bulging disc
- Pinched nerve
More than 75% of us suffer from neck pain at some point. Luckily, many of us only experience mild neck pain that can often subside without requiring extensive treatment. However, many other Florissant, MO neck pain patients are not so lucky. Up to 85% of people who have neck pain will experience a episode within one to five years and in 37% of those with neck pain, the condition becomes chronic, with neck pain lasting more than one year.
Neck pain is a prevalent problem that can clearly happen to anyone in Florissant, MO. However, there are certain risk factors that make an individual more susceptible to neck pain. Risk factors for neck pain include:
- Occupation – Neck pain is most common among office workers, healthcare workers, and manual laborers. However, when considering the relationship between occupations and neck pain, there is an even stronger relationship between individuals who suffer from neck pain and workers who are stressed and unhappy at work, irrespective of the industry or occupation.
- Weight – With comparatively less muscle strength and more stress on the joints and skeletal system, those who are overweight or obese are more likely to experience neck pain.
- Posture – We have a term for it these days: “text neck.” Poor posture is to blame for a myriad of neck and spine issues including neck pain. For every 15 degrees of forward bend in the neck, the strain on the cervical spine nearly doubles.
- Smoking – Smoking can be blamed for much pain in the neck, back, and other joints due to its ability to can precipitate disc degeneration.
- Stress – Stress has significant impact on the human body. Stress-related neck pain is not simply being tense. The physiological stress response also enlarges blood vessels in the muscles, restricts neck and shoulder movement, and heightens the perception of pain.
Physical Therapy for Neck Pain in Florissant, MO
It is paramount that you seek out the proper treatment and care for your neck pain as soon as possible. Physical therapy is an accepted and popular treatment methods for neck pain sufferers. This is with good reason, as evidence supports it as a highly effective neck pain treatment strategy. Though in some cases more extensive medical treament beyond physical therapy may be required, physical therapy is a much safer choice than surgery or medication.
Physical therapy is an easily accessible, low-risk, non-invasive form of treatment that addresses the cause of your Florissant, MO neck pain. It offers neck pain patients a way to heal as safely and as thoroughly as possible. Through a combination of hands-on manual therapies, exercise, and education, Florissant, MO physical therapy treatment for neck pain helps you experience life without pain and regain complete range of motion. Florissant, MO physical therapy patients can also benefit by stretching and strengthening muscles affecting the neck, shoulder, shoulder blade, and core, all while implementing preventative practices to avoid the risk of future pain or injury, like postural training and changes to the work-station.
With a quality Florissant, MO physical therapy program designed to your unique neck condition and goals, you will soon be able to once again enjoy your usual mobility, flexibility, strength, and range of motion. When you seek treatment with Axes Physical Therapy, the care we provide for your neck pain care near Florissant, MO will address the cause of your neck pain and your priorities for managing the condition. Your dedicated Florissant, MO physical therapist may design your neck pain treatment plan to include some of the following strategies:
- Stretching – stretches and exercises to improve the strength and range of motion of the muscles connected to the cervical spine
- Core, shoulder, and back strengthening – conditioning to improve the strength of muscles that support and stabilize the cervical spine and upper body, including those in the torso, shoulders, and scapular regions
- Aerobic activity – low impact cardiovascular exercise that increase body strength and blood flow
- Traction – creating space between the vertebrae decreases pressure and allows inflamed and irritated nerves and discs to heal
- Postural correction – training and demonstration to develop better posture, supplementing with modification devices like lumbar support cushions when necessary
- Joint mobilization and manipulation – your physical therapist performs this manual therapy by manipulating the joint to mimic normal joint and tissue movement to gradually restore normal joint function and range of motion
- Trigger point dry needling – directly targeting irritated or strained muscle tissue areas releases and relaxes the irritated tissue, which allows the muscles to reclaim their range of motion
- Massage – releasing tension through the massage and hands-on manipulation of soft tissues
- Therapeutic modalities – things like ultrasound therapy and electrical nerve and muscle stimulation therapies can supplement other therapies
- Heat and ice – use of heat and/or ice can manage pain and swelling
- Ergonomic modifications and devices – to prevent complication and further neck pain, support devices can be used for sleeping, sitting, working at a desk, etc
- Posture correction – training to correct posture to minimize the amount of pressure and strain the neck is subjected to, helping to manage pain and prevent ongoing Florissant, MO neck pain issues
Symptoms of Neck Pain
To truly understand the nature of your neck pain and formulate the best treatment plan in Florissant, MO, your Axes Florissant, MO physical therapist will perform an examination to evaluate your condition. Pain, of course, is the primary symptom of neck pain. However, the spine is a brilliantly complex and versatile structure within the human body and the cervical spine, or the neck portion of the spine, is no exception. Different neck pain causes may present different symptoms and present certain types of pain.
Symptoms that can distinguish the cause of your neck pain are:
- Numbness or tingling in the arms or hands
- Upper extremity weakness
- Pain that extends to the upper back, shoulders, arms, or hands
- Stiffness and immobility
- Increased pain when sneezing, coughing, reaching, etc
- Comparatively limited range of motion
- Difficulty sleeping
- Pain that escalates at a certain period of the day, like in the morning or at night at bedtime
Your neck pain may be described with words such as:
Your Florissant, MO physical therapist will ask you questions designed to understand the cause of your neck pain. In addition, they may assess factors including strength, flexibility, range of motion, posture, and functional ability through an examination. By serving as your dedicated Florissant, MO neck pain physical therapist, our mission is to gain as complete a picture as possible of your neck pain before beginning your care so that we can provide the safest and most effective treatment available.
Care for your Neck Pain near Florissant, MO | Axes Physical Therapy
If you suffer from persistent or recurring neck pain, you are not alone. Axes recognizes that neck pain is a complex condition that affects many of us in Florissant, MO and throughout the United States and we are here to help. As an Axes patient, you will receive accessible, low-risk, evidence-based neck pain care near Florissant, MOby the hands of licensed physical therapists which advanced degrees and training in the treatment of the spine. It is our patient-centered philosophy, dedicated approach to our patients, and the talented physical therapists in our practice that make it possible for you to find meaningful, lasting relief from your neck pain near Florissant, MO.
It is time that you were able to live pain-free and with the freedom to do the activities you enjoy. Our family of twelve locally owned and operated physical therapy facilities offer neck pain care of unrivaled quality in the Florissant, MO area location that is convenient for you. Discover the effect of Axes’ quality physical therapy treatment on your neck pain by starting your treatment promptly. Contact us today or fill out an appointment request form now.


Ratchet, Tarp, or Pull straps getting old or weathered? Load control is something we take serious here at J&J Trailer and Equipment Sales! With all the options of straps, tie downs, track, and anchors we are sure to have what you need.
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Johann Jakob Mechtl
Das Göttliche Gericht Machet dem Sünder schwär die abscheuliche Sieben Todt- ...
4 Mor. 600 h, Oettingen im Rieß 1721
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For Immediate Release
Monday, February 23, 2015
Statement of AFSCME Pres. Lee Saunders: Strengthening Retirement Investments Is Key to Retirement SecurityWashington, D.C. —
AFSCME Pres. Lee Saunders issued the following statement in response to the Obama administration’s move to strengthen retirement investment protections:
“Every American worker and retiree should be confident that their retirement savings will be there so they can live with dignity after a lifetime of labor, without being shortchanged by financial advisers giving inappropriate or conflicted advice in exchange for lucrative commissions.
“Today the Obama administration took a necessary step to ensure that tens of millions of working families will receive investment advice that directly benefits them rather than large profitable Wall Street firms.
“AFSCME applauds the administration’s efforts to limit conflicts of interest and increase accountability and more importantly protect consumers who seek retirement investment advice. We encourage the Office of Management and Budget (OMB) to review and finalize action on this long overdue consumer protection proposed rule.”


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President Park Geun-hye’s job approval rating dropped to an all-time low this week, a new poll said Friday, a development underscoring challenges faced by the nation’s top leader.
The survey of 1,009 adults found only 29 percent of those polled approved of the way Park had been handling her job, down 1 percentage point from the previous week, according to Gallup Korea.
It marked the first time that Park’s approval rating had slid below 30 percent.
President Park Geun-hye attends an award ceremony for public servants at Cheong Wa Dae on Friday. (Yonhap)
Among those who disapproved of Park’s performance, 16 percent blamed Park for lack of communication and another 16 percent said they did not support Park due to the government’s new tax settlement scheme, according to Gallup Korea.
Park has long been branded by her critics as “uncommunicative” for allegedly refusing to listen to what critics and others have to say and insisting only on what she believes to be right.
On Monday, Park expressed regret for causing trouble over the new tax settlement scheme that has sparked strong public backlash.
The new scheme is designed to collect less taxes throughout the year and return less overcharges during tax settlements at the start of a new year.
Under the new scheme, some people with an annual income of less than 55 million won ($50,700) are required to pay additional taxes this year.
Before the revision, many wage earners were able to get tax refunds, which were commonly referred to as a bonus or extra wage.
The new tax settlement scheme has angered the public, prompting the government and the ruling party to push for revision of the tax code to ensure that it offers additional tax refunds to eligible taxpayers.
The survey, conducted between Tuesday and Thursday, has a margin of error of plus or minus 3.1 percentage points. (Yonhap)

domain: politics


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Palm Beach Schools Diverting Money from Kids to College Board Testing
You will not see this story in papers from Florida or anywhere else. I guess I must continue the muckraking tradition of Jacob Riis, Upton Sinclair and John Spargo. In order to force all 11th grade students to take the SAT during the school day- the Palm Beach County School Board has
stolen taken 3% from the budget of each high school.
Each school gets money based on how many students pass AP, IB and AICE tests. This money has been used to fill many gaps left by budget woes. Some schools haves used the funds to buy books and support their performing arts programs.
Now- 3% will be spirited away to David Coleman.
Is this a misuse of funds? Is this immoral? Illegal? Unethical? You can decide.
I can already predict the results of testing. Just like last year- wealthy schools on top and poor schools at the bottom. As I have argued before- you might as well give the New York Times Crossword Puzzle. The SAT is generally a test of income, family and access to books growing up.
Saturday testing has never been a problem. School districts offer the equity of access canard all the time when forcing kids to take this test. It smacks of paternalism. The SAT was never designed to be FORCED on students. Would not forcing kids to take this test make it invalid? I spoke to an English teacher in Palm Beach who told me it took THREE class periods to get kids signed up for this test.
The public should be outraged. My only question is, “Will Palm Beach deliver the money to David Coleman at the College Board using unmarked bills?”


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Month: August 2014
Had you had enough of the 24/7 Ferguson TV show? Yes you did. That’s the eighth deadly sin: media numbing to apathy. Worse, believing Ferguson is just a Missouri shame — Ferguson is serial historical murder incorporated, gone viral.
We’ve left the scent of blood in the air for those with economic muscle to further embellish their power over 99.9% of the world.
Unseen power repressing the populace to maintain controlled work force for living large bottom line, and barrel bottom wannabes short-cutting get rich scams are not exceptional:
- Store Hackers and Hotel Pizza Delivery Credt Card Scams
- Wisconsin Walker’s Union busting
- Chinese Jet flyover flipping American airpower
- Russian Humanitarianism for Ukraine, laced with artillery shells
- Rubio & Ryan turning away Dreamers
- ISIS Recruiting with internet beheading, Gaza and American bombs
- Obama sued on vacation as Rick Perry indicted with WH runs
19 days ago Michael Brown joined the ranks of unarmed black men killed by uniformed white men, using re-gifted power tools to leave blood in our streets. Giving permission to kill minorities to those with a little power seeking more, recasts the OK Corral mindset.
Hands up: bang you’re dead — Livestream – for all doomed to relive it in American neighborhoods every day & night — and for those who think it’s their place to keep those people out of our place: 1600 Pennsylvania Ave.
Being preoccupied with trying to stay afloat since 2009, we may have been too underemployed and media distracted to notice the troubling evolution of American hypocrisy by moral supremacists.
- Killing the unarmed hemorrhages our humanity
- Death by police state emits a residue of decay
- Corporate desertion scents the air with indifference
- Open Carry taunts our Constitution
And the perpetrators are as old as Cain, as near as foreign sweat shops fueled by American consumer gluttony and as anti-American as banks dumping pin pricked fines onto shareholder families.
But there is no gift more devastating to the state of the union, than a government reluctant to use the long arm of the law to make Wall Street speak truth to Main Street People power.
Allowing federal legislators and jurists to revitalize civil inequities, we can only expect good people of Fergusons all across the nation to rise up and take back the dignity, respect and bank balances that have been stolen from them.
If together we choose to reverse Citizens United by ballot, overruling Supremes with Constitutional Amendment;
If Ferguson chooses to turn the other cheek and love thy neighbor as thyself, while running for political office to replace the preservers of segregation;
If urban centers arrest deportation of the innocent and desertion by Wall Street traitors to private islands, instead of relocation camps, we will be spared more blood in our city streets.
The truth is, we can no longer afford to deny our country the cultural wealth of experiences, knowledge and talent of Female Americans, African-Americans, Asian-Americans Hispanic-Americans and Native-Americans.
Thus, United We Stand, we discover our power is in
- Educating children and young adults for free
- Cancelling Citizens United
- Circumventing Climate Change
- Curing Cancer
- Elevating equal pay for women and equal opportunity for all
- Eradicating Voter ID Jim Crow
- Maximizing minimum wage
- Providing infrastructure that better withstands earthquakes, hurricanes, tornados and terrorists
- Restoration of Rights
- Restraining political redistricting
Then we might make an impact on the international perception that the promise of our Stature of Liberty is, For Whites Only.
However, for those who just can’t find the power to be color blind — elect a white person in 2016 — just be certain, it’s a woman.
Darren Wilson is innocent until proven guilty, but we must vote believing, so was the black man, named Brown.
Join Us: Become one of the reasonable voices heard round the world
Is Ferguson’s state of emergency: militarized police brutality, government racial imbalance, street arms raised unity and looting inspired curfew, an America reminiscent of 1861, a GHW Bush prediction, “if the American people ever find out what we have done, they would chase us down the street and lynch us, or a Robin Williams cry for help?
One thing is clear, whether Colorado Open Carry, Internet mouse clicked petitions or prosecuting Governors, Americans have been awakened!
Assaulting First Amendment rights to peacefully assemble in protest, didn’t begin with policeman Darren Wilson shooting unarmed robbery suspect, Michael Brown, multiple times.
It began with
- Attorney General John Ashcroft’s administrative degree to quarantine dissent, which then standardized designated protest areas as our new First Amendment M. O.
- President GW Bush’s political events banning and arresting those peacefully wearing anti-Bush T-Shirts
- Our Pentagon buying surplus war weaponry from those who profit from our being at war, and then giving the surplus munitions to police departments all over America
- DWB – FWA – SWB – and Trayvon Martin’s crime: WWB
- Assassinating Kennedy, King and Bobby
- An Uniformed Oakland California thug pepper spraying sitting peaceful demonstrators
- People being left to die in New Orleans after Katrina
- Michael Brown’s body left in the street for hours
Indiscriminately death casts a tall shadow, so our lives are our weapons for listening, advocating, voting, learning and sharing: the systemic stigmatizing of those deemed different is as old and prescient as
- Salem and McCarthy witch hunts;
- war on women history: no voting rights, being institutionalized for enjoying sex with their husbands, having three states block ERA ratification
- Veterans dying waiting for healthcare
- the cataclysmic void in our treatment and understanding of Depression – in 2014, deserting those with Mental Health issues, to the life of a 19th century outcast
Any death diminishes us, especially if unable to extrapolate Mental Health issues, and Ferguson, to all of America. Either we are the prime-time stereotypes and redundant news media cutouts, or we are who we love and emulate; what we vote for and fight against – we are our brothers’ keeper or not my problem.
However death comes: Lauren Bacall & Charles Keating, a NYC choke-hold or Ohio shot for being black, a war weary Anne Sexton, Seymour Hoffman and Robin Williams – it guts our six degrees of separation.
I worked with Charles Keating on Another World, but the closest I got to Robin Williams was his co-stars: literally bumping into Nathan Lane in NYC one night, after his brilliance in The Producers, and chatting on the set of Lincoln with Sally Field – yet losing him feels too akin to my losing those who changed my life: Francis Hughes, PhD, Father Gilbert Hartke and iconic actress, Mercedes McCambridge.
I knew the addiction Mercedes was successfully resisting, before her son, John Lawrence Fifield, killed himself after murdering his wife and two daughters.
Her understandable withdrawal and retreat taught me:
- Stigmatizing Mental Illness and our ignorance of Depression, Schizophrenia and Alzheimer’s are as deadly as a militarized police state.
- Ferguson is America in miniature, from which we can learn to honestly live together as equals, or continue Supreme Court building a house divided against itself.
- Not all battles are civil wars between Christians and non, rich and poor, white and black, but increasingly, too many are.
Revelation: conservative top 1%, your armed biased law enforcement & private prisons — despite Koch-head billions and clichéd regurgitation of propped up puppets still not prosecuted for their 2008 Wall Street economic devastation on the world, people power re-elected a black looking chess player for President…
Join Us: Become one of the reasonable voices heard round the world.