#Proline FC

[aliasswali9]

Stanbic Uganda Cup: Vitalis, Ssemujju Inspires BUL FC To A 2-0 Win Over Proline At MTN Omondi Stadium.

Stanbic Uganda Cup: Vitalis, Ssemujju Inspires BUL FC To A 2-0 Win Over Proline At MTN Omondi Stadium.

First half goals from Tabu Vitalis and Joseph Ssemujju inspired BUL Football Club to an emphatic 2-0 win at Big League outfit Proline Football Club in the first leg of their Stanbic Uganda Cup quarter-final tie. Tabu Vitalis’ early goal and Joseph Ssemujju’s stunner in the 37 minutes were the difference on Friday, between the teams as BUL FC took a commanding 2-0 advantage ahead of the return…

[kon-igi]

in cosa è diverso il vaccino Johnson rispetto agli altri, per non necessitare di una seconda dose?

C’è bisogno di un piccolo preambolo.

La differenza tra la SCIENZA e la RELIGIONE è che alla prima ci credi perché ne comprendi i meccanismi mentre con la seconda sei costretto a farlo in modo fideistico.

Quindi decidi tu i vaccini in quale categoria ricadano.

(mi piacerebbe mettere il pulsante ‘salta tutto il testo citato’ ma non esiste e quindi la ‘spiegazione’ è da scrollare fino in fondo) 

Vaccine design

The S protein of SARS-CoV-2 corresponding to positions 21,536–25,384 in SARS-CoV-2 isolate Wuhan-Hu-1 (GenBank accession number: MN908947) was codon-optimized for expression in human cell lines. S designs were either based on the native SP, replacement of the native SP by the tissue plasminogen activator (tPA) SP or a SP upstream of the native signal, resulting in a sequence encoding SARS-CoV-2 amino acids 2-1273 and tPA leader as described for MERS-CoV spike protein13,39. In some constructs the furin cleavage site was abolished by amino acid changes R682S and R685G, or proline substitutions (K986P, V987P) were introduced.

Protein expression and purification

A plasmid encoding the SARS-CoV2 S-2P protein12 with the wt SP and with the wt SP replaced by the tPA SP and with a C-tag for purification were codon-optimized and synthesized at GenScript. The constructs were cloned into pCDNA2004. The expression platform used was the Expi293F cells. The cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 6 days at 37 °C and 10% CO2. The culture supernatant was harvested and spun for 5 min at 300 × g to remove cells and cellular debris. The supernatant was subsequently sterile filtered using a 0.22 µm vacuum filter and stored at 4 °C until use. The C-tagged SARS-CoV2 S trimers were purified using a two-step purification protocol by 5 mL CaptureSelect™ C-tag Affinity Matrix (ThermoFisher Scientific). Proteins were further purified by size-exclusion chromatography using a HiLoad Superdex 200 16/600column (GE Healthcare).

Antibodies and reagents

SAD-S35 was purchased from ACROBiosystems (cat# SAD-S35-100ug). ACE2-Fc (ACE2) was made according to Liu et al. 201840. For CR3022, CR301541, CR3046, and S30934 the heavy and light chain were cloned into a single IgG1 expression vector to express a fully human IgG1 antibody. The antibodies were made by transfecting the IgG1 expression construct using the ExpiFectamine™ 293 Transfection Kit (ThermoFisher) in Expi293F (ThermoFisher) cells according to the manufacturer specifications. Antibodies were purified from serum-free culture supernatants using mAb Select SuRe resin (GE Healthcare) followed by rapid desalting using a HiPrep 26/10 Desalting column (GE Healthcare). The final formulation buffer was 20 mM NaAc, 75 mM NaCl, 5% Sucrose pH 5.5. Convalescent serum (SER-0743-00001) was obtained from Sanquin, the Netherlands.

Cell-based ELISA

HEK293 cells were seeded at 2 × 105 cells/mL in appropriate medium in a flat-bottomed 96-well microtiter plate (Corning). The plate was incubated overnight at 37 °C in 10% CO2. After 24 h, transfection of the cells was performed with 300 ng DNA for each well and the plate was incubated for 48 h at 37 °C in 5% CO2. Two days post transfection, cells were washed with 100 μl/well of blocking buffer containing 1% (w/v) BSA (Sigma), 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM Tris pH 8.0 in 1× PBS (GIBCO). After washing, nonspecific binding was blocked, using 100 μl/well of blocking solution for 20 min at 4 °C. Subsequently, cells were incubated in 50 μl/well blocking buffer containing primary antibodies ACE2-Fc (5 µg/mL, 1 µg/mL and 0.2 µg/mL)(1 µg/mL for radar plot), S309 (1 µg/mL), SAD-S35 (1 µg/mL), CR3015 (5 µg/mL), CR3022 (5 µg/mL), CR3046 (5 µg/mL), and convalescent serum (1:400) for 1 hr at 4 °C. The plate was washed three times with 100 μl/well of the blocking buffer, three times with 100 μl/well of washing buffer containing 1 mM MgCl2, 1.8 mM CaCl2 in 1× PBS and then incubated with 100 μl/well of the blocking buffer for 5 min at 4 °C. After blocking, the cells were incubated with 50 μl/well of secondary antibodies HRP conjugated Mouse Anti-Human IgG (Jackson, 1:2500) or HRP Conjugated goat anti-mouse IgG (Jackson, 1:2500) then incubated 40 min at 4 °C. The plate was washed three times with 100 μl/well of the blocking buffer, three times with 100 μl/well washing buffer. 30 μl/well of BM Chemiluminescence ELISA substrate (Roche, 1:50) was added to the plate, and the luminosity was immediately measured using the Ensight Plate Reader.

Flow cytometry

MRC-5 cells (0.4 × 106 cells/well) were seeded in 6-well plates and after overnight growth transduced with Ad26 vectors encoding SARS-CoV-2 S transgenes at 5000 vp/cell for 48 h. Harvested cells were washed with PBS and stained with LIVE/DEADTM Fixable Violet Dead Cell Stain Kit (Invitrogen). For SARS-CoV-2 surface staining, cells were washed twice with PBS and then incubated with ACE2-Fc (1 μg/ml), convalescent serum (1:400), and mAbs S309, SAD-S35, CR3022, CR3015 and CR3046 (1 μg/ml) for 30 min in FACS buffer (PBS with 0.5% BSA). Cells were washed twice with FACS buffer and stained with goat anti-Human IgG Alexa Fluor 647 (Invitrogen) or goat anti-Mouse IgG Alexa Fluor 647 (Invitrogen) secondary antibody for 30 min in FACS buffer. Cells were washed twice with FACS buffer and fixed with 1× BD CellFIX (BD Biosciences) for 15 min. Cells were washed once with FACS buffer and resuspended in FACS buffer before analysis on a FACS Canto instrument (BD Biosciences). Data were analyzed with FlowJoTM Software (Becton, Dickinson and Company) and plotted as median fluorescence intensity of the MRC-5 single, live cell population (Fig. S6).

BioLayer interferometry (BLI)

Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37 °C and 10% CO2. The culture supernatant was harvested and spun for 5 min at 300 × g to remove cells and cellular debris. The spun supernatant was subsequently sterile filtered using a 0.22 μm vacuum filter and used as the analyte in the experiment. A solution of ACE2-Fc at a concentration of 10 μg/mL was used to immobilize the ligand on anti-hIgG (AHC) sensors (FortéBio cat#18-5060) in 1× kinetics buffer (FortéBio cat#18-1092) in 96-well black flat bottom polypropylene microplates (FortéBio cat#3694). The experiment was performed on an Octet HTX instrument (Pall-FortéBio) at 30 °C with a shaking speed of 1000 rpm. Activation was 60 s, immobilization of antibodies 600 s, followed by washing for 300 s and then binding the S proteins for 1200 s. Data analysis was performed using the FortéBio Data Analysis 8.1 software (FortéBio). Binding levels were plotted as nm shifts at 1200 s after S protein binding.

Differential scanning fluorometry (DSF)

0.2 mg of purified protein in 50 µl PBS pH 7.4 (Gibco) was mixed with 15 µl of 20 times diluted SYPRO orange fluorescent dye (5000 x stock, Invitrogen S6650) in a 96-well optical qPCR plate. A negative control sample containing the dye only was used for reference subtraction. The measurement was performed in a qPCR instrument (Applied Biosystems ViiA 7) using a temperature ramp from 25–95 °C with a rate of 0.015 °C per second. Data were retrieved continuously. The negative first derivative was plotted as a function of temperature. The melting temperature corresponds to the lowest point in the curve.

Mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS/MS) was used to determine the N-terminus on either purified soluble protein or on a pull-down of the full-length S protein from cell membranes using either Mab CR3022 or ACE2-Fc. The purified soluble proteins were subjected to direct digest, whereas the membrane-bound spike protein samples were either subjected to direct digest or in gel digestion. The experiments were performed on C18 nRP-LC connected to a ESI-Q-orbitrap mass spectrometer. Data processing for the different proteins was performed using Biopharma Finder 3.1 (Thermo Scientific). Each data file was compared to its corresponding amino acid sequence. Peptides were filtered by mass accuracy, confidence and structural resolution and reported. In order to pick up low abundant peptides, the thresholds for the MS noise level and the S/N were lowered 100-fold in comparison to the normal processing method.

Cell–cell fusion assay

Cell–cell fusion assays were performed to ascertain the relative fusogenicity of the different S protein variants. For fluorescent readout, full-length wild-type SARS-CoV-2 S protein and variants thereof, human ACE2, human TMPRSS2 and GFP were co-expressed from pcDNA2004 plasmids in HEK293 cells using Trans-IT transfection reagent according to the manufacturer’s instructions. Transfections were performed on 70% confluent cell monolayers in 6-well plates. Transfected cells were incubated at 37 °C and 10% CO2 for 24 h before imaging on an EVOS cell imaging system (Thermofisher). Overlays between brightfield and GFP channels were made in ImageJ.

The fluorescent fusion assay was adapted to allow quantitative measurement of cell–cell fusion by leveraging the NanoBiT complementation system (Promega). Donor HEK293 cells were transfected with full-length S and the 11 S subunit in 96-well white flat bottom TC-treated microtest assay plates. Acceptor HEK293 cells were transfected in 6-well plates (Corning) with ACE2, TMPRSS2 and the PEP86 subunit, or just the PEP86 subunit (‘No spike’) as negative control. Eighteen hour after transfection, the acceptor cells were released by 0.1% trypsin/EDTA and added to the donor cells at a 1:1 ratio for 4 h. Luciferase complementation was measured by incubating with Nano-Glo® Live Cell Reagent for 3 min, followed by readout on an Ensight plate reader (PerkinElmer).

Ad26 vectors

Replication-incompetent, E1/E3-deleted Ad26 vectors were engineered using the AdVac system42, here using a single plasmid technology containing the Ad26 vector genome including a transgene expression cassette. The codon-optimized SARS-COV-2 Spike genes (as described in vaccine design) were inserted into the E1-position of the Ad26 vector genomes under transcriptional control of the human cytomegalovirus promoter and the SV-40 polyadenylation sequence. Rescue and manufacturing of the Ad26 vectors was performed in the complementing cell line PER.C6 TetR43,44. Virus particle (vp) titers in the Ad26 vector preparations were quantified by measurement of optical density at 260 nm45 and infectivity was assessed by quantitative potency assay (QPA)46, using PER.C6 TetR cells. PCR including subsequent sequencing of PCR products has confirmed the identity and integrity of the SARS-COV-2 Spike genes (primers used are listed in Table S1). Ad26 vector-mediated expression of SARS-COV-2 Spike genes was confirmed by western blot analysis of cell-culture lysates from infected MRC-5 cells (Fig. 2d). Bioburden levels and endotoxin levels met the preset release criteria for animal experiments.

SDS-PAGE and western blotting

Twenty-four-well plates were seeded with MRC-5 cells (1.25 × 105 cells/well), and after overnight growth transduced with Ad26 vectors encoding SARS-CoV-2 S transgenes. Cell lysates were harvested 48 h post transduction and, after heating for 5 min at 85 °C, samples were loaded under non-reduced conditions on a precast 3–8% Tris-Acetate SDS-PAGE gel (Invitrogen). Proteins were transferred to a nitrocellulose membrane using an iBlot2 dry blotting system (Invitrogen), and membrane blocking was performed overnight at 4 °C in Tris-buffered saline (TBS) containing 0.2% Tween 20 (V/V) (TBST) and 5% (W/V) Blotting-Grade Blocker (Bio-Rad). Following overnight blocking, the membrane was incubated for 1 h with 2.8 µg/ml CR3046. in TBST-5% Blocker. CR3046 is a human monoclonal antibody directed against SARS-CoV-1 Spike. After incubation, the membrane was washed three times with TBST for 5 min and subsequently incubated for 1 h with 1:10,000 IRDye 800CW-conjugated goat anti-human secondary antibody (Li-COR) in TBST-5% Blocker. Finally, the PVDF membrane was washed three times with TBST for 5 min, and after drying developed using an ODYSSEY® CLx Infrared Imaging System (Li-COR). All samples derived from one experiment and were processed in parallel. The uncropped blot is provided in Fig S7.

Animals

Animal experiments were approved by the Central Authority for Scientific Procedures on Animals (Centrale Commissie Dierproeven) and conducted in accordance with the European guidelines (EU directive on animal testing 86/609/EEC) and local Dutch legislation on animal experiments.

Female BALB/c or C57BL6 mice (specific pathogen-free), aged 8–12 weeks at the start of the study were purchased from Charles River laboratories (Sulzfeld, Germany). Mice were immunized via the intramuscular (i.m.) route with 100 μl vaccine (50 μl per hind leg) under isoflurane anesthesia. Intermediate blood samples were collected via submandibular bleeding route. At the end of the experiment, under anesthesia, animals were exsanguinated by heart puncture and sacrificed by cervical dislocation. Blood was processed for serum isolation and spleens were collected. Spleens were processed into single cell suspensions for cellular assays (when applicable).

Virus neutralization assay

Neutralization assays against live SARS-CoV-2 were performed using the microneutralization assay previously described by Algaissi and Hashem47. Vero-E6 cells [CRL-1580, American Type Culture Collection (ATCC)] were grown in Eagle’s minimal essential medium (EMEM; Lonza) supplemented with 8% fetal calf serum (FCS; Bodinco BV), 1% penicillin-streptomycin (Sigma–Aldrich, P4458) and 2 mM L-glutamine (PAA). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Clinical isolate SARS-CoV-2/human/NLD/Leiden-0001/2020 (Leiden L-0001) was isolated from a nasopharyngeal sample and its characterization will be described elsewhere (manuscript in preparation). The NGS-derived sequence of this virus isolate is available under GenBank accession number MT705205 and shows 1 mutation in the Leiden-0001 virus compared to the Wuhan sequence resulting in Asp614 > Gly at position 614 of the Spike protein. Isolate Leiden-0001 was propagated and titrated in Vero-E6 cells using the tissue culture infective dose 50 (TCID50) endpoint dilution method and the TCID50 was calculated by the Spearman-Kärber algorithm as described48. All work with live SARS-CoV-2 was performed in a biosafety level 3 facility at Leiden University Medical Center.

Vero-E6 cells were seeded at 12,000 cells/well in 96-well tissue culture plates 1 day prior to infection. Heat-inactivated (30 min at 56 °C) serum samples were analyzed in duplicate. The panel of sera were two-fold serially diluted in duplicate, with an initial dilution of 1:10 and a final dilution of 1:1280 in 60 μL EMEM medium supplemented with penicillin, streptomycin, 2 mM L-glutamine and 2% FCS. Diluted sera were mixed with equal volumes of 120 TCID50/60 µL Leiden -0001 virus and incubated for 1 h at 37 °C. The virus-serum mixtures were then added onto Vero-E6 cell monolayers and incubated at 37 °C in a humidified atmosphere with 5 % CO2. Cells either unexposed to the virus or mixed with 120 TCID50/60 µL SARS-CoV-2 were used as negative (uninfected) and positive (infected) controls, respectively. At 3 days post-infection, cells were fixed and inactivated with 40 µL 37% formaldehyde/PBS solution/well overnight at 4 °C. The fixative was removed from cells and the clusters were stained with 50 µL/well crystal violet solution, incubated for 10 min and rinsed with water. Dried plates were evaluated for viral cytopathic effect. Neutralization titer was calculated by dividing the number of positive wells with complete inhibition of the virus-induced cytopathogenic effect, by the number of replicates, and adding 2.5 to stabilize the calculated ratio. The neutralizing antibody titer was defined as the log2 reciprocal of this value. A SARS-CoV-2 back-titration was included with each assay run to confirm that the dose of the used inoculum was within the acceptable range of 30 to 300 TCID50.

Pseudotyped virus neutralization assay

MLV pseudotyped with SARS-COV-2 S protein was produced as previously described49 with some minor changes. In short, Platinum-GP cells (cell Biolabs, Inc) were transfected with a plasmid encoding the codon-optimized SARS-COV-2 Spike gene from strain Wuhan-Hu-1 (GenBank: MN908947) carrying a 19-aa cytoplasmic tail truncation, a GAG-Pol packaging vector and an MLV vector encoding a luciferase reporter gene using Lipofectamine 3000 transfection reagent (Life Technologies) according to manufacturer’s protocol. Cells were incubated overnight at 37 °C 10% CO2 with OPTIMEM transfection medium. Next day, medium was replaced by OPTIMEM supplemented with 5% FBS and 1% PenStrep and incubated for 48 h prior to harvest. The harvested pseudotyped MLV particles were stored at −80 °C prior to use. In the neutralization assay, soluble ACE2-Fc and mAbs CR3015, CR3022, CR3046 and SAD-S35 (ACROBiosystems) were two-fold serial diluted (n = 3) in DMEM without phenol red supplemented with 1% FBS and 1% PenStrep and incubated with an equal volume of pseudotyped MLV. After 30 min incubation, the mixture was inoculated onto susceptible Vero-E6 cells in MW96 well plates. Luciferase activity was measured after 40 h of incubation by addition of an equal volume of substrate NeoLite (Perkin Elmer) followed by luminescence readout on the EnSight Multimode Plate Reader (Perkin Elmer). The percentage of infectivity was calculated as ratio of luciferase readout in the presence of mAbs normalized to luciferase readout in the absence of mAb.

Direct coat ELISAs

IgG binding to SARS-CoV-2 Spike antigen was measured by ELISA with the full-length in-house produced Spike protein COR200099. COR200099 is an in-house produced SARS-CoV-2 Spike protein, stabilized by two point mutations in the S1/S2 junction that knocks out the furin cleavage site, and by two newly introduced prolines in the hinge region in S2. In addition, the transmembrane and cytoplasmic regions have been replaced by a foldon domain for trimerization mutations, allowing the protein to be produced as soluble protein (see S.dTM.PP Fig. 3C). Briefly, 96-wells Perkin Elmer white ½ area plates were O/N coated with COR200099 protein. Next day plates were washed, blocked for 1 h and subsequently incubated for 1 h with 3-fold serially diluted serum samples in block buffer in duplicate. After washing, plates were incubated for 1 h with goat anti-mouse IgG-HRP in block buffer, washed again and developed using ECL substrate. Luminescence readout was performed using a BioTek Synergy Neo instrument.

For IgG1 and IgG2a ELISAs, a similar protocol was followed as described above, but respectively using Goat anti-Mouse IgG1-HRP and Goat anti-Mouse IgG2a-HRP as secondary antibodies.

ELISpot assay

IFN-γ ELISpot was performed on splenocytes of mice isolated after sacrifice using mouse IFN-γ ELISpot-plus kit (Mabtech). Splenocytes were obtained by disaggregation of spleens with the gentleMACS dissociator. IFN-γ ELISpot assay was performed by stimulating splenocytes from individual mice for 18 h with two different peptide pools (pool 1; peptides 1-156, and pool 2; peptides 157-313) consisting of in total 313 15-mers peptides overlapping by 11 amino acids together covering the full-length Spike protein at a final concentration of 1 μg/peptide/mL. Results shared in this manuscript are the sum of stimulation with peptide pool 1 and pool 2. PMA/ionomycin stimulation was used as a positive control (data not shown); medium was used as negative control and used to calculate the lower limit of detection. Stimulation was done overnight in duplicate wells containing 0.5–2.5 × 105 cells per well.

Multiplex ELISA

The Th1/Th2 multiplex ELISA assay was performed on splenocytes obtained after sacrifice. Splenocytes were stimulated by 48 h culturing in the presence of two Spike 15-mer peptide pools (pool 1 and pool 2). Resulting supernatants were diluted 4-fold and measured for the presence of Th1 (IFN-γ) and Th2 cytokines (IL-10, IL-4, and IL-5) using a 10-plex multiplex ELISA proinflammatory panel (mouse) kit (Meso Scale Discovery, V-PLEX Proinflammatory Panel 1 Mouse Kit cat# K15048D). Results shared in this manuscript are the sum of stimulation with peptide pool 1 and pool 2. Ratios of Th1/Th2 cytokines (IFN-γ/IL-10, IFN-γ/IL-4, and IFN-γ/IL-5) were calculated on basis of cytokine measurements in supernatants by dividing the Th1 cytokine (IFN-γ) with the respective Th2 cytokines. Multiplex ELISA measurements were done on supernatant diluted either 2-fold or 4-fold.

ICS assay

The intracellular cytokine staining assay (ICS) was performed on splenocytes obtained two weeks after sacrifice. Splenocytes were obtained by disaggregation of spleens with the gentleMACS dissociator. ICS assay was performed by stimulating splenocytes from individual mice for 16 h, 106 cells per well, with two different peptide pools (pool 1; peptides 1-156, and pool 2; peptides 157-313) consisting of in total 313 15-mers peptides overlapping by 11 amino acids together covering the full-length Spike protein at a final concentration of 0.2 μg/peptide/well. Stimulation with peptide pools was done at 37 °C, 10% CO2 for 1 h in presence of Rat-anti-Mouse CD49d (1:500, BD; cat #553153) and Hamster-anti-Mouse CD28 (1:500, BD; cat#553294). Protein transport was blocked (1:1000; BD GolgiPlugTM cat#51-230KZ) overnight at 37 °C, 10% CO2. Cells were washed twice with PBS and stained during 20 min at room temperature in the dark, according to manufacturers’ instructions, with a violet viability dye (1:5000; Invitrogen Violet ViD cat#L34955). Cells were washed twice with 0.5% BSA in PBS (PBA) and Fc receptors were blocked during 10 min in the fridge in the dark (1:50; BD Mouse Fc block cat# 553142). Cells were washed once with PBA and stained during 30 min in the fridge in the dark with anti-CD3e FITC (BD, cat#553062), anti-CD4 PerCP-Cy5.5 (BD, cat#550954) and anti-CD8α APC-H7 (BD, cat#557654). Cells were washed twice with PBA and permeabilized/fixated during 20 min in the fridge in the dark (BD Cytofix/CytopermTM cat#51-2090KZ/554722), after which 1× BD Perm/WashTM buffer (BD cat#51-2091KZ/554723) was added. Cells were stained during 30 mins in the fridge, in the dark with anti-IFN-γ PE (BD, cat#554412), anti-TNFa PE-Cy7 (557644) and anti-IL-2 APC (BD, cat#554429). Cells were washed twice with Perm/Wash buffer, resuspended in PBA, and fluorescence was measured on the BD FACSCantoTM II and analyzed with BD FACSDivaTM software: Flow Jo version 10.06.1. Cells were gated on single cells, excluding dead cells, and gated for lymphocytes (Fig S8a). CD8-CD4+ and CD8+ T cells were then gated on IFN-γ, IL-2, and TNF-α (Fig S8b).

Statistical analysis

Statistical differences between immunization regimens were evaluated two-sided for S-specific binding antibodies as measured by ELISA, NAb titers as measured by VNA, IFN-γ producing cells as measured by ELISpot and cytokine production by MSD and ICS assays. Comparisons between Ad26.S, Ad26.tPA.S, Ad26.tPA.SS, Ad26.tPA.WT.S, Ad26.S.PP, adjuvanted S protein, tPA.S, tPA.S.PP, S and S.PP groups were made using the exact Wilcoxon rank-sum test, Cochran–Mantel–Haenszel test, Mann–Whitney U test, t-test from ANOVA, or z-test from Tobit ANOVA. Results were corrected for multiple comparisons by Bonferroni correction; 3-fold Bonferroni correction Fig. 5a, b, 2-fold Bonferroni correction Fig. 5c, d.

Lo ammetto... SONO STATO STRONZO ma era per farti capire che la mia è la semplificazione di una semplificazione di una semplificazione.

Molto sinteticamente, il vaccino Johnson & Johnson sfrutta una tecnologia specifica di ancoraggio dell’encoding della proteina S (Ad26) a un vettore virale modificato (Adenovirus) simile a quello del vaccino AstraZeneca ma, evidentemente, questa metodica è più efficace nello stimolare una risposta anticorpale che da subito raggiunge livelli stimati tra il 64% e il 72% senza bisogno di una seconda dose di boost.

Perché non te l’ho detto subito?

Perché è bene non dimenticare che anche dietro a una banale compressa di paracetamolo ci sono una serie di azioni, interazioni e reazioni ‘scientifiche’ che in un mondo ideale sarebbe bello che tutti conoscessero ma che allo stato attuale funzionano come la comunione con l’ostia benedetta...

Credeteci e verrete salvati.

[bigeyeug]

StarTimes decorates Big League final with prizes

StarTimes decorates Big League final with prizes

By Reporter The StarTimes FUFA Big League 2020 / 2021 season officially ended on 17th August 2021 after a series of football lockdown due to Covid 19 pandemic. In a colorful football event that was organized by FUFA at the technical center in Njeru. Arua Hill Sports Club commonly known as ‘Kongolos’ took the day with a convincing win against Tooro United Football Club. Gaddafi FC against Proline…

[mubahood360]

AS Kigali Arrive In Uganda, Before Their CAF Confederation Cup Match Against KCCA At St. Mary's Stadium, Kitende

AS Kigali Arrive In Uganda, Before Their CAF Confederation Cup Match Against KCCA At St. Mary’s Stadium, Kitende

AS Kigali is Rwanda’s flag bearers in the CAF Confederation Cup twice in a row after winning the Peace Cup in 2019. Last season, The cup was not played and completed due to COVID-19 which gave AS Kigali the chance to represent the country a second time in a row even though they had finished sixth in the league. They were eliminated at this stage last year by Uganda’s Proline FC 3-2 on…

[zonefoot]

Coach David Mutono is set to sue his former bosses of Kyetume FC over unpaid salaries. Mutono who was an assistant coach at the Mukono-based UPL side last season, says that he demands arrears around Sh10m in total. The CAF B licensed coach was a stand-in at crucial times including a vital win at Proline FC at Lugogo when coach Jackson Mayanja reportedly left for Tanzania. [ 223 more words ] https://zonefoot.net/2020/07/31/uganda-coach-threatens-to-sue-kyetume-over-unpaid-arrears/

[africazilla]

Uganda Premier League Match Day 18 Best XI

Uganda Premier League Match Day 18 Best XI

Uganda Premier League

Match 18 Results

KCCA FC 1-0 Tooro United FC

Police FC 1-0 Kyetume FC

Express FC 3-0 Proline FC

Vipers SC 0-1 SC Villa

Wakiso Giants FC 1-0 Onduparaka FC

Busoga United FC 1-0 Mbarara City FC

Maroons FC 2-0 BUL FC

URA FC 1-0 Bright Stars FC

Match day 18 saw Police FC, Busoga United FC and URA FC extend their perfect run this year registering three wins in as many…

[baudronsport-blog]

StarTimes FUFA Big League | Matchweek 13 | Results | Live Kira United FC 1-0 Kitara FC | Michael Senyonjo 5' Proline FC 3-0 Kireka United FC | Kabugu 49', Otieno (O.G) 87', Kiwanuka 90' https://www.instagram.com/p/BuMGv30HSCO/?utm_source=ig_tumblr_share&igshid=h69jed4e35h

[aliasswali9]

SFBL MD1 Wrap: MYDA, Kitara Register Wins, Maroons Held To A Goalless Draw By Calvery As Proline Salvage A Point At Nyamityobora

SFBL MD1 Wrap: MYDA, Kitara Register Wins, Maroons Held To A Goalless Draw By Calvery As Proline Salvage A Point At Nyamityobora

There were wins for Kitara Football Club and Malaba Youth Development Association Football Club while Maroons were held to a goalless draw by Calvary as Proline salvaged a point at Nyamityobora after settling for a goalless draw during their respective Startimes FUFA Big League matchday one encounters played on Friday afternoon. MYDA FC 3-2 Ndejje University: MYDA FC kicked off their FUFA Big…

[footballghana]

Nana Afia Kobi Ampem Ladies to unveil Mary Hasford as new CEO today

Ghana Women’s lower-tier side, Nana Afia Kobi Ampem Ladies has appointed Mary Hasford as the club’s new Chief Executive Officer (CEO), footballghana.com can confirm.

The Women’s Division One League is one of the top sides and well-run clubs in the country.

As the owner of the club, Madam Evelyn Nsiah Asare is taking advantage of the Coronavirus period to put things in order and has together with the top hierarchy of the club settled on a new CEO.

Mary Hasford, a known face who has been working in the football space for a long time has been given the mantle with her appointment scheduled for Friday, August 21, 2020, on the social media pages of Nana Afia Kobi Ampem Ladies.

She has in the past worked for Proline FC as a Financial Secretary [2005-2006], and she also served as the welfare and equipment officer of the Black Princesses team from 2006 to 2012,

In addition, she has worked for Ashtown Ladies and Soccer Angels Ladies FC which has seen her gather a lot of experience in football administration.

Madam Mary Hasford’s officially unveiling comes off at 08:00GMT on the social media platforms of Nana Afia Kobi Ampem Ladies.

More on the newly appointed CEO below:

  source: https://footballghana.com/

[wincatcherses]

Kagwa: Wakiso Giants sella el fichaje del extremo del Police FC

Los Purple Sharks han presentado su segundo fichaje mientras se esfuerzan por fortalecer el equipo antes de la nueva temporada. Los Wakiso Giants han confirmado la firma del extremo Pius Kagwa del Police FC de la Liga Premier de Uganda (UPL) antes de la nueva temporada. Los Purple Sharks están fortaleciendo su escuadrón antes de la nueva temporada después de que lograron asegurar su estatus de máximo nivel en la recién concluida temporada 2019-20. El club ha confirmado en su sitio web oficial la llegada del extremo, que se siente cómodo en ambos lados en un contrato de tres años. "En un intento por fortalecer nuestro escuadrón, los Tiburones Púrpuras han firmado al extremo Pius Kagwa de la Policía", confirmó el club. "El extremo altamente calificado que se siente cómodo en ambos flancos se une a nosotros en un contrato de tres años con la opción de extenderlo por un año más y se convierte en nuestro segundo fichaje en la ventana de transferencia después del mediocampista Ibrahim Kasule alias Owen Kasule". Al firmar para el equipo, Kagwa dijo: “Estoy emocionado de unirme a uno de los mejores clubes del país en este momento. Me lo he pasado muy bien en Police y me gustaría agradecer a la gerencia, a los ex compañeros de equipo y a mis entrenadores por convertirme en el jugador que soy hoy. "Todavía hay mucho que aprender para mí, pero estoy listo para el desafío y no puedo esperar para comenzar". Kagwa también ha aparecido para Synergy FC en la liga inferior antes de mudarse para fichar a la Policía en la máxima categoría. Kagwa se convierte en el segundo fichaje para Wakiso después de que el club trajo a bordo a Kasule del Kampala Junior Team en un contrato de tres años y con la opción de extenderlo por un año más. Kasule, de 22 años, había acudido previamente a Nansana United y Buddu Ssaza, donde emergió como el jugador más valioso en 2018. También ganó el MVP durante la University Football League, donde logró capitanear a la Lawrence University en 2019 y también ganó el Mejor Centrocampista de la temporada en la misma competencia. El artículo continúa abajo Wakiso jugará en la máxima categoría durante otra temporada después de que evitaron el descenso en la campaña 2019-20 cuando la liga terminó debido a los efectos causados ​​por la pandemia de coronavirus. Wakiso terminó la liga en el puesto 10 con la Federación de la Asociación de Fútbol de Uganda (Fufa) coronando a Vipers SC, que lideraba la tabla de la liga, con el ex campeón KCCA FC en segundo lugar. Maroons FC, los campeones de la Copa de Uganda, Proline FC y Tooro United fueron relegados, Myda FC y UPDF obtuvieron puestos en el primer nivel, mientras que Kiboga, Kataka, Kitara y Ndejje se enfrentarán en los play-offs para producir el tercer equipo que honrará el primer nivel en la temporada 2020/21.

from Noticias Wincatchers https://noticias.wincatchers.com/2020/07/14/kagwa-wakiso-giants-sella-el-fichaje-del-extremo-del-police-fc/

[zonefoot]

Despite scooping the Most Valuable Player (MVP) award, Kampala Capital City Authority (KCCA FC) forward Allan Okello believes that his footballing journey has just begun and that he still has a lot to do. The teenage sensational bagged the award when he saw off competition from his club teammate Mustafa Kizza and Bright Anukani of Proline FC. “It is just the beginning and the struggle continues,” Okello posted on his official facebook page before adding. [ 273 more words ] https://zonefoot.net/2019/12/17/uganda-i-still-have-a-lot-to-do-okello/

[africazilla]

Uganda Premier League Matchday 16 Best XI

Uganda Premier League Matchday 16 Best XI

Matchday 16 Games

URA FC 2-0 SC Villa

KCCA FC 1-0 Mbarara City FC

Maroons FC 1-2 Proline FC

Bright Stars FC 1-0 BUL FC

Wakiso Giants FC 1-2 Police FC

Vipers SC 1-0 Kyetume FC

Busoga United FC 2-0 Express FC

Tooro United FC Vs Onduparaka FC (Not played)

The second round of the 2019/20 Uganda Premier League officially got underway last week with seven games played across different grounds.…

[baudronsport-blog]

StarTimes FUFA Big League | Matchweek 12 | Friday, 22nd February 2018 | 4:00 PM | Live Proline FC vs Kireka United FC - StarTimes Stadium, Lugogo Kira United FC vs Kitara FC - Namboole Stadium https://www.instagram.com/p/BuLqQ5bHXb9/?utm_source=ig_tumblr_share&igshid=vcjrpzepj2ww

[aliasswali9]

Transfer News: KCCA FC Confirm Signing Proline's Budding Teenager

Transfer News: KCCA FC Confirm Signing Proline’s Budding Teenager

StarTimes Uganda Premier League side Kampala City Council Authority Football Club have announced the signing of promising teenager Mato Rogers. Mato joins Morley Byekwaso’s charges from FUFA Big League entity Proline Football Club ahead of the coming campaign. He has signed a four-year employment contract with the Kasassiro Boys that will keep him at MTN Omondi until 2025. The Club have…

[soccernetghana]

2019/2020 football season ends in Uganda, Vipers SC are declared champions

The Federation of Uganda Football Association (FUFA) has decided to abandon the 2019/2020 with Vipers SC being declared champions.

The FUFA executive committe decided to effect Article 18 of the statutes since more than 75% of the league matches had been played.

Vipers SC were topping the log standings with a 4-point lead after 25 matches remaining just five to conclude the season.

The decision is taken as result of the widespread of the global pandemic coronavirus.

The Uganda Premier League and the other leagues were suspended in March as a result of fighting against the deadly disease.

The big losers are Proline FC as they have been relegated after competing in the CAF Confederation Cup for the first time this season where they reached the play-off stage.

source: https://ghanasoccernet.com/

[footballghana]

COVID-19 impact on African leagues

  The coronavirus pandemic has continued to cause major upsets on world sports and African football has not been left behind, with the effects already rolling down. Here is how the pandemic has so far affected African leagues.

  Mauritius

Mauritius became the first African country to end its football season due to the pandemic after an initial indefinite postponement on March 19.

The Football Association, through General Secretary Didier Gnanapragassa communicated to the clubs the decision to wind down the season throughout the country over uncertainties as to when the pandemic will be over.

A decision is yet to be made on its representatives in next year’s CAF Clubs competitions.

Date of decision: 6 April 2020

Kenya

The Football Kenya Federation has decided to end football across all its seven tiers of the men’s game as well as the two tiers of the Women Leagues. Under a Force Majeure clause in its rules, the champions of each league will be decided by the standings at mid-season.

Gor Mahia are crowned the Premier League champions for a 19th time with Chemelil Sugar and Sony Sugar relegated while Nairobi City Stars are champions of the second tier and promoted to the Premier League alongside Bidco United.

The two tiers of the women’s leagues have consequently been cancelled as they had only played one round of matches into the new season.

Date of decision: 30 April 2020

Angola

After a meeting with the top tier clubs, the Angolan Football Federation also made a decision on it’s leagues and different from Kenya, they decided to cancel the season with five rounds of matches left to play.

Petro de Luanda were leading the top tier with 54 points with Primero de Agosto second. A decision was reached that the two clubs will represent Angola in the Total CAF Champions League.

For the Confederations Cup representative, a decision will be reached at a later date, depending on finances and the coronavirus situation.

Date of decision: 30 April 2020

Guinea

The President of the Guinean Professional Football League (LGFP) General Mathurin Bangoura announced that the top two tiers of Guinean football will not be able to be completed because of the pandemic.

With the uncertainty on when the pandemic will be over, Bangoura said a unanimous decision was reached to cancel the season with no champions, promotion or relegation.

The league had reached the halfway point with Horoya leading the standings with 29 points, four ahead of second placed Wakirya. The Federation is set to make a decision on the representatives for CAF Competitions.

Date of Decision: 30 April 2020

Burkina Faso

The Emergency Committee of the Burkina Faso Football Federation has made the decision to cancel the top tier season with six rounds of matches left to play.

There will be no champions or relegation in the top tier, but with the second tier already completed, the top two teams will be promoted which means the next season of the top tier league will have 18 teams

At the same time, Rahimo FC has been selected to represent Burkina Faso in the Total CAF Champions League while Salitas FC will play in the CAF Confederation Cup.

Date of decision: 4 May 2020

Ethiopia

The Executive Committee of the Ethiopia Football Federation, in consultation with government organs and the league administrators has announced the cancellation of all tiers of football in the Eastern Africa Country with no champions declared, promotion or relegation as well.

The league was halted early March as the coronavirus pandemic began to gather pace with Fasil Kenema top of the standings with 30 points, Mekelle Enderta second with 28 points, same as third placed St, George.

Consequently, EFF has announced that Ethiopia will not have a representative at next season’s continental club championship.

Date of decision: 5 May 2020

Congo

The Congo Football Federation has named AS Otoho as champions of the 2019-20 season after cancelling all football due to the coronavirus.

The Executive Committee made the decision after a meeting to assess the situation and it was unanimously agreed that it would be difficult to resume sporting activities soon.

Otoho who were leading the standings with a 14-point gap with six matches remaining before the season was halted will play in the Total CAF Champions League while second placed Diables Noirs will play in the CAF Confederation Cup.

Date of decision: 5 May 2020

Liberia

The Liberia Football Association has also announced the cancellation of all its football leagues with no champions, relegation or promotion after a meeting of the Executive Committee.

With nine rounds of matches left, Mighty Barolle were leading the standings with 23 points, one ahead of BEA Mountain and two ahead of third placed MC Breweries. The league was still open with only four points separating the leaders and the eighth placed team.

Meanwhile the Liberia FA will consult on a play-off between the top four to determine representatives at next season’s CAF Competitions.

Date of decision: 5 May 2020

Cameroon

Cameroon became the latest African country to void its football season due to the pandemic and a meeting between the Federation (FECAFOOT) and the respective clubs came to a consensus that it would be impossible to complete the campaign.

In accordance with the discussions held, PWD Bamenda have been named champions of the Elite One League and will represent Cameroon in the 2020-21 Total CAF Champions League.

At the same time, Yaounde’s Louves Minproff Club were named champions of the Cameroon Women’s Football Division One League.

Date of decision: 12 May 2020

DR Congo

The Executive Committee of the Democratic Republic of Congo Football Federation has called an end to all its football leagues due to the sweeping effects of the COVID-19 pandemic

TP Mazembe who led the standings by the time the league was suspended have been named champions and will represent DR Congo in the 2020-21 Total CAF Champions League alongside AS Vita who were second, five points behind.

Meanwhile, AS Maniema Union and DC Motema Pembe who were third and fourth respectively in the standings will represent the Central African country in the Total CAF Confederation Cup.

Date of decision: 14 May 2020

Uganda

Citing a Force Majeure clause in its competition, the Federation of Ugandan Football Associations (FUFA) has resolved to end the season due to COVID-19.

Vipers led the standings with 54 points, four ahead of second placed Kampala Capital City Authority (KCCA) after 25 rounds of matches. Vipers will represent Uganda in next season’s Total CAF Champions League while KCCA will play in the Total CAF Confederation Cup.

Meanwhile, Maroons, Proline and Tooro United have been relegated to the second tier FUFA Big League while UPDF Football Club and MYDA Football Club have earned promotion.

At the same time, the Women’s Premier League has been cancelled.

Date of decision: 20 May 2020

South Sudan

South Sudan has become the latest CAF Member Association to call an end to its season due to the COVID-19 pandemic that has currently caused a stoppage to sports events all over the world.

Apart from calling to an end the Premier League, the SSFA has also cancelled all football activities in the country while there will be no promotion or relegation across all leagues and as thus South Sudan will not have a team representing it at the 2020-21 Total CAF Champions League.

However, Alrabita Juba FC have earned themselves a ticket to the CAF Confederations Cup having won the Cup tournament which had been completed before football was called off due to the virus.

Date of decision: May 20, 2020

Burundi

Burundi’s Primus Ligue became the first African league to re-start as the world of sports begins to slowly pick up the ruins from the stoppages caused by the COVID-19 pandemic. The league-restarted on Thursday with a match between Musongati and Athletico Olympic.

The match, staged at the Stade Ingoma in Gitaga ended in a 3-3 draw with full attendance including the president of the Burundi Football Federation. The result saw Musongati move to 54 points, just one behind leaders Le Messager Ngozi.

This weekend, action will shift to the Cup fixtures. The competition is currently in the semi-final stage. The final is scheduled for the 13th of June. League matches will resume on the 30th of this month with the final round scheduled for June 28.

Date of resumption: 21 May 2020

Tanzania

In Tanzania, the Government has announced that sports activities in the country will be allowed to resume from June 1.

The Tanzanian league managers are thus preparing for resumption after stoppage since March 17.

With 10 rounds of matches left, giants Simba SC were leading the Ligi Kuu Bara standings with 71 points, a healthy 17 ahead of second placed Azam FC. Just six wins out of the remaining 10 matches will confirm them as champions.

Expected date of resumption: 1 June 2020

Rwanda

Army side APR have been crowned Rwanda Premier League champions for a record 18th time after the Rwandese Football federation (FERWAFA) called an end to the season due to the COVID-19 pandemic.

The decision was reached upon by the FERWAFA Executive Committee in accordance to Article 33 of the FERWAFA Statutes and Article 28 of the FERWAFA Internal rules and regulations.

FERWAFA handed APR the title based on the standings after Match Day 23, and subsequently, results from Match Day 24 have been cancelled as not all teams had played round 24 games. APR led the standings with 57 points.

Date of Decision: 22 May 2020

Togo

Togo became the latest African country to announce an end to its national championships due to the COVID-19 situation. The Togolese Football Federation has formally announced the end to the 2019-20 season, with football having been suspended over the last two months.

The Togolese Federation has been holding consultations with government authorities over the last few weeks, to see if it was safe to return to action. The decision was finally reached, to cease all football activities permanently.

Consequently, the Kozah Sports Association (ASKO) have been named champions of the first division as they led the standings with 39 points, six ahead of second placed Unisport when football was halted with 20 rounds of matches played. There were six match days remaining before the season ended.

Date of decision: 26 May 2020

Tunisia

The Tunisian Football Federation has announced plans to allow Ligue One teams to resume individual training sessions from June 4, while group sessions can start in a maximum of 15 days as they started plans to get the season back on track.

The Tunisian football league has been suspended since mid-March 2020 due to COVID-19. According to the federation, they are considering a return to competitions in August to complete the current campaign.

Meanwhile, the Ligue Two season has been called off, with the exception of teams who were up for promotion and relegation, with a play-off tournament set to be organized for that purpose.

Botswana

Jwaneng Galaxy FC have been declared champions after the Botswana Football Association (BFA) made the decision to annul the season due to the challenges posed by COVID-19.

A meeting of the BFA Executive Committee on June 14 made the decision to end the campaign with the league standings remaining as they were when the season was halted in March.

Galaxy led the standings with 41 points, one ahead of second placed Township Rollers and third placed Orapa United with 10 rounds of matches left. They will thus represent Botswana at next season’s Total CAF Champions League.

Date of Decision: 14 June 2020

Source: cafonline.com 

source: https://footballghana.com/