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kon-igi · 4 years ago

Note

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.

#Anonymous

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