Paul.xu: Created page with "S The full-length spike protein sequence was modified to remove the polybasic cleavage site, which is recognized by furin and to add a pair of stabilizing mutations. The..."

2020-04-25T16:43:45Z

Created page with "S The full-length spike protein sequence was modified to remove the polybasic cleavage site, which is recognized by furin and to add a pair of stabilizing mutations. The..."

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The full-length spike protein sequence was modified to remove the polybasic cleavage site, which is recognized by furin and to add a pair of stabilizing mutations. These two modifications were included to enhance the stability of the protein based on published literature. At amino acid P1213 the sequence was fused to a thrombin cleavage site, a T4 foldon sequence for proper trimerization and a C-terminal hexahistidine tag for purification. 5 mg/liter in 293 cells

RBD

sequence (amino acid 319 to 541,  MFVF….TSGS----RVQP….CVNF) with a C-terminal hexahistidine tag. 25-50 mg/liter in 293 cells culture

96 well plates (Immulon 4 HBX; Thermo Scientific) were coated overnight at 4°Celsius with 50 ul per well of a 2 ug/ml solution of each respective protein suspended in PBS (Gibco). The next morning, the coating solution

Recombinant proteins were analyzed via a standard SDS-PAGE 292 gel to check protein integrity. One ug of protein was mixed with 2X Laemmli buffer containing 5% beta-mercaptoethanol (BME) at a ratio of 1:1. Samples were heated at 100 °Celsius for 15 minutes and then loaded onto a polyacrylamide gel (5% to 20% gradient; Bio-Rad). Gels were stained with SimplyBlue SafeStain (Invitrogen) for 1-2 hours and then de-stained in distilled water overnight.

Human samples

Human plasma and serum samples were obtained from a number of different sources. First, de-identified samples from the University of Melbourne (n=3, taken on day 2, 4 and 6 after

symptom onset) and University of Helsinki (n=1, day 20 after symptom onset, neutralizing titers 1:160)13 301 were used as positive controls. For those, human experimental work was conducted according to the Declaration of Helsinki Principles and according to the Australian National Health and Medical Research Council Code of Practice. All participants provided written informed consent prior to the study. The studies were approved by the Alfred Hospital (ID #280/14) and University of Melbourne (ID #1442952.1,

1955465.2) Human Research Ethics Committees, and under research permit for project TYH2018322 of Helsinki University Hospital Laboratory. Second, banked human samples were collected from study participants enrolled in several ongoing IRB approved longitudinal observational study protocols of the Mount Sinai Personalized Virology Initiative.

The pre-pandemic serum panel comprised samples selected based on the date of collection (e.g., fall 2019) and whether participants had a documented history of viral infection (e.g., dengue virus, hantavirus, Chikungunya virus, coronavirus NL63). All participants agreed to sample banking and future research use. Self-reported ethnicities of the individuals from which samples were tested included Caucasian, Asian, African American and Hispanic. Samples included sera from a participant with acute NL63 infection as determined by the Biofire Respiratory panel. We included serum collected at day 3 post symptom onset as well as convalescent serum from the same person (day 30 post symptom onset). These samples served as negative controls given that they were collect prior to SARS-CoV-2 spread in the US. Six subjects were 20-29, 19 were 30-39, 13 were 40-49, 7 were 50-59 years old and six were 60

or older. For the mRBD ELISAs sera from additional nine subjects were tested (30-39: 2; 40-49: 4; 50-59: 2; 60+: 1). The pre-pandemic panel was complemented by a collection of plasma samples collected from

50 HIV-1 infected individuals between 2008 and 2011. The COVID19 panel comprised serum and plasma samples from individuals with severe, mild or asymptomatic SARS-CoV-2 infections. Seven paired serum and plasma samples from patients with COVID19 were used for comparison purposes. These samples were collected between 7 and 31 days post symptom onset. Normal human immunoglobulin (NHIG) The following NHIG preparations, each prepared from >1000 blood/ plasma donors and intended for

intravenous use for medical conditions, were tested in an ELISA to determine if they have reactivity against SARS-CoV-2 spike or RBD: Octagam (M934A8541), Gamunex-c (B2GMD00943, A1GLD01882, B3GLD01223, A1GLD01902, B2GLD01972, B3GGD00143, A1GKE00012 (2 different vials), B2GKD00863, B2GJE00033 (3 different vials)), Gammagard liquid (LE12T292AB, LE12V238AB, LE12V278AD),

Gammagard S/D (LE08V027AB, 4 different vials), Gammagard liquid (C19G080AAA, LE12V071AD, LE12V230AB, LE12V115AC, LE12V205AB, LE12VE25AB, LE12V115AC).

ELISA The ELISA protocol was adapted from previously established protocols 34,35337 . Ninety-six well plates (Immulon 4 HBX; Thermo Scientific) were coated overnight at 4°Celsius with 50 ul per well of a 2 ug/ml solution of each respective protein suspended in PBS (Gibco). The next morning, the coating solution

was removed and 100 ul per well of 3% non-fat 340 milk prepared in PBS with 0.1% Tween 20 (TPBS) was added to the plates at room temperature (RT) for 1 hour as blocking solution. Serum samples were heated at 56°C for 1 hour before use to reduce risk from any potential residual virus in serum. Serial dilutions of serum and antibody samples were prepared in 1% non-fat milk prepared in TPBS. The blocking solution was removed and 100 ul of each serial dilution was added to the plates for 2 hours at RT. Next, the plates were washed thrice with 250ul per well of 0.1% TPBS. Next, a 1:3000 dilution of goat anti-human IgG-horseradish peroxidase (HRP) conjugated secondary antibody (ThermoFisher

Scientific) was prepared in 0.1% TPBS and 100 ul of this secondary antibody was added to each well for 1 hour. Plates were again washed thrice with 0.1% TBS. Once completely dry, 100 ul of SigmaFast OPD (o349

phenylenediamine dihydrochloride; Sigma-Aldrich) solution was added to each well. This substrate was left on the plates for 10 minutes and then the reaction was stopped by addition of 50 μL per well of 3 M

hydrochloric acid (HCl). The optical density at 490 nanometers was measured via a Synergy 4 (BioTek) plate reader. The background value was set at and optical density (OD) 490nm of 0.11 and area under the curve (AUC) was calculated. AUC values below 1 were assigned a value of 0.5 for graphing and calculation purposes. Data were analyzed using Prism 7 (Graphpad). In some cases endpoint titers were calculated, the endpoint titer being the last dilution before reactivity dropped below and OD 490nm of below 0.11. To determine the impact of heat treatments, paired samples that were heat treated or not

treated were analyzed. NHIGs were run similar as serum/plasma samples but with a starting dilution at a concentration of 100 ug/ml. Three non-SARS-CoV-2 reactive human mAbs and CR302220-22358 , a human mAb reactive to the RBD of both SARS-CoV-1 and SARS-CoV-2 were used as controls. To assess the distribution of the different antibody isotypes/subclasses in the samples that reacted well

in our standard ELISA, another ELISA was performed with different secondary antibodies 29361 . These antibodies include anti-human IgA (α-chain-specific) HRP antibody (Sigma A0295) (1:3,000), anti-human IgM (μ-chain-specific) HRP antibody (Sigma A6907) (1:3,000), anti-human IgG1 Fc-HRP (Southern Biotech 9054-05) (1:3,000), anti-human IgG2 Fc-HRP (Southern Biotech #9060-05) (1:3,000), anti-human IgG3hinge-HRP (Southern Biotech 9210-05) (1:3,000), and anti-human IgG4 Fc-HRP (Southern Biotech 9200-05).
[[Category:Virus]]

Expression of SARS-CoV-2 spike protein - Revision history

Paul.xu: Created page with "S The full-length spike protein sequence was modified to remove the polybasic cleavage site, which is recognized by furin and to add a pair of stabilizing mutations. The..."