An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen
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SciScore for 10.1101/2021.06.29.21259605: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: All individuals gave their writing informed consent for the use of their serum. 2.2.
IACUC: The experimental protocols were approved by the Ethical Committee on Animal Experimentation of the Center for Genetic Engineering and Biotechnology (CIGB, Havana, Cuba) and the Center for Production of Laboratory Animals (CENPALAB, Bejucal, Cuba).
Euthanasia Agents: The immunogen content per 500 µL: 50 µg of C-RBD-H6 PP protein adjuvated with 0.3 mg of aluminum hydroxide gel (Alhydrogel ®) in phosphate buffer (0.28 mg of disodium hydrogen phosphate, 0.31 mg of sodium dihydrogen phosphate dihydrate, 4.25 mg of sodium chloride).Sex as a biological variable Six- to eight-week-old female BALB/c … SciScore for 10.1101/2021.06.29.21259605: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: All individuals gave their writing informed consent for the use of their serum. 2.2.
IACUC: The experimental protocols were approved by the Ethical Committee on Animal Experimentation of the Center for Genetic Engineering and Biotechnology (CIGB, Havana, Cuba) and the Center for Production of Laboratory Animals (CENPALAB, Bejucal, Cuba).
Euthanasia Agents: The immunogen content per 500 µL: 50 µg of C-RBD-H6 PP protein adjuvated with 0.3 mg of aluminum hydroxide gel (Alhydrogel ®) in phosphate buffer (0.28 mg of disodium hydrogen phosphate, 0.31 mg of sodium dihydrogen phosphate dihydrate, 4.25 mg of sodium chloride).Sex as a biological variable Six- to eight-week-old female BALB/c mice, and male and female SD rats were used for the study and housed in the animal facility. Randomization Three transformants from the plate with the highest concentration of Zeocin™ were randomly picked, purified by further streaking on YPD-Zeocin™, and used to prepare small seed banks. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Culture supernatants were analyzed at 96 h post-induction by SDS-PAGE and Western blotting with an anti-His6 antibody (Promega, USA). 2.3. Fermentation: Fermentation was carried out in a 75-liters Chemap fermenter (Germany) with a working volume of 50 L of fermentation medium containing per liter of culture 8.8 mL of 85 % phosphoric acid, 6.92 g MgSO4.7H2O, 1.23 g (NH4)2SO4, 16.77 g K2HPO4, 0.46 g CaCl2.2H2O, 32.3 mL of 98% glycerol, 4.61 g yeast extract, 4 mL histidine solution, 5 mL of 400X vitamin base solution (1 g L−1 myo-inositol, 0.8 g L−1 anti-His6suggested: NoneAfter the plates were blocked with 2 % skim milk, 0.05 % Tween 20, serially diluted mouse, rat or NHP sera or control monoclonal antibodies SS-1, SS-4, SS-7 and SS-8 (Center for Genetic Engineering and Biotechnology, Sancti Spiritus, Cuba), were added and incubated at 37 °C for 2 h in 0.2 % skim milk, 0.05 % Tween 20 in PBS, followed by six washes with PBS containing 0.05 % Tween 20 SS-8 ( Center for Genetic Engineering and Biotechnology , Sancti Spiritus , Cuba) ,suggested: NoneBound antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse IgG (SIGMA, USA), anti-Rat IgG (SIGMA, USA anti-mouse IgGsuggested: Noneanti-Rat IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Briefly, 0.25 µg of RBD protein produced in HEK293T cells (Center for Molecular Immunology, Havana) was used to coat 96-well microtiter plates (Corning Costar, Acton, MA) in 0.1 M sodium carbonate buffer (pH 9.6) at 4 °C overnight. HEK293Tsuggested: NoneAfterward, Vero E6 cell were overlaid with virus suspensions. Vero E6suggested: RRID:CVCL_XD71)Experimental Models: Organisms/Strains Sentences Resources Animals and immunization schedules: Three different animal species were used for evaluation of immunogenicity of the C-RBD-H6 PP protein: BALB/c mice, Sprague–Dawley (SD) rats, and African green monkeys (Chlorocebus aethiops sabaeus). Sprague–Dawleysuggested: NoneSix- to eight-week-old female BALB/c mice, and male and female SD rats were used for the study and housed in the animal facility. BALB/csuggested: RRID:IMSR_ORNL:BALB/cRl)Recombinant DNA Sentences Resources Construction of Pichia pastoris strains expressing RBD from SARS-CoV-2 (C-RBD-H6 PP): A sequence coding for residues 331-530 of the Spike protein of SARS-CoV-2 strain Wuhan-Hu-1 (NCBI Acc. No. YP_009724390) with the appropriate N- and C-terminal extensions was codon-optimized for S. cerevisiae using J-Cat [17] and cloned in-frame with the KEX2 cleavage site of the pre-pro MATα sequence of pPICZαA (Invitrogen, USA), placing it under transcriptional control of the P. pastoris AOX1 promoter. pPICZαAsuggested: NoneSoftware and Algorithms Sentences Resources Surface plasmon resonance experimental procedure: The interaction between mFc-ACE2 fusion protein and the recombinant C-RBD-H6 PP was monitored by SPR using a BIACORE X (GE Health-care) at 25 °C in a multi-cycle mode. GE Health-caresuggested: NoneThe equilibrium dissociation constant (binding affinity, KD) was estimated with the BIAevaluation® software (GE Healthcare) using the Langmuir 1:1 interaction model. BIAevaluation®suggested: (BIAevaluation Software, RRID:SCR_015936)Statistical analysis: Prism 8.4.3 software was used to generate dose-response curves for ELISA test and to calculate EC50 values when needed. Prismsuggested: (PRISM, RRID:SCR_005375)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 49 and 54. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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