Extracellular vesicle-based vaccine platform displaying native viral envelope proteins elicits a robust anti-SARS-CoV-2 response in mice

Article activity feed

1. 

   ScreenIT

   Oct 30, 2020

   SciScore for 10.1101/2020.10.28.357137: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Ethics statement: All animal studies were performed in accordance with national regulations and approved prior to experimentation by the Ethics Committee for Animal Testing Languedoc-Roussillon (national agreement CEEA-036) and French Ministry of Research with the reference APAFIS #24869 (10 April 2020).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Mice and study design: Female BALB/cAnNCrl mice, 6 weeks old, were purchased from Charles River-Italy and placed into four groups of n=6.
   Cell Line Authentication	Contamination: HEK293T cells were routinely tested and found negative by MycoAlert™ mycoplasma …

   SciScore for 10.1101/2020.10.28.357137: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Ethics statement: All animal studies were performed in accordance with national regulations and approved prior to experimentation by the Ethics Committee for Animal Testing Languedoc-Roussillon (national agreement CEEA-036) and French Ministry of Research with the reference APAFIS #24869 (10 April 2020).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Mice and study design: Female BALB/cAnNCrl mice, 6 weeks old, were purchased from Charles River-Italy and placed into four groups of n=6.
   Cell Line Authentication	Contamination: HEK293T cells were routinely tested and found negative by MycoAlert™ mycoplasma detection kit (Lonza Nottingham, Ltd.)

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Tetraspanin kit chips (EV-TETRA-C), with capture antibodies against CD81, CD63, CD9 and control IgG (MIgG), were used to detect EVs.	CD81 suggested: None CD63 , CD9 suggested: None control IgG suggested: None
   EVs (1010 EVs/ml) were coated on chips, then probed with detection antibodies provided with the kit (CD81/CF55, CD63/CF647, CD9 CF488A) according to the manufacturer’s protocol.	CD9 suggested: None
   The immunodetection of SARS-CoV-2 spike protein was performed with primary antibodies against either S1 domain (SARS-CoV-2 Spike S1, rabbit monoclonal antibody [HL6], GTX635654, Genetex), or S2 domain (SARS-CoV-2 Spike S2, mouse monoclonal antibody [1A9], GTX632604, Genetex), or CilPP (in-house antibody raised in rabbit).	GTX635654 suggested: (GeneTex Cat# GTX635654, RRID:AB_2888548) in-house suggested: None
   Membranes were then incubated with the corresponding secondary HRP-conjugated antibodies (donkey anti-mouse or anti-rabbit HRP, #715-035-150 or #711-035-152 from Jackson ImmunoResearch); the signals were detected using an enhanced chemiluminescence detection kit (Super Signal West Pico Plus, 34580, ThermoFischer Scientific) and membranes imaged with ChemiDoc Imaging System (Bio-Rad).	anti-rabbit HRP suggested: (Jackson ImmunoResearch Labs Cat# 711-035-152, RRID:AB_10015282)
   The same anti-S1 and anti-S2 antibodies as well secondary antibodies were also used to detect the Spike protein on EVs by ELISA (see protocol below).	anti-S2 suggested: None
   S1/S2 and antigen specific IgG ELISA: SARS-CoV-2 specific antibody titers of sera were determined by ELISA.	antigen specific IgG suggested: None
   This was followed by 3 washes with 200 μl of 1X PBS per well and incubation with 100 μl per well of secondary donkey anti-mouse HRP conjugated antibody diluted 1:10,000 in 3% BSA in 1X PBS.	anti-mouse HRP suggested: None
   Saturation was performed with 3% BSA in 1X PBS and followed by incubation with dilutions of either anti-S1 or S2 antibodies.	anti-S1 suggested: None
   Secondary HRP-conjugated donkey anti-rabbit and anti-mouse antibodies were used, respectively.	anti-rabbit suggested: None anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   HEK293T cells were routinely tested and found negative by MycoAlert™ mycoplasma detection kit (Lonza Nottingham, Ltd.)	HEK293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mice and study design: Female BALB/cAnNCrl mice, 6 weeks old, were purchased from Charles River-Italy and placed into four groups of n=6.	BALB/cAnNCrl suggested: RRID:IMSR_CRL:028)
   Software and Algorithms
   Sentences	Resources
   Luc.R-E-luciferase reporter vector from NIH-AIDS Reagent Program [24] and exchanged the luciferase gene by the Nano Luciferase gene (named hereafter pNL4-3.	NIH-AIDS Reagent Program suggested: None
   Protein sequence alignment: Multiple sequence alignment was performed with Clustal Omega tool (EMBL-EBI).	Clustal Omega suggested: (Clustal Omega, RRID:SCR_001591)

   ---

   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: We detected the following sentences addressing limitations in the study:

   Despite the fact that they are well characterized, it is clear now that one of their major limitations is the natural, pre-existing immunity against these vectors [33, 34]. Likely, all primarily vaccinated hosts will mount an anti-adenovirus immune response that will hamper a strong anti-SARS-CoV-2 response after a second injection. As a consequence, adenoviral vectors from other species or with a lower seroprevalence in humans must be explored as vaccine carriers [30, 32, 35]. One of the major concerns with emerging viruses is the threat of re-infections in the general population lacking a long-lasting immunity contributing to the virus becoming endemic, which in turn may be linked with increased mutational-rate, as in the case of influenza virus, necessitating yearly vaccination campaigns. Due to the high immunogenicity of viral vectors themselves multiple immunizations with such platforms could prove challenging, if not inefficient. EV antigenic display can overcome this problem. Moreover, it has been demonstrated that an antigen embedded in the membrane of EVs is more immunogenic and triggers a higher humoral response in vivo when compared with human (Ad5) and non-human (ChAdOx1) adenoviral vectors, both proposed as vaccine platforms against SARS-CoV-2 [36]. The immune responses observed upon CoVEVax administration in vivo are directed against both S1 an S2 parts of the Spike protein, which is important for a broad coverage of the vaccine in regard to the high conservatio...

   ---

   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: We did not find any issues relating to colormaps.

   ---

   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.

   ---

   Read the original source
2. 

   Version published to 10.1101/2020.10.28.357137v1 on bioRxiv

   Oct 28, 2020