Assessment of immunogenicity and protective efficacy of ZyCoV-D DNA vaccine candidates in Rhesus macaques against SARS-CoV-2 infection

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   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2021.02.02.429480: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Ethical review: The study was approved by the Institutional Project Review Committee, and Institutional Biosafety Committee, ICMR-National Institute of Virology (NIV), Pune.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	A placebo group of 4 animals (2 male and 2 female) was also included in the study (Fig. 8).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Detection of anti-SARS-CoV-2 IgG antibodies using Spike-protein based-ELISA: Ninety-six well microtiter plates were coated with 100 µL/well of diluted S1 protein (1µg/ml with Phosphate buffer saline) incubated for …

   SciScore for 10.1101/2021.02.02.429480: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Ethical review: The study was approved by the Institutional Project Review Committee, and Institutional Biosafety Committee, ICMR-National Institute of Virology (NIV), Pune.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	A placebo group of 4 animals (2 male and 2 female) was also included in the study (Fig. 8).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Detection of anti-SARS-CoV-2 IgG antibodies using Spike-protein based-ELISA: Ninety-six well microtiter plates were coated with 100 µL/well of diluted S1 protein (1µg/ml with Phosphate buffer saline) incubated for 16-18 hours at 2-8°C.	anti-SARS-CoV-2 IgG suggested: None
   anti-monkey IgG HRP-antibodies 1:5000 (Thermoscientific, USA) were added and incubated for 1 hour at 37°C.	anti-monkey IgG suggested: None
   Immunophenotyping of PBMCs: For surface phenotyping, freshly isolated PBMCs from immunized/ SARS-CoV-2 challenged NHPs adjusted to 1 × 106 cells per test were incubated with anti NHP T/B/NK cocktail and anti CD4 PECF594 monoclonal antibodies for 30 min at 4°C.	anti NHP suggested: (MABTECH Cat# 3465-72-100T, RRID:AB_2888642) anti CD4 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Post an hour of incubation at 37°C, the virus–serum mixtures (0.1ml) were added onto the Vero CCL-81 cell monolayers and incubated at 37°C with 5% CO2for 1 hour.	Vero CCL-81 suggested: None
   Vero CCL-81 cells were grown to confluent monolayer in 24-well plate maintained in MEM supplemented with 10 %FBS (HiMedia, Mumbai), penicillin (100 U/ml) and streptomycin (100 mg/ml).	CCL-81 suggested: None
   Software and Algorithms
   Sentences	Resources
   PRNT50 titer was calculated using a log probit regression analysis by SPSS (SPSS Inc., Chicago, IL).	SPSS suggested: (SPSS, RRID:SCR_002865)
   Cytokine levels were measured on a BD FACSCaliburTM flow cytometry (BD Biosciences) using BD CellQuestTM Pro software.	BD CellQuestTM Pro suggested: None
   Data analysis: Clinical, virological, serological and immunological data were analysed using GraphPad Prism software version 8.4.3 (GraphPad, San Diego, California).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   A limitation of our study is that the control animals were followed only till 7 days. Viral clearance was observed from ZyCoV-D vaccinated macaques starting from week 1 and complete clearance was observed by second weeks (31). There was significant increase in the percent lymphocytes and IL-8 in group IV suggestive of the vaccine induced host immune response/lymphoproliferation to infection. The present study indicated the transient decrease in the % CD8+ T cell population on 1 DPI and 3 DPI. By now, it is well established that lymphopenia due to SARS-CoV-2 infection is biased towards CD8+ T cells. A recent study on rhesus macaque post challenge with SARS-CoV-2 demonstrated neutropenia, which could be associated with comparatively low IL-8 level in the control group (19). Our study demonstrated an elevated level of serum IL-5, a Th2 cytokine is primarily associated with eosinophilia in group II and IV, which subsided by 11 DPI. There is compelling experimental evidence that eosinophils have potential antiviral activity. Other DNA vaccine candidates like GX-19 produced a Th1 cell response with TNF-α, IFN-γ & IL-2 in vaccinated macaques whereas INO-4800 induced only IFN-γ (26, 27). In conclusion, the ZyCoV-D vaccine candidate, 2mg by NFIS elicited significant SARS-CoV-2 specific IgG, NAbtiters and lesser viral loads in animals post challenge demonstrating its protective efficacy.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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.

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2. 

   Version published to 10.1101/2021.02.02.429480v1 on bioRxiv

   Feb 3, 2021