SARS-CoV-2 infected cells present HLA-I peptides from canonical and out-of-frame ORFs

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1. 

   Version published to 10.1101/2020.10.02.324145v2 on bioRxiv

   Oct 26, 2020
2. 

   ScreenIT

   Oct 4, 2020

   SciScore for 10.1101/2020.10.02.324145: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The lysates were then centrifuged at 4,000 rpm for 22min at 4°C and the supernatants were transferred to another set of six eppendorf tubes containing a mixture of pre-washed beads (Millipore Sigma, GE17-0886-01) and 50 ul of an MHC class I antibody (W6/32) (Santa Cruz Biotechnology, sc-32235).	GE17-0886-01 suggested: None 50 ul of an MHC class suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   We generated stable HEK293T and A549 cells …

   SciScore for 10.1101/2020.10.02.324145: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The lysates were then centrifuged at 4,000 rpm for 22min at 4°C and the supernatants were transferred to another set of six eppendorf tubes containing a mixture of pre-washed beads (Millipore Sigma, GE17-0886-01) and 50 ul of an MHC class I antibody (W6/32) (Santa Cruz Biotechnology, sc-32235).	GE17-0886-01 suggested: None 50 ul of an MHC class suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   We generated stable HEK293T and A549 cells expressing human ACE2 and TMPRSS2 by transducing them with lentivirus particles carrying these two cDNAs.	HEK293T suggested: None
   To generate the virus P2 stock, we infected Vero E6 cells with the P1 stock at a multiplicity of infection (MOI) of 0.1 plaque forming units (PFU)/cell and harvested the culture medium three days later using the same protocol as for the P1 stock.	Vero E6 suggested: RRID:CVCL_XD71)
   RNA-Seq: A549 and HEK293T cells were seeded into 6-well plates at a density of 5 x 105 cells per well (one well per condition).	A549 suggested: None
   Software and Algorithms
   Sentences	Resources
   We then added 1 ml per well of the overlay medium containing 2X DMEM (Gibco: #12800017) supplemented with 4% FBS and mixed at a 1:1 ratio with 1.2% Avicel (DuPont; RC-581) to obtain the final concentrations of 2% and 0.6% for FBS and Avicel, respectively.	Gibco suggested: None
   LC-MS/MS data interpretation: Peptide sequences were interpreted from MS/MS spectra using Spectrum Mill (v 7.1 pre-release) to search against a RefSeq-based sequence database containing 41,457 proteins mapped to the human reference genome (hg38) obtained via the UCSC Table Browser (https://genome.ucsc.edu/cgi-bin/hgTables) on June 29, 2018, with the addition of 13 proteins encoded in the human mitochondrial genome, 264 common laboratory contaminant proteins, 553 human non-canonical small open reading frames, 28 SARS-CoV2 proteins obtained from RefSeq derived from the original Wuhan-Hu-1 China isolate NC_045512.2 (https://www.ncbi.nlm.nih.gov/nuccore/1798174254)(Wu et al., 2020), and 23 novel unannotated virus ORFs whose translation is supported by Ribo-seq (Finkel et al., 2020b) for a total of 42,337 proteins.	UCSC Table Browser suggested: None
   RNA sequencing reads alignment: Sequencing reads were mapped to SARS-CoV-2 genome (RefSeq NC_045512.2) and human transcriptome (Gencode v32).	RefSeq suggested: (RefSeq, RRID:SCR_003496)
   Alignment was performed using Bowtie version 1.2.2 (Langmead et al., 2009) with a maximum of two mismatches per read.	Bowtie suggested: (Bowtie, RRID:SCR_005476)
   The raw RNA sequencing data generated in this study have been submitted to the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE159191.	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

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   Results from OddPub: Thank you for sharing your data.

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

   It is plausible that lacking peptides from N with typical binding motifs represents a sensitivity limitation of our method, the differences of antigen processing machinery within our cell lines or the time points post infection, as earlier or later time points may reveal additional HLA-I epitopes. Recent studies in COVID-19 patients indeed detected positive CD8+ T cell responses against pooled peptides from N (Grifoni et al., 2020a; Le Bert et al., 2020). However, T cell reactivity toward N was lower than S, M and nsp6 (accounting for 12%, 26%, 22% and 15% of the reactivity, respectively)(Grifoni et al., 2020a) even though their source protein abundance is at least 10-fold lower (Fig. 2B). Together with these clinical observations, our findings support the notion that N may be less immunogenic than expected based on its high expression level, and that ORF9b and nonstructural proteins should be considered in multiepitope vaccine pools and immune monitoring efforts that currently dominated by highly abundant structural proteins like N and M. Although only nine HLA-I alleles were profiled in our study, we show that these data combined with in silico epitope prediction can rapidly prioritize peptides that cover a wide range of HLA-I alleles. Importantly, two of the HLA-I alleles, A*02:01 and B*07:02, have high frequencies across subpopulations and are prioritized for in-vitro and in-vivo studies (Ferretti et al.; Grifoni et al., 2020a), allowing a direct comparison between our re...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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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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3. 

   Version published to 10.1101/2020.10.02.324145v1 on bioRxiv

   Oct 2, 2020