This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research Article
SARS-CoV-2 candidate vaccine ChAdOx1 nCoV-19 infection of human cell lines reveals a normal low range of viral backbone gene expression alongside very high levels of SARS-CoV-2 S glycoprotein expression.
David A. Matthews
Universty of Bristol
Corresponding Author
ORCiD: https://orcid.org/0000-0003-4611-8795
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Declarations:
Declarations
- The author has confirmed that a statement listing potential conflicts of interest or lack thereof is included in the text.
View Research Highlight
Research Highlight
created by Research Square
The COVID-19 pandemic, caused by the virus SARS-CoV-2, has driven a global effort to develop a vaccine. This study describes the characteristics of a vaccine candidate, ChAdOx1. ChAdOx1 is created by inserting SARS-CoV-2 genes into the DNA of an adenovirus, a common type of virus that does not typically cause illness in humans. Large quantities of the viral vaccine are grown in a human kidney cell line, and the vaccine can then be administered to people to generate an immune response (without causing illness) before they are infected by SARS-CoV-2. One issue with adenovirus vaccines is that because humans interact with adenoviruses often, the immune system may react to cells expressing the adenovirus’ genes before genes from the virus of interest (in this case, SARS-CoV-2) can drive an immune response. In this study, the researchers aimed to test whether ChAdOx1 produced enough of a critical viral protein (SARS-CoV-2 S) to generate an effective immune response, and they also tested the quantity of adenovirus genes expressed, as too much could prevent an anti-SARS-CoV-2 immune response. Using two different cell lines to test the vaccine, they found that ChAdOx1 led to high quantities of the SARS-CoV-2 S protein and minimal expression of adenoviral genes. This suggests that the vaccine has the potential to promote an immune response against SARS-CoV-2.
View Research Highlight
Background: ChAdOx1 nCoV-19 is a recombinant adenovirus vaccine candidate against SARS-CoV-2. Although replication defective in normal cells, 28kbp of adenovirus genes are delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene.
Methods: We used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine candidate.
Results: The expected SARS-CoV-2 S coding transcript dominated in all cell lines. We also detected rare S transcripts with aberrant splice patterns or polyadenylation site usage. Adenovirus vector transcripts were almost absent in MRC-5 cells but in A549 cells there was a broader repertoire of adenoviral gene expression at very low levels. Proteomically, in addition to S glycoprotein, we detected multiple adenovirus proteins in A549 cells compared to just one in MRC5 cells.
Conclusions: Overall the ChAdOx1 nCoV-19 vaccine’s transcriptomic and proteomic repertoire is as expected. The combined transcriptomic and proteomics approaches provide an unparalleled insight into the behaviour of this important class of vaccine candidate and illustrate the potential of this technique to inform future viral vaccine vector design.
Keywords
ChAdOx1 nCoV-19, adenovirus, SARS-CoV-2, vaccine, transcriptome, proteome
Figure 1
Figure 2
Figure 3
Due to technical limitations, tables 1-4 are only available as a download in the supplemental files section.
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1chadox293features.txt
List of features on the viral genome
- Additionalfile2Hsapiens.GRCh38.cdna.fasta
Human transcript list wiht modified headers that contain ENSG ENST and ENSP data
- Additionalfile3proteinssearchlist.fasta
Finalised protein search list
- Additionalfile4chadoxMRC5characterisedtranscripts.xlsx
Description of transcripts found mapped to the vacine genome in MRC5 cells
- Additionalfile5chadoxA549characterisedtranscripts.xlsx
Description of transcripts found mapped to the vacine genome in A549 cells
- Additionalfile6chadox293characterisedtranscripts.xlsx
Description of transcripts found mapped to the vacine genome in 293 cells
- Additionalfile7A549Chadox1Totalproteinsidentified.xlsx
Protiens identified by MS/MS in A549 cells infected with ChAdOx1 nCoV19
- Additionalfile8A549Chadox1PhosphoProteins.xlsx
PhosphoProtiens identified by MS/MS in A549 cells infected with ChAdOx1 nCoV19
- Additionalfile9MRC5Chadox1Totalproteinsidentified.xlsx
Protiens identified by MS/MS in MRC5 cells infected with ChAdOx1 nCoV19
- Additionalfile10MRC5Chadox1PhosphoProteins.xlsx
PhosphoProtiens identified by MS/MS in A549 cells infected with ChAdOx1 nCoV19
- Additionalfile11A549Chadox1TotalPSMsidentified.xlsx
PSMs identified by MS/MS in A549 cells infected with ChAdOx1 nCoV19
- Additionalfile12A549Chadox1PhosphopeptidesPSMs.xlsx
Phospho peptide PSMs identified by MS/MS in A549 cells infected with ChAdOx1 nCoV19
- Additionalfile13MRC5Chadox1TotalPSMsidentified.xlsx
PSMs identified by MS/MS in MRC5 cells infected with ChAdOx1 nCoV19
- Additionalfile14MRC5Chadox1PhosphopeptidesPSMs.xlsx
Phospho peptide PSMs identified by MS/MS in MRC5 cells infected with ChAdOx1 nCoV19
- Additionalfile15A549transcriptsandproteomics.xlsx
Transcriptomic and proteomics data for A549 in one table
- Additionalfile16MRC5transcriptsandproteomics.xlsx
Transcriptomic and proteomics data for MRC5 in one table
- Tables.docx
2 comments flagged for moderation
If you posted a comment and think it was mistakenly removed, please contact customer support at [email protected].
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 20 Oct, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Learn more about our company.
This is a preprint. It has not completed peer review.
Research Article
SARS-CoV-2 candidate vaccine ChAdOx1 nCoV-19 infection of human cell lines reveals a normal low range of viral backbone gene expression alongside very high levels of SARS-CoV-2 S glycoprotein expression.
David A. Matthews
Universty of Bristol
Corresponding Author
ORCiD: https://orcid.org/0000-0003-4611-8795
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 20 Oct, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Declarations:
Declarations
- The author has confirmed that a statement listing potential conflicts of interest or lack thereof is included in the text.
View Research Highlight
Research Highlight
created by Research Square
The COVID-19 pandemic, caused by the virus SARS-CoV-2, has driven a global effort to develop a vaccine. This study describes the characteristics of a vaccine candidate, ChAdOx1. ChAdOx1 is created by inserting SARS-CoV-2 genes into the DNA of an adenovirus, a common type of virus that does not typically cause illness in humans. Large quantities of the viral vaccine are grown in a human kidney cell line, and the vaccine can then be administered to people to generate an immune response (without causing illness) before they are infected by SARS-CoV-2. One issue with adenovirus vaccines is that because humans interact with adenoviruses often, the immune system may react to cells expressing the adenovirus’ genes before genes from the virus of interest (in this case, SARS-CoV-2) can drive an immune response. In this study, the researchers aimed to test whether ChAdOx1 produced enough of a critical viral protein (SARS-CoV-2 S) to generate an effective immune response, and they also tested the quantity of adenovirus genes expressed, as too much could prevent an anti-SARS-CoV-2 immune response. Using two different cell lines to test the vaccine, they found that ChAdOx1 led to high quantities of the SARS-CoV-2 S protein and minimal expression of adenoviral genes. This suggests that the vaccine has the potential to promote an immune response against SARS-CoV-2.
View Research Highlight
Background: ChAdOx1 nCoV-19 is a recombinant adenovirus vaccine candidate against SARS-CoV-2. Although replication defective in normal cells, 28kbp of adenovirus genes are delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene.
Methods: We used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine candidate.
Results: The expected SARS-CoV-2 S coding transcript dominated in all cell lines. We also detected rare S transcripts with aberrant splice patterns or polyadenylation site usage. Adenovirus vector transcripts were almost absent in MRC-5 cells but in A549 cells there was a broader repertoire of adenoviral gene expression at very low levels. Proteomically, in addition to S glycoprotein, we detected multiple adenovirus proteins in A549 cells compared to just one in MRC5 cells.
Conclusions: Overall the ChAdOx1 nCoV-19 vaccine’s transcriptomic and proteomic repertoire is as expected. The combined transcriptomic and proteomics approaches provide an unparalleled insight into the behaviour of this important class of vaccine candidate and illustrate the potential of this technique to inform future viral vaccine vector design.
Figure 1
Figure 2
Figure 3
Due to technical limitations, tables 1-4 are only available as a download in the supplemental files section.