SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications
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Abstract
The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications.
Objectives
In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful tools for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity.
Methods
Five antibody candidates were selected out of a naïve camelid library by phage display and expressed as full length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron).
Results
All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50).
Conclusion
Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy.
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SciScore for 10.1101/2022.03.24.485614: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Heavy chain Ab design, expression and purification: For the generation of a full length heavy-chain-only antibody, the different VHHs were amplified with SL13/14 and cloned into the vector pNEM_camhAb. pNEM_camAb was designed in our lab as vector for eukaryotic protein expression with a camelid Fc fragment (CH2-CH3 domain) resulting in hcAbs. CH2-CH3 domainsuggested: NoneWestern Blot analyses and ELISA: To investigate the different hcAb candidates in Western Blot, 1 μg SARS-CoV-2 Spike … SciScore for 10.1101/2022.03.24.485614: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Heavy chain Ab design, expression and purification: For the generation of a full length heavy-chain-only antibody, the different VHHs were amplified with SL13/14 and cloned into the vector pNEM_camhAb. pNEM_camAb was designed in our lab as vector for eukaryotic protein expression with a camelid Fc fragment (CH2-CH3 domain) resulting in hcAbs. CH2-CH3 domainsuggested: NoneWestern Blot analyses and ELISA: To investigate the different hcAb candidates in Western Blot, 1 μg SARS-CoV-2 Spike protein (antibodies-online, ABIN6952734) per lane was applied onto a 4-12% SDS polyacrylamide gradient gel. antibodies-online , ABIN6952734suggested: NoneAfter washing the membrane, a horseradish peroxidase (HRP) conjugated secondary antibody (ABIN1981272, antibodies-online) was applied and incubated for 3 h at RT. antibodies-onlinesuggested: NoneExperimental Models: Cell Lines Sentences Resources In specific, VSV*ΔG either bearing the SARS-CoV-2 (D614G) or SARS-CoV-2 B.1.1.529 (Omicron) Spike protein was incubated with a two-fold dilution of hcAbs and subsequently used to inoculated VeroE6 cells. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Expi293 cells were grown at 37°C, 8% CO2 with 130-150 rpm on a Rotamax120 platform shaker ( Expi293suggested: RRID:CVCL_D615)Recombinant DNA Sentences Resources Amplified VHHs were cloned into phagemid vector pADL-22c (Antibody Design Labs). pADL-22csuggested: NonePositive clones were sequenced and become heavy chain only antibodies by cloning into the expression plasmid pNEM_camAb. pNEM_camAbsuggested: NoneThe expression vector for the Omicron spike (based on isolate hCoV19/Botswana/R40B58_BHP_3321001245/2021; GISAID Accession ID: EPI_ISL_6640919) was generated by Gibson assembly using five overlapping DNA strings (Thermo Fisher Scientific, sequences available upon request), linearized (BamHI/XbaI digest) pCG1 plasmid and GeneArt™ Gibson Assembly HiFi Master Mix (Thermo Fisher Scientific). pCG1suggested: NoneVLPs): Human codon optimized sequences of genes encoding the S and E structural proteins of SARS-CoV-2 were synthesized by BioCat GmbH (Heidelberg, Germany) and subcloned into the pcDNA3.1 expression plasmid using the NheI 5’ and XhoI 3’ restriction site, respectively. pcDNA3.1suggested: RRID:Addgene_79663)Another plasmid (pEXP-M+N) for the dual expression of the human codon optimized sequences of the M and N protein was generated by Vectorbuilder Inc. (VB200528-1033wpt). pEXP-M+Nsuggested: NoneThe DNA mix was composed of pcDNA3.1-Spike (FKO), pcDNA3.1-E-Protein and pEXP-M+N-protein at a ratio of 6:2:3 diluted in Opti-MEM I Medium. pcDNA3.1-Spikesuggested: NonepcDNA3.1-E-Proteinsuggested: NonepEXP-M+N-proteinsuggested: NoneSoftware and Algorithms Sentences Resources VLPs): Human codon optimized sequences of genes encoding the S and E structural proteins of SARS-CoV-2 were synthesized by BioCat GmbH (Heidelberg, Germany) and subcloned into the pcDNA3.1 expression plasmid using the NheI 5’ and XhoI 3’ restriction site, respectively. BioCatsuggested: (BioCAT, RRID:SCR_001440)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: 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.
- No funding statement was detected.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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