Structure-selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern
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Abstract
Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to affect the quality of the antibody response focusing it to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and three variants of concern (VOC), B.1.351, B.1.617.2, and BA.1. We demonstrate that immunogens built on structure selection can be used to influence the quality of the antibody response by focusing it to a conserved site of vulnerability shared between wildtype virus and VOCs, resulting in neutralizing antibodies across variants.
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SciScore for 10.1101/2021.10.01.462840: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Procedures were per protocol approved by the Institutional Animal Care and Use Committee (IACUC) and in compliance with Association for Assessment Accreditation of Laboratory Animal Care (AAALAC) International guidelines.
Field Sample Permit: SARS-CoV-2 viruses: All work with SARS-CoV-2 was conducted in Biosafety Level-3 conditions at the University of California San Diego following the guidelines approved by the Institutional Biosafety Committee.Sex as a biological variable Mice and immunizations: Twelve-week-old female C57Bl/6 (H-2b) were bred at the University of California, San Diego animal facility where they were kept throughout the performance of the experiment. Randomization not … SciScore for 10.1101/2021.10.01.462840: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Procedures were per protocol approved by the Institutional Animal Care and Use Committee (IACUC) and in compliance with Association for Assessment Accreditation of Laboratory Animal Care (AAALAC) International guidelines.
Field Sample Permit: SARS-CoV-2 viruses: All work with SARS-CoV-2 was conducted in Biosafety Level-3 conditions at the University of California San Diego following the guidelines approved by the Institutional Biosafety Committee.Sex as a biological variable Mice and immunizations: Twelve-week-old female C57Bl/6 (H-2b) were bred at the University of California, San Diego animal facility where they were kept throughout the performance of the experiment. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Human monoclonal antibodies B38, CC12.1 and CC6.30 have been described previously (Rogers et al., 2020; Wu et al., 2020). CC6.30suggested: NoneAfter 1 day incubation (37 °C) cells were fixed with 4% formaldehyde and stained with antibody against nucleocapsid protein. antibody against nucleocapsid protein .suggested: NoneExperimental Models: Cell Lines Sentences Resources Briefly, ~100 focus forming units (ffu) of SARS-CoV-2 were incubated with or without serially diluted antibodies for 1 hour in DMEM with 1% FBS at 37 °C before adding to 100% confluent TMPRSS2-Vero cell monolayers in 96 well plates. TMPRSS2-Verosuggested: NoneExperimental Models: Organisms/Strains Sentences Resources Mice and immunizations: Twelve-week-old female C57Bl/6 (H-2b) were bred at the University of California, San Diego animal facility where they were kept throughout the performance of the experiment. C57Bl/6suggested: NoneRecombinant DNA Sentences Resources The ZUC1.1 plasmid lacks Amp resistance gene and SV40 sequences, and is optimized for human use. ZUC1.1suggested: NoneSoftware and Algorithms Sentences Resources One week later, supernatants were clarified by centrifugation, BioLock was added, passaging through a 0.22 μuM sterile filter, and proteins were first purified on an ÄKTA go system (Cytivia) using a 5mL StrepTrap-HP column equilibrated with TBS buffer (25mM Tris pH 7.6, 200mM NaCl, 0.02% NaN33) and eluted using TBS buffer supplemented with 5mM d-dDesthiobiotin (Sigma Aldrich). BioLocksuggested: NoneBest fit curves generated in PRISM 9 determined the serum dilution which achieved 50% focus reduction (FRNT50). PRISMsuggested: (PRISM, RRID:SCR_005375)Quantification of data and statistical analysis: Data plotting and statistical analyses were done using GraphPad Prism version 7.0. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)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:Our study has limitations. First, the sample size was deliberately low since the study was designed to identify (a) pDNA with ability to prime RBM-specific B cells that could be re-expanded during a recall response, and (b) yield consistent virus neutralization. In spite of this limitation we managed to identify the immunogen yielding the overall more consistent response. The second is that the dose of Spike protein used in the booster immunization was probably excessive as this protein is highly immunogenic. This may have masked the true potential of the memory response. Future experiments will need to assess memory responses against booster immunization with lower doses of antigen. Finally, although the study was successful in identifying an optimal prime-boost combination resulting in neutralizing antibodies effective against wildtype virus and B.1.351 and B.1.617.2 variants, these results cannot be extrapolated to predict transmission inhibition in vivo. Control of the COVID-19 pandemic rests on an effective immunological intervention to curb the spread of infection by vaccination to induce durable transmission-blocking immunity. The initial evidence gathered in this study suggests that a pDNA-protein (prime-boost) approach was successful in focusing the antibody response to a narrow site of the RBM ridgeline that overlaps with the RBM supersite recently described (Wang et al., 2021a) and proved effective against wildtype virus and two VOCs. This suggests that binding and...
Results from TrialIdentifier: No clinical trial numbers were referenced.
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).
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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