Taylor Makela Journal Week 11

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Taylor Makela Week 11

Purpose

The purpose of this assignment was to assess our selected article--Isolation of Potent SARS-CoV-2 Neutralizing Antibodies and Protection from Disease in a Small Animal Model--in terms of accuracy, availability, and reliability. Since we are using this paper as the basis of our final research project, it is important to evaluate the journal, publisher, authors, and of course the article itself. This assignment also served as a means for us to begin to look deeper at our selected article and learn more about SARS-CoV-2 neutralizing antibodies in small animal models.

Electronic Lab Notebook

Searching the Scientific Literature Part 2: Evaluating Scientific Sources

  1. We began to evaluate our assigned article in three areas: availability, the journal, and the article metadata. Again, a citation for the article in APA format is provided, this time including the DOI.
    • Rogers, T. F., Zhao, F., Huang, D., Beutler, N., Burns, A., He, W. T., ... & Yang, L. (2020). Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science, 369(6506), 956-963. DOI: 10.1126/science.abc7520
    1. Provide a link to the abstract of the article on PubMed
    2. Provide a link to the full text of the article in PubMed Central
    3. Provide a link to the full text of the article (HTML format) from the publisher website.
    4. Provide a link to the full PDF version of the article from the publisher website.
    5. Who owns the rights to the article? Look at the first page of the PDF version of the article for the © symbol. Generally, either the journal/publisher or the authors will hold the copyright.
      • The PDF states that the rights to the article are owned by the authors, and the exclusive licensee is under the American Association for the Advancements of Science
    6. How is the article available to you:
      • Is the article available “open access” (look for the words “open access” or the “unlocked” icon on the article website or the first page of the PDF) If YES, stop here.
        • The article website states that "this is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited."
    7. Is the article available online-only or both in print and online? Look on the journal website for a “subscription” link. If that page talks about subscribing to the print edition, then it is available in print. If not, it is available online-only.
      • I did not see a "subscription" link for our specific article, however, the journal that the article is published in is Science Magazine, which does have subscription options for their different magazines.
  2. Evaluating the source--the journal
    1. Who is the publisher of the journal?
      • The article was published by the American Association for the Advancements of Science
    2. Is the publisher for-profit or non-profit?
      • The American Association for the Advancements of Science is a non-profit organization.
    3. Is the publisher a scientific society (some scientific societies partner with a for-profit publisher, some act as their own non-profit publisher)
      • Yes, the publisher is a scientific society -- the American Association for the Advancements of Science.
    4. Does the publisher belong to the Open Access Publishers Association?
      • Yes, the American Association for the Advancements of Science belongs to the Open Access Publishers Association, and is considered a "Professional Publisher (Large)" on their website.
    5. What country is the journal published in?
      • The journal is published in America.
    6. How long has the journal been in operation? (e.g., browse the archive for the earliest article published)
      • The journal has been in operation since 1880!
    7. Are articles in this journal peer-reviewed?
      • Yes, the articles in Science are peer-reviewed.
    8. Provide a link to the scientific advisory board/editorial board of the journal.
    9. What is the journal impact factor (look to see if it is provided on the journal home page; often you can also find it through a Google search)?
      • According to Web of Science, Science had a 2019 impact factor of 41.846 and five-year impact factor of 44.374
  3. Evaluating the source--the article
    1. Is the article a review or primary research article?
      • The article is a primary research article.
    2. On what date was the article submitted?
      • The article was submitted on May 12, 2020.
    3. On what date was the article accepted?
      • The article was accepted on June 11, 2020.
    4. Did the article undergo any revisions before acceptance?
      • There were no additional revisions listed before acceptance.
    5. When was the article published?
      • The article was published on June 15, 2020.
    6. What is the approximate elapsed time between submission and publication?
      • There was approximately a year and one month between submission and publication of this article.
    7. What are the institutions with which the authors are affiliated?
      • According to PubMed, the authors are affiliated with the following institutions:
        • Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA.
        • Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, CA 92037, USA.
        • IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA.
        • Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, CA 92037, USA.
        • IAVI, New York, NY 10004, USA.
        • Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA 92037, USA.
        • Center for Viral Systems Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
        • Department of Pathology, George Washington University, Washington, DC 20052, USA.
        • Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
        • Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA. dsok@iavi.org jjardine@iavi.org burton@scripps.edu.
        • IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA. dsok@iavi.org jjardine@iavi.org burton@scripps.edu.
        • Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, MA 02139, USA.
    8. Have the authors published other articles on this subject? (How will you find this out?)
      • Upon doing an author search on PubMed, I discovered that Rogers published several other articles relating to SARS-CoV-2 animal models and antibodies, and Zhao also published several other articles regarding SARS-CoV-2 antibodies and patients.
    9. Is there a conflict of interest for any of the authors?
      • In the acknowledgments section of the article it is noted that there are competing interests for "D.R.B., D.H., J.G.J., E.L., T.F.R., D.S., and F.Z., all of which are listed as inventors on pending patent applications describing the SARS-CoV-2 antibodies."
    10. Make a recommendation--based just on the information you have gathered so far, is this a good article to evaluate further? Why or why not?
      • Based on the information gathered thus far, this appears to be a great article to evaluate further. The article was published by a reputable publisher which belongs to a scientific society, and the journal in which the article is published is a very well established journal that has been around for over 100 years. Additionally, the article has been peer-reviewed and since the authors have published articles on similar subjects.

Preparation for Journal Club 2

The papers assigned for Journal Club 2:

Biological terms I did not know the definitions of when I first read our journal club article:

  1. Prophylaxis: The prevention of disease, preventative treatment
  2. Cohort: A group of animals of the same species identified by a common characteristic, which are studied over a period of time as part of a scientific or medical investigation.
  3. Titer: An empirical measure of avidity of an antibody
  4. Simpson's Rule: Used in statistics to confirm data resulting from the combination of unequal size of group that has been pooled into a single data set
  5. Endemic: Native to a particular area or region or an exclusive characteristic of a thing, place, or concept
  6. Epitope: The part of an antigenic molecule to which the T cell receptor responds, a site on a large molecule against which an antibody will be produced and to which it will bind
  7. Monoclonal: Used of a cell line whether within the body or in culture to indicate that it has a single clonal origin
  8. Intraperitoneal: Within the peritoneal cavity, the area that contains the abdominal organs
  9. Heterologous: Of, or relating to, tissues or cytologic elements not normally found parts of the body of an individual, or that are derived from a different species
  10. Tropism: A movement or growth response of a cell or an organism to a stimulus, which may either be positive or negative depending on the source and kind of stimulation

Each of the terms were defined using an online biological dictionary. My sources for the definitions were cited by providing the proper citation (for a book) or the URL to the page with the definition for online sources. Each definition must have it's own citation, to a book or URL. I made an in text citation of the (name, year) format next to the definition, and then listed the full citation in the References section of my journal page.

Article Outline

Abstract and Introduction
  • The prevention and treatment of COVID-19 is a health priority across the world
  • Enrolled a cohort of SARS-CoV-2, recovered participants, developed assays to examine antibody response, screened more than 1800 antibodies, and determined an animal model
  • Isolated nAbs to two epitopes on the RBD and to non-RBD epitopes on the spike protein
  • Passive transfer of an nAb provides Syrian hamsters with protection against disease
  • This research suggests that nAbs potentially play a role in the prophylaxis and therapy of COVID-19
    • The nAbs also provide the protective epitopes that can help design a vaccine
    • An nAb for another virus, respiratory syncytial virus, is used clinically to protect susceptible infants
    • nAbs also prevent death from the Ebola virus, which is why nAbs have been suggested for use in outbreaks
  • nAbs with immense potency (aka super-antibiotics) are isolated by mining antibody response from a collection of infected donors
  • This article presents a manor of isolating highly potent nAbs to SARS-CoV-2 and how their "in vivo efficacy" in a Syrian hamster model suggests their potential clinical use
  • Plasma and peripheral blood mononuclear cells (PBMCs) were collected from a cohort of donors who previously tested positive for COVID-19
  • Developed pseudo virus and live replicating assays using angiotensin-converting enzyme 2
  • Evaluated convalescent serum responses for neutralization activity against SARS-CoV-1 and SARS-CoV-2
  • Eight donors were then selected
  • Corresponding variable genes to single antigen-specific memory B cells were sorted, recovered, and cloned
Development of Viral Neutralization Assays
  • To evaluate plasma neutralization activity against SARS-CoV-2, a replication-competent virus and a pseudo-virus (PSV) platform were created
  • Began by using vero-E6 cells as target cells for neutralization assays, however, HeLa cell lines proved to better express the ACE2 receptor, and was chosen as the better target cell
  • The live replicating virus assay used the following strain of SARS-CoV-2: USA-WA1/2020
  • The PSV assay used murine leukemia virus-based PSV (MLV-PSV) and was established for both SARS-CoV-1 and SARS-CoV-2
  • The assays used a single-cycle infectious viral particles with a firefly luciferase reporter for high-throughput testing
  • Since coronaviruses assemble in the endoplasmic reticulum - Golgi intermediate compartment, the C terminus of the spike protein for SARS-CoV-1 has an endoplasmic reticulum retrieval signal
    • SARS-CoV-2 also has this retrieval signal, which is shown in the alignment of the SARS-CoV-1 and SARS-CoV-2 spike proteins
  • To improve the exocytosis of the virus, various shortened SARS-CoV-1 and SARS-CoV-2 spike proteins with a removed ER retrieval signal were expressed
Establishment of a SARS-CoV-2 Cohort
  • A cohort of 17 donors in San Diego, California who were previously infected with SARS-CoV-2 was established
    • Cohort was 47% male, with an average age of 50
  • Infection was confirmed through a positive PCR via a nasopharyngeal swab
  • All participants had COVID-19 symptoms, ranging in severity from mild to severe
    • All participants recovered
  • The donors plasma was tested for SARS-CoV-1 and SARS-CoV-2 RBD and spike proteins, binding to cell surface expressed spikes, and for neutralization in both types of assays (PSV and live replicating virus)
  • The binding concentration of antibodies for SARS-CoV-2 spike protein varied widely
    • The binding concentration of antibodies for the RBD was around an order of magnitude less
  • The concentration of antibodies for SARS-CoV-1 spike protein were significantly less than those for SARS-CoV-2 spike protein, and concentration of antibodies for SARS-CoV-1 RBD were only found in a small number of donors
  • The neutralizing concentration of antibodies in the PSV assay also varied widely for the SARS-CoV-2 spike protein, and were low for SARS-CoV-1
  • There was a correlation between RBD binding and PSV neutralization
  • There was a positive correlation between live replicating virus neutralization and cell surface spike binding
  • The concentrations of antibodies in the replicating virus and PSV assays were very similar
Antibody Isolation and Preliminary Function Screens for Down-Selection
  • Before single-cell sorting, cryopreserved PBMCs from 8 donors were stained for memory B cell markers, SARS-CoV-2 antigen baits, and AviTag biotinylated RBD
  • 3,160 antigen-positive memory B cells were sorted to recover heavy and light chain pairs for mAb production
  • 2,045 antibodies were cloned and expressed, yielding 65% PCR recovery of paired variable genes and >86% recovery of fully functional cloned genes
  • Bulk-transformed ligation products for the heavy and light chains were transfected tested for binding to spike protein and RBD, as well as for neutralization in the SARS-CoV-2 PSV assay with HeLA-ACE2 target cells
  • 92% of transfected pairs expressed IgG
    • 43% of these bound only to the spike protein
    • 0.1% bound only to RBD
    • 5.9% bound to both the spike protein and RBD
  • To eliminate non-specific or polyreactive supernatants, the supernatants were also tested for binding to an HIV antigen
  • Supernatants were tested for neutralization activity using SARS-CoV-2 and SARS-CoV-1 pseudoviruses
  • Small fraction of the binding antibodies showed neutralization activity
    • Activity was equally distributed between RBD+/S+ and S+ only binders
      • Shown in donors CC6, CC12, and CC25

Data indicates that the infection generates a strong response against the non-RBD region of the spike protein, but only a small fraction of that response is neutralizing

  • There are fewer RBD-binding antibodies, however, a larger fraction of these antibodies neutralize the SARS-CoV-2 pseudo-virus
  • The antibodies that tested positive for neutralization were then confirmed through sequence and were then advanced for additional characterization
    • 33 antibodies were selected for further characterization from donors CC^, CC12, and CC25
      • 25 distinct lineages were identified, 23 of which contained a single member
  • Vh1 and VH3 gene families were prominent in these antibodies, but there was diversity in CDR3 lengths
Functional Activity of Down-Selected Antibodies
  • The identified antibodies were next evaluated for epitope specificity using bilayer interferometry with spike and RBD proteins as capture antigens
  • Only antibodies that bind to a noncompeting will be detected in the assay
  • The data revealed 3 epitope bins for RBD designated as RBD-A, RBD-B, and RBD-C, as well as 3 epitope bins for the spike protein designated as S-A, S-B, and S-C
  • The mAb CC12.19 appeared to compete with antibodies targeting RBD-B and S-A
  • Other antibodies in the S-A epitope bin competing with CC12.19 either showed binding to either RBD-SD1 and RBD-SD1-2 but not RBD, or showed no binding to RBD or RBD-SD constructs
  • This data suggests that there are 2 competing epitopes with the S-A bin
    • One is confined to the non-RBD region of the spike protein
    • The other includes some element of RBD-SD1-2
  • Next, the mAbs were evaluated for neutralization activity for SARS-CoV-2 and SARS-CoV-1 pseudo-viruses
  • The strongest neutralizing antibodies were CC6.29 and CC6.30, both of which are directed to the RBD-A epitope
    • Neutralize SARS-CoV-2 pseudo=virus with an IC50 of 2 ng/ml and 1 ng/ml, respectively
  • Antibodies directed to the RBD-B epitope typically had a higher IC50 and most plateaued under 100% neutralization
  • CC6.33 was the only antibody that had high neutralization for both pseudo-viruses
  • Antibodies that do not bind to RBD showed low potency for neutralization
  • Next performed cell surface competition experiments to determine whether RBD-A epitope could reach the ACE2 binding site
  • Antibodies were premixed with RBD proteins or biotinylated spike proteins to target antigen
  • Mixture was incubated with HeLa-ACE2 and percent competition against ACE2 was recorded
  • Antibodies targeting RBD-A competed best against ACE2
  • Poor correlation between neutralization potency and affinity
    • Correlation is higher when only looking at antibodies targeting RBD-A
  • Data suggests that RBD-A is the preferred target for evoking neutralizing antibodies, and that affinity for RBD-A and mAbs will presumably result in an increase in neutralization potency
  • Investigated the activity of 5 nAbs for 6 viral variants
    • CC6, CC12, and CC25 neutralized all variants
Passive Transfer of Neutralizing Antibodies and SARS-CoV-2 Challenge in Syrian Hamsters
  • Two mAbs were selected for passive transfer in a Syrian hamster
  • The first experiment tested nAb CC12.1, which targets RBD-A
  • The second experiment tested nAb C12.23, which targets S-B
  • Antibody to dengue virus, Den3 was used as a control
  • Anti-SARS-CoV-2 nAbs were distributed at 5 different concentrations, starting at 2 mg per animal
  • Collected sera from each animal 12 hours post infusion of the antibody
  • The animals were then given a dose of 1 x 10^6 plaque forming units (PCU) of SARS-CoV-2
  • Hamsters were weighed daily
  • On day 5, lung tissue was collected to measure viral load
  • The control animals, on average, lost 13.6% of their body weight by day 5 of the virus challenge
  • Animals that received the neutralizing RBD-A antibody at a dose of 2 mg (500 μg) had no weight loss
  • Animals that received the neutralizing RBD-A antibody at a dose of 125 μg had an average weight loss of 8%
  • Animals that received the neutralizing RBD-A antibody at a dose of 31 μg/ml had an average weight loss of 15.8%
  • Animals that received the neutralizing RBD-A antibody at a dose of 8 μg/ml had an average weight loss of 16.7%
  • This weight loss trend did not have statistical significance
  • Also measured the antibody serum concentrations right before intranasal virus challenge to determine the antibody serum concentrations needed for protection against SARS-CoV-2
  • The data showed that an antibody serum concentration of ~22 μg/ml of nAb provides full protection
    • An antibody serum concentration of 12 μg/ml is adequate for a 50% reduction in disease
  • Still need to determine the effective antibody concentration required at the site of infection to protect from disease

Discussion

  • Over 1000 mAbs were isolated from three convalescent donors by memory B cell selection using either RBD or SARS-CoV-2 S recombinant proteins
  • Approximately half of the isolated mAbs could be expressed and effectively bind to RBD and/or S proteins
  • Only a small fraction of the Abs were neutralizing
  • A variety of nAbs were isolated to different sites on the spike protein
  • The most potent Abs are targeted to a site that overlaps the ACE2 binding site
  • One of the Abs neutralized SARS-CoV-1 PSV
  • Antibodies that are directed to the RBD but not competitive with soluble ACE2 are generally less potent neutralizers and typically show incomplete neutralization
  • RBD-B is the one exception to this
  • RBD-A nAbs that compete with ACE2 are the best/most preferred for therapeutic and prophylactic applications, as well as reagents to define nAb epitopes for vaccine design
  • Two variants showed notable resistance to individual nAbs to the WA1 strain, suggesting that neutralization resistance needs to be considered when planning for clinical applications of nAbs
  • The efficacy of a potent anti-RBD nAb in vivo in Syrian hamsters was promising and suggests that human studies should be conducted
  • There are still limitations to animal models such as the differences in Fc receptors and effector cells in hamsters and humans
  • For future human studies, the following should be considered: improving the potency of protective nAbs through the enhancement of binding affinity to RBD epitopes, reducing Fc receptor binding as an attempt to minimize potential antibody-dependent enhancement (ADE), and improving half-life
  • The described nAbs have low somatic hypermutation (SHM), with typically one or two mutations in the VH gene and one or two mutations in the VL gene
  • As far as the SARS-CoV-2 vaccine, the results suggest a focus on the RBD
  • Strong nAb response has been demonstrated through the immunization of mice with a multivalent presentation of RBD

Journal Club Presentation

Presentation Slides (PDF)


Scientific Conclusion

This assignment gave me a better understanding of how to properly evaluate a source to be used for scientific research based on reliability, accuracy, and availability. Furthermore, I learned the importance of not only evaluating the article itself, but also the the authors, publisher, and journal. Lastly, this assignment allowed me to review our selected article and prepared me to begin working on our final research project. I look forward to continuing to learn about neutralizing antibodies, small animal models, and SARS-CoV-2 as we work on our project.

Acknowledgments

  • I acknowledge my partners Nida Patel, Anna Horvath, and Aiden Burnett who I met with on zoom for several hours and consulted via a text message group chat regarding syntax, formatting, and content questions.
  • I acknowledge that I paraphrased and referenced our journal club article (Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model), especially within the article outline section of this journal.
  • I acknowledge that I copied and modified the protocol shown on the Week 11 Assignment Page for this course.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source. Taylor Makela (talk) 02:09, 25 January 2021 (PST)

References

Template Links

Assignment Pages

Individual Journal Pages

Class Journal Pages

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