Anna Horvath Week 11

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Purpose

To understand the relevance of a scientific article about neutralizing antibodies in SARS-CoV-2 in Syrian hamsters. A cohort study type was utilized in order to test the efficacy of these antibodies in preventing the binding of the spike protein to the RBD.

Methods/Results

Searching the Scientific Literature Part 2: Evaluating Scientific Sources

  1. Now we will begin to evaluate your assigned article in three areas availability, the journal, and the article metadata. Again, provide a citation for the article in APA format, this time including the DOI.
    • Rogers TF, Zhao F, Huang D, Beutler N, Burns A, He WT, Limbo O, Smith C, Song G, Woehl J, Yang L, Abbott RK, Callaghan S, Garcia E, Hurtado J, Parren M, Peng L, Ramirez S, Ricketts J, Ricciardi MJ, Rawlings SA, Wu NC, Yuan M, Smith DM, Nemazee D, Teijaro JR, Voss JE, Wilson IA, Andrabi R, Briney B, Landais E, Sok D, Jardine JG, Burton DR. Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science. 2020 Aug 21;369(6506):956-963. doi: 10.1126/science.abc7520.
    1. Provided a link to the abstract of the article on PubMed.
    2. Provided a link to the full text of the article in PubMed Central.
    3. Provided 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? Looked at the first page of the PDF version of the article for the © symbol.
      • The authors own the right to this article.
    6. How is the article available:
      • 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.
        • Yes, according to the website, Science is an open access journal from AAAS. All content is available from the date of publication.
    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.
      • The article is available in print as well. This edition can be requested directly in the website.
  2. Evaluating the source--the journal
    1. Who is the publisher of the journal?
      • The American Association for the Advancement of Science, or the AAAS, is the publisher of the journal.
    2. Is the publisher for-profit or non-profit?
      • AAAS is a non-profit.
    3. Is the publisher a scientific society (some scientific societies partner with a for-profit publisher, some act as their own non-profit publisher)
      • AAAS acts as their own non-profit publisher. They include a lot of scientific societies under their own umbrella.
    4. Does the publisher belong to the Open Access Publishers Association?
      • Yes, the publisher belongs to the Open Access Publishers Association. It is listed as a Professional Publisher (Large).
    5. What country is the journal published in?
      • It 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, articles in the journal are all 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)?
      • 41.845 is the journal's impact factor.
  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 May 12, 2020.
    3. On what date was the article accepted?
      • The article was accepted June 11, 2020.
    4. Did the article undergo any revisions before acceptance?
      • There are no listed revisions before acceptance.
    5. When was the article published?
      • The article was first published June 15, 2020.
    6. What is the approximate elapsed time between submission and publication?
      • About a month elapsed between submission and publication.
    7. What are the institutions with which the authors are affiliated?
      • The authors are affiliated with: 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.
      • Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology
    8. Have the authors published other articles on this subject? (How will you find this out?)
      • Rogers and Zhao, the first two authors on the paper, have both published multiple articles on the topic. Rogers focuses on animal models and COVID and Zhao focuses on antibodies and COVID.
    9. Is there a conflict of interest for any of the authors?
      • According to the competing interests section of the paper, D.R.B., D.H., J.G.J., E.L., T.F.R., D.S., and F.Z. are inventors on pending patent applications describing the SARS 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?
      • Yes, this is a good article to evaluate because it is published in a very well known journal. It has been peer-reviewed and the authors have published similar work in other journals, meaning they understand the topic well.

Preparation for Journal Club 2

The paper for Journal Club 2:

  • Group 1: Taylor, Nida, Anna, Aiden
    • 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. https://science.sciencemag.org/content/369/6506/956.full

Biological Terms Defined

  • Made a list of at least 10 biological terms for which I did not know the definitions when I first read the article. Defined each of the terms. Cited sources for the definitions by providing the proper citation (for a book) or the URL to the page with the definition for online sources. 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 your journal page.
  1. 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 (Biology Online, 2020)
  2. Neutralizing antibodies: an antibody that is responsible for defending cells from pathogens, which are organisms that cause disease. They are produced naturally by the body as part of its immune response, and their production is triggered by both infections and vaccinations against infections. (Lois Zoppi, 2020)
  3. Prophylactic: an agent that prevents the development of a condition or disease (Oxford Dictionary of Biochemistry and Microbiology, 2020)
  4. Potency: the measure of the activity of a drug in a biological system (Biology Online, 2020)
  5. Convalescent: serum from patients recently recovered from a disease; useful in preventing or modifying by passive immunization the same disease in exposed susceptible individuals (Biology Online, 2020)
  6. Titers: a measure of the concentration or activity of an active substance, e.g. an antibody, in a solution, usually expressed as the highest dilution of the solution in which the activity can be detected. By convention, if the highest dilution giving activity is 100‐fold, the titre is said to be 100 (Oxford Dictionary of Biochemistry and Microbiology, 2020)
  7. Luciferase: any of the group of enzymes that act on the oxidation of luciferin of bioluminescent organisms (Biology Online, 2020)
  8. Sera: plural noun of serum, which is the clear portion of any bodily fluid (Biology Online, 2020)
  9. Subtherapeutic: less than adequately treated; taking a drug with a blood level below a desired treatment range (Free Medical Dictionary, 2020)
  10. Preponderance: the quality or fact of being greater in number, quantity, or importance (Oxford Dictionary of Biochemistry and Microbiology, 2020)

Outline

Abstract and Introduction
  • Ways to prevent and treat COVID-19 are a priority right now.
  • Enrolled recovered SARS-CoV-2 patients
    • Used neutralization assays to test their antibody responses
    • Screened 1,800 antibodies
  • Isolated potent neutralizing antibodies (nAbs) to two epitopes on the RBD and to non-RBD epitopes on the spike protein
  • Neutralizing antibodies to the cause of SARS-CoV-2 can help guide vaccine design
    • A nAb to respiratory syncytial virus (RSV) is used in clinical settings already and helps to protect infants from the illness.
    • nAbs prevent death from Ebola
  • nAbs can be isolated by mining the antibody response of infected donors
  • Outstanding potency can extend antibody half-life and bring down the cost
  • What is the importance or significance of this work?
    • The importance/significance of this work is that the authors isolate a potent nAb to SARS-CoV-2 and demonstrate its efficacy in small animals. This means it is likely useful in medical interventions in humans.
  • They isolated and characterized monoclonal antibodies (mAbs) from convalescent donors.
  • A cohort of swab-postive COVID-19 donors were used for peripheral blood mononuclear cell (PBMC) and plasma collection
  • Also developed live replicating and pseudovirus neutralization assays using a HeLa-ACE2 (angiotensin-converting enzyme 2) cell line
    • This allowed them to have reproducible virus titers
  • Serum responses were evaluated against both SARS-CoV-1 and SARS-CoV-2
    • From here, 8 donors were selected for mAb
  • Single-antigen -specific memory B cells were sorted
    • These were then cloned
    • This enabled antibody expression and characterization
  • What were the limitations in previous studies that led them to perform this work?
    • No previous studies have looked at SARS-CoV-2 antibody response before. They knew from previous literature that it was helpful in creation of vaccines and better understanding the virus.
  • How did they overcome these limitations?
    • They overcame these limitations by following previous studies, which used small animal models, to perform this same test for SARS-CoV-2.
  • What is the main result presented in this paper?
    • The main result is that the nAbs they used has a potential role in prophylaxis and could be a form of therapy for SARS-CoV-2. This could lead to the development of vaccines.
Methods and Results
  • Development of viral neutralization assays
  • Platforms for evaluating plasma neutrilization activity
    • Using replication-competent virus
    • Using another pseudovirus (PSV)
  • HeLa-ACE2 target cells gave reproducible titers and were therefore used during the study
  • Live replicating virus assay used was USA-WA1/2020 (BEI Resources NR-52281)
  • PSV assay was established
    • Used both SARS-CoV-1 and SARS-CoV-2 with murine leukemia virus–based PSV (MLV-PSV)
    • This buds at the plasma membrane, unlike the coronavirus, which does so at the endoplasmic reticulum
  • Establishment of a SARS-CoV-2 cohort
  • Made a cohort of 17 donors who had SARS-CoV-2
    • From San Diego, California
    • Cohort was 47% female
    • Average age was 50 years old
  • Determined if they an infection by using a PCR test from their nasopharyngeal swab
  • All donors were symptomatic
    • Severity ranged from mild to severe
    • One case had intubation
    • All members of the cohort eventually recovered
  • Donor plasma tested in
    • ability to bind recombinant SARS-CoV-2 and SARS-CoV-1 S and receptor-binding domain (RBD) proteins
    • binding to cell surface spikes
    • neutralization in live and replicating PSV assays
  • Binding titers to SARS-CoV-2 spike protein varied
    • Titers against SARS-CoV-1 S protein were less than those for SARS-CoV-2 S protein
    • Titers against SARS-CoV-1 RBD were only detected in a small number of the cohort donors sampled
  • Antibody isolation and preliminary functional screens for down-selection
  • Cryopreserved PBMCs from eight of the donors were stained
    • Tested for memory B cell markers and AviTag RBD and SARS-CoV-2 S antigens
  • 3160 antigen-positive (Ag+) memory B cells were sorted
  • 2045 antibodies were cloned and expressed
    • 65% PCR recovery of paired variable genes
  • Heavy and light chain were tested for binding to both RBD and S
  • 92% of the pairs resulted in IgG expression
    • 43% only bound to S
    • 5.9% bound to both S and RBD
    • 0.1% only bound to RBD
  • Some binding antibodies showed neutralization activity
  • Viral infection creates a strong response against the non-RBD region of the S protein
    • Only a small portion is neutralizing
  • Fewer proportion of RBD-binding antibodies
    • Larger proportion of them neutralize SARS-COV-2 antibodies
  • three donors continually used
    • CC6
    • CC12
    • CC25
  • 33 antibodies were characterized from the three donors
    • VH1 and VH3 gene families were present within their Abs
  • Functional activity of down-selected antibodies
  • Antibodies were evaluated for epitope specificity
  • Only antibodies that bound to the noncompeting sites were detectable
  • Three epitope bins for RBD and S
    • RBD-A, RBD-B, and RBD-C
    • S-A, S-B, and S-C
  • None of the epitopes showed binding to the N-terminal domain (NTD)
  • Two competing epitopes in the S-A epitope bin
    • One in the non-RBD region of the S protein
    • One that includes RBD subdomain 2 (RBD-SD1-2)
  • Looked at mAb neutralization activity against SARS-CoV-2 and SARS-CoV-1 pseudoviruses
  • Most effective neutralizing antibodies were those on RBD-A epitope
    • Two antibodies, CC6.29 and CC6.30, used
    • Found to neutralize SARS-CoV-2 pseudovirus with an IC50 of 2 ng/ml and 1 ng/ml
  • Antibodies used in RBD-B had a higher IC50 and plateaued below 100% neutralization
  • CC6.33 against RBD-B and showed complete neutralization of SARS-CoV-2
    • IC50 of 39 ng/ml
    • Neutralized SARS-CoV-1 with an IC50 of 162 ng/ml
  • CC6.33 was the only antibody that showed effective neutralization of both pseudoviruses
  • Antibodies targeting the RBD-A epitope competed best against the ACE2 receptor
    • Preferred target for neutralizing antibodies
    • Increasing affinity of mAbs to RBD-A will likely increase the neutralization potency
  • All five nAbs neutralized the D614G variant
  • Passive transfer of neutralizing antibodies and SARS-CoV-2 challenge in Syrian hamsters
  • Used two mAbs in a Syrian hamster animal model
  • Tested nAb CC12.1, which targets the RBD-A epitope
  • Tested nAb C12.23, which targets the S-B epitope
  • Dengue virus, Den3, used as control
  • Looked at dose-dependency by using 5 different concentrations of anti-SARS-CoV-2 nAbs
  • Typically, Syrian hamsters fight off the virus after 1 week of SARS-CoV-1 infection
  • Lung tissue were collected every day to measure disease due to infection
  • Control hamsters
    • Received Den3
    • Lost an average of 13.6% of their body weight by day 5 after virus
  • Neutralizing RBD-A hamsters
    • Dose of 2 mg or 500 μg lost no weight
    • Dose of 125 μg lost 8% of their body weight
    • Dose of 31 μg/ml lost 15.8% of body weight
    • Dose of 8 μg/ml lost 16.7% of body weight
    • No statistical significance from control
  • Larger animal group sizes should potentially be used
  • S-B epitope showed no protection at any concentration compared to controls
  • Antibody serum concentration of about 22 μg/ml of nAb enables full protection from intranasal virus
    • Antibody serum concentration of 12 μg/ml shows a 50% reduction of disease
Discussion
  • Half of the 1,000 mAbs isolated could be expressed
    • They were able to bind effectively to S and the RBD proteins
  • The most potent Abs are targeted to the place that overlaps with the ACE2 binding site
  • Only one Abs, the one directed to RBD-D was capable of neutralizing the SARS-CoV-1 PSV
  • Abs directed to the RBD were less potent neutralizers
    • Showed less than 100% neutralization
  • There was also low potency and neutralization to the S protein
    • This may be due to S protein heterogeneity
  • RBD-A nAbs were able to compete with ACE2
    • Most preferred for prophylactic treatments
  • Efficacy of RBD nAb in vivo in Syrian hamsters means that human studies on this would be promising
    • Lots of limitations in animal models
  • Failure of non-RBD S nAb in animal model is consistent to the other results discovered
  • Human studies should consider:
    • enhance binding affinity of nAbs to the RBD epitope
    • improve half-life
    • reduce Fc binding to ensure there are no antibody-dependence
  • If there is antibody dependence, the vaccine would need to ensure that the epitopes are defined
Implications

The nAbs they used has a potential role in prophylaxis and could be a form of therapy for SARS-CoV-2. This could lead to the development of vaccines. The results suggest that this vaccine development should focus on the RBD, as there are strong nAb responses visible.

Future Directions

Future directions of the study are moving from the small animal model to humans. This would likely improve the efficacy of the study and provide a better understanding of the implications it can have in vaccine development.

Critical Evaluations

This article did a good job of explaining relatively complicated topics in a streamlined way. There was a very clear organization, breaking down the methods and then directly presenting the results in the same section. I think that the figures the authors added are very effective at showing their results. The limitation is that they only ended up using three donors and 33 antibodies, so it might not potentially be applicable for the larger population.

Figures 2 and 3a

  • Figure 2
    • Figure 2A. Lists the demographics of the seventeen participants from University of Califorina, San Diego in the cohort study.
      • Cohort studies are those in which people free of disease are divided by exposure or non-exposure. Both groups followed to determine incidence
      • All seventeen participants had SARS-CoV-2 with a varying range of symptoms
      • The cohort was 47% female and the average age was 50 years old
    • Figure 2B. Cohort participants were tested for binding to SARS-CoV-1 and SARS-CoV-2 S proteins
      • CC6, CC12, CC25 were the three participants chosen for further study across all experiments
      • Plasma tested by ELISA. Background binding of plasma to bovine serum albumin–coated plates
        • ELISA is a plate-based assay technique that is designed for detecting substances such as peptides, proteins, antibodies, and hormones. It couples an antibody to an antigen in the assay.
    • Figure 2C. Cohort participants were tested for binding to RBD subunits
      • Uses the same ELISA assay that was employed in Figure B
    • Figure 2D. Depicts a graph of the plasma tested for neutralization of pseudotyped (PSV) SARS-CoV-1 and SARS-CoV-2 virions
      • CC6 patient was the best at neutralizing
      • Better percent neutralization of SARS-CoV-2 than SARS-CoV-1
    • Figure 2E. Looks at the correlation between the pseudovirus SARS-CoV-2 neutralization from the previous exam and RBD subunit ELISA binding area under the curve
      • Used an r2 with a correlation of 0.529 and a p-value of 1.4 x 10^-3.
      • Of the patients, CC25 lay the closest to the fit line depicted.
  • Figure 3
    • Figure 3A. Antibody down-selection process from three donors, CC6, CC12, and CC25, presented as bubble plots.
      • Cloned is the largest bubble because it is the replication of a fragment of DNA placed in an organism so that there is enough to analyze or use in protein production.
      • Expressed is slightly smaller because it looks at the full use of the information in a gene via transcription and translation leading to production of a protein
      • Binders are those that bound either to S, RBD, or RBD and S
      • Neutralizers are those that were able to neutralize either S, RBD, or RBD and S
      • Between binding and neutralization, S only had the largest increase

Journal Club Presentation

Presentation Slides (PDF)

Scientific Conclusion

The first exercise helped me to understand whether a journal or particular article is from a reputable source and if it is appropriate to use in a scientific context. Previously, I understood how to find a journal article on different websites and how to find basic information about the publisher. Through this exercise, I learned how to find the journal impact factor and how to look for who sponsors a journal. The journal club article explained the meaning of nAb antibodies and how the RBD-A nAbs were able to compete with ACE2. After testing in Syrian hamsters, the authors concluded that the preliminary results give it the potential in the development of vaccines after it is tested in humans.

Acknowledgements

  • I spoke with my partners, Aiden Burnett, Taylor Makela, and Nida Patel over Zoom to discuss our journal article.
  • I copied and modified the procedures shown on the Week 11 page.
  • I used the Rogers et al paper for the journal club outline and presentation.
  • I used definitions from the following sources: Biology Online Dictionary, Dictionary.com, Free Medical Dictionary, Lois Zoppi's paper and Oxford Dictionary of Biochemistry and Molecular Biology.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Anna Horvath (talk) 20:53, 16 November 2020 (PST)

References

Biology Online. (2020). Convalescent. Retrieved 16 November 2020, from https://www.biologyonline.com/dictionary/convalescents

Biology Online. (2020). Epitope. Retrieved 16 November 2020, from https://www.biologyonline.com/dictionary/epitope

Biology Online. (2020). Luciferase. Retrieved 16 November 2020, from https://www.biologyonline.com/dictionary/luciferase

Biology Online. (2020). Potency. Retrieved 16 November 2020, from https://www.biologyonline.com/dictionary/potency

Biology Online. (2020). Sera. Retrieved 16 November 2020, from https://www.biologyonline.com/dictionary/sera

Free Medical Dictionary. (2020). Subtherapeutic. Retrieved 16 November 2020, from https://medical-dictionary.thefreedictionary.com/subtherapeutic

Lois Zoppi, B. (2020). What are Neutralizing Antibodies?. Retrieved 16 November 2020, from https://www.news-medical.net/health/What-are-Neutralizing-Antibodies.aspx

Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Titre. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-19642

Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Preponderance. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780195392883.001.0001/m_en_us1280416?rskey=cuBwdW&result=6

Oxford Dictionary of Biochemistry and Molecular Biology. (2006). Prophylactic. Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. Retrieved November 14, 2020, from https://www-oxfordreference-com.electra.lmu.edu/view/10.1093/acref/9780198788454.001.0001/acref-9780198788454-e-7392?rskey=ClWg5E&result=1

Rogers, T. F., Zhao, F., Huang, D., Beutler, N., Burns, A., He, W. T., Limbo, O., Smith, C., Song, G., Woehl, J., Yang, L., Abbott, R. K., Callaghan, S., Garcia, E., Hurtado, J., Parren, M., Peng, L., Ramirez, S., Ricketts, J., Ricciardi, M. J., … Burton, D. R. (2020). Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science (New York, N.Y.), 369(6506), 956–963. https://doi.org/10.1126/science.abc7520

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