Potent neutralizing equine antibodies raised against recombinant SARS-CoV-2 spike protein for COVID-19 passive immunization therapy

  1. Luis Eduardo R. Cunha
  2. Adilson A. Stolet
  3. Marcelo A. Strauch
  4. Victor A. R. Pereira
  5. Carlos H. Dumard
  6. Andre M. O. Gomes
  7. Patrícia N. C. Souza
  8. Juliana G. Fonseca
  9. Francisco E. Pontes
  10. Leonardo G. R. Meirelles
  11. José W. M. Albuquerque
  12. Carolina Q. Sacramento
  13. Natalia Fintelman-Rodrigues
  14. Tulio M. Lima
  15. Renata G. F. Alvim
  16. Federico F. Marsili
  17. Marcella Moreira Caldeira
  18. Luisa M. Higa
  19. Fábio L. Monteiro
  20. Russolina B. Zingali
  21. Guilherme A. P. de Oliveira
  22. Thiago M. L. Souza
  23. Amilcar Tanuri
  24. Andréa C. Oliveira
  25. Herbert L. M. Guedes
  26. Leda R. Castilho
  27. Jerson L. Silva

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Abstract

We used the trimeric spike (S) glycoprotein (residues 1-1208) in the prefusion conformation to immunize horses for production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by anti-spike ELISA were above 1:1,000,000, and neutralizing antibody titer was 1:14,604 (average PRNT 90 ), which is 140-fold higher than the average neutralizing titer of plasma from three convalescent COVID-19 patients analyzed for comparison. Using the same technology routinely used for industrial production of other horse hyperimmune products, plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding a F(ab’) 2 preparation with PRNT 90 titers 150-fold higher than the neutralizing titers in human convalescent plasma. Repeating the hyperimmunization in a second group of horses confirmed the very high neutralizing titers in serum and in a GMP clinical F(ab’) 2 lot. Virus-neutralizing activity in samples from mice that received the F(ab’) 2 preparation was detected even three days after injection, indicating an appropriate half-life for therapeutic intervention. These results supported the design of a clinical trial (identifier NCT04573855 ) to evaluate safety and efficacy of this horse F(ab’) 2 preparation.

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    SciScore for 10.1101/2020.08.17.254375: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIACUC: The protocol was approved by the Animal Care and Use Committee from IVB under permission no. 003.
    IRB: Sample collection from human subjects: Samples of human convalescent plasma collected at the State Hematology Institute Hemorio followed a protocol approved by the local ethics committee (CEP Hemorio; approval #4008095), as described previously15.
    Randomizationnot detected.
    BlindingThe readers were blind with respect to the sample ID.
    Power Analysisnot detected.
    Sex as a biological variableFive 3- to 5-year-old healthy horses (3 males and 2 females) from the IVB farm, weighing approximately 350 kg each, were used for the production of polyvalent sera.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The IgG antibody titer was defined as the highest dilution of serum yielding an absorbance ratio greater than 2 in the same dilution (λ490 of sample/λ490 of negative control).
    IgG
    suggested: None
    Neutralizing titers in plasma of mice injected with equine F(ab’)2: In order to investigate if neutralizing activity against SARS-CoV-2 can be observed in animals treated with the fragments, equine F(ab’)2 (pilot lot) was injected by the intraperitoneal route and neutralizing titers were measured in the plasma of mice. 11 Balb/C mice were divided into 4 groups, which received different doses of the antibody fragments: 50 μg (n = 3); 25 μg (n = 3); 10μg (n = 3) and negative control (saline) (n = 2).
    SARS-CoV-2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Production and purification of recombinant SARS-CoV-2 spike (S) glycoprotein: The cell line HEK293-COV2-S was generated by Alvim et al. (2020)15 and stably expresses the soluble ectodomain of the spike protein of SARS-CoV-2 in the prefusion trimeric conformation14.
    HEK293-COV2-S
    suggested: None
    SARS-CoV-2 was prepared in Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Then, the samples were transferred to 96-well plates with monolayers of Vero cells (2 × 104 cells/well) with serial dilutions of sample for 1 h at 37°C.
    Vero
    suggested: RRID:CVCL_ZW93)
    Experimental Models: Organisms/Strains
    SentencesResources
    Neutralizing titers in plasma of mice injected with equine F(ab’)2: In order to investigate if neutralizing activity against SARS-CoV-2 can be observed in animals treated with the fragments, equine F(ab’)2 (pilot lot) was injected by the intraperitoneal route and neutralizing titers were measured in the plasma of mice. 11 Balb/C mice were divided into 4 groups, which received different doses of the antibody fragments: 50 μg (n = 3); 25 μg (n = 3); 10μg (n = 3) and negative control (saline) (n = 2).
    Balb/C
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Software and Algorithms
    SentencesResources
    Statistical analyses were performed with GraphPad Prism 7®.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Statistical significance was calculated using Graphpad Prism® 7 software, by one-way ANOVA and Tukey post-hoc test to the confidence levels indicated in Figure S2.
    Graphpad Prism®
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Overall, these data confirm the promising potential of equine hyperimmune products as COVID-19 countermeasures over human convalescent plasma, considering the high potency and safety record of F(ab’)2 products as well as eventual limitations related to human plasma availability and eventual risks of adventitious agent transmission by human plasma products. The advantage of using the fully folded trimeric S protein in the prefusion conformation to elicit equine polyclonal antibodies against SARS-CoV-2 is the main novelty of our work. In a recent report, Liu et al. reported that highly neutralizing monoclonal antibodies discovered from B cells of convalescent COVID-19 patients targeted epitopes in the RBD and in the N-terminal domain (NTD), as well as quaternary epitopes of the trimeric spike protein20. Thus, the use of the trimeric spike protein combines the advantages of higher immunogenicity than smaller protein fragments (such as the RBD used in other works12,13) with low biosafety concerns compared to those that would apply in case of inoculating the whole virus (as used for SARS-CoV10). Fig. 7 illustrates our whole strategy and emphasizes the potential binding of F(ab’)2 fragments to the S protein on the viral surface. The presence of neutralizing activity in mice three days after F(ab’)2 injection (Fig. 6) supports its use for passive immunization treatment of hospitalized patients. Because multiple organs can be affected by SARS-CoV-2, resulting in a worse prognosis, th...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04573855Not yet recruitingTREATMENT WITH ANTI-SARS-COV-2 IMMUNOGLOBULIN IN PATIENTS WI…

    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.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2020.08.17.254375 on bioRxiv