A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 Delta variant

  1. Nitin Hingankar
  2. Suprit Deshpande
  3. Payel Das
  4. Zaigham Abbas Rizvi
  5. Constantinos Kurt Wibmer
  6. Poppy Mashilo
  7. Mohammed Yousuf Ansari
  8. Alison Burns
  9. Shawn Barman
  10. Fangzhu Zhao
  11. Sohini Mukherjee
  12. Jonathan L. Torres
  13. Souvick Chattopadhyay
  14. Farha Mehdi
  15. Jyoti Sutar
  16. Deepak Kumar Rathore
  17. Kamal Pargai
  18. Janmejay Singh
  19. Sudipta Sonar
  20. Kamini Jakhar
  21. Jyotsna Dandotiya
  22. Sankar Bhattacharyya
  23. Shailendra Mani
  24. Sweety Samal
  25. Savita Singh
  26. Pallavi Kshetrapal
  27. Ramachandran Thiruvengadam
  28. Gaurav Batra
  29. Guruprasad Medigeshi
  30. Andrew B. Ward
  31. Shinjini Bhatnagar
  32. Amit Awasthi
  33. Devin Sok
  34. Jayanta Bhattacharya

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Abstract

Although efficacious vaccines have significantly reduced the morbidity and mortality of COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of five neutralizing mAbs from an Indian convalescent donor, out of which two (THSC20.HVTR04 and THSC20.HVTR26) showed potent neutralization of SARS-CoV-2 VOCs at picomolar concentrations, including the Delta variant (B.1.617.2). One of these (THSC20.HVTR26) also retained activity against the Omicron variant. These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein and prevent virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as a cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness.

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  1. Version published to 10.1371/journal.ppat.1010465
  2. ScreenIT

    SciScore for 10.1101/2021.12.25.474152: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The study protocol was approved by the Institute Ethics Committees of all participating institutions.
    IACUC: Animals and ethics statement: Prior to the conduct of experiments to assess protective efficacy of the novel mAbs in small animals, approvals on the protocols involving dosing and animal challenge were obtained from the institutional animal ethics committee (approval # IAEC/THSTI/159), institutional biosafety committee (approval # 324/2021) and DBT Review Committee on Genetic Manipulation (RCGM; DBT RCGM approval #: IBKP UAC: TRARDSAB0214).
    Euthanasia Agents: Except for the unchallenged control group (n=3), animals in all other groups were challenged with 105 PFU of SARS-CoV2 (Wuhan and Delta isolates) intranasally on Day 0, administered through a catheter 25 µl/ nare under anesthesia by using ketamine (150mg/kg) and xylazine (10mg/kg) inside ABSL3 facility (Chan et al., 2020; Rizvi et al., 2021b; Sia et al., 2020; Winkler et al., 2020).
    Sex as a biological variablenot detected.
    RandomizationMice randomly allotted to different groups (n=5) viz, infection control and those received SARS-CoV-2 specific (THSC20.HVTR04 and THSC20.HVTR26) and non-specific (HIV CAP256.VRC26.25) monoclonal antibodies as IgG were housed in different cages.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Briefly, cryo-preserved PBMCs were first thawed at 37°C in a water bath and washed with an RPMI medium containing 10% fetal bovine sera (FBS) following incubation with fluorescently-labeled antibodies (BD Biosciences) against cell surface markers for CD3 (PE-Cy7); CD8 (PE-Cy7); CD14(PE-Cy7); CD16 (PE-Cy7); CD19 (BV421); CD19 (BV421); IgD (PerCP-Cy5.5); IgG (APC-H7) in addition to labelled RBD as an antigen in FACS buffer containing PBS (pH7.4), 1% FBS, and 1.0 mM EDTA on ice.
    CD3
    suggested: None
    CD8
    suggested: (BioLegend Cat# 391503, RRID:AB_2721611)
    CD14
    suggested: (BioLegend Cat# 348805, RRID:AB_2889063)
    CD16
    suggested: None
    CD19
    suggested: None
    Capture ELISA for the detection of IgG expression: Maxisorp high protein binding 96 well ELISA plate (Nunc, Thermo Fisher Scientific.) was coated with 2μg/mL goat anti-human Fc antibody (Thermo Fisher Scientific) and incubated overnight at 4°C.
    anti-human Fc
    suggested: None
    This was followed by addition of alkaline phosphatase-conjugated goat anti-human F(ab’)2 antibody (Thermo Fisher Scientific) Inc.) at 1:1000 dilution in 1% bovine serum albumin (BSA) incubated for an hour at room temperature.
    anti-human F(ab’)2
    suggested: None
    Streptavidin ELISA for anti SARS- CoV-2 (RBD) antibody detection: 2μg/mL of Streptavidin (G-Biosciences) was coated onto each wells of Nunc maxisorp high protein-binding 96 well ELISA plate (Thermo Fisher Scientific Inc.) and incubated overnight at 4°C.
    anti SARS- CoV-2 ( RBD
    suggested: None
    Cells were fixed and stained with anti- spike RBD antibody (Sino Biologicals) followed by HRP-conjugated anti-rabbit antibody (Invitrogen) and incubated with TrueBlue substrate (Sera Care).
    anti- spike RBD
    suggested: None
    anti-rabbit
    suggested: None
    Fragment Goat Anti-Human IgG, F(ab’)₂ fragment specific antibody (Jackson ImmunoResearch Inc.) for 45 min.
    Anti-Human IgG
    suggested: None
    CC12.1 (SARS-CoV-2 mAb), and CAP256.VRC26.25 antibody (HIV-1 bnAb) were used as positive and negative controls respectively for this experiment.
    CAP256.VRC26.25
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Confirmed heavy and light chain plasmid DNA were co-transfected in 293T cells (ATCC) using Fugene transfection reagent (Promega) in 24 well plates for preparing antibody supernatant for initial screening for their expression and antigen specificity as detailed in the following section.
    293T
    suggested: None
    Preparation and purification of IgG: The IgGs representing the mAbs were produced in either HEK 293T (ATCC) or Expi293 (Thermo Scientific) cells
    HEK 293T
    suggested: None
    Plasmid DNA expressing variable heavy and light IgG chains were transiently transfected into HEK293T or Expi293 cells using polyethylenimine (PEI).
    Expi293
    suggested: RRID:CVCL_D615)
    Neutralization assay was carried out using HeLa-hACE2 cells for the infection of SARS-CoV-2 wild type and variant pseudoviruses.
    HeLa-hACE2
    suggested: None
    0.5 million HeLa and HeLa-ACE2 cells were incubated in separate wells with RBD alone without mAbs for use as background and positive control, respectively.
    HeLa-ACE2
    suggested: None
    HeLa and HeLa-ACE2 cells stained with SARS-CoV-2 RBD alone were used as background and positive control separately.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Solubilized CHO cell membrane protein (SMP) was coated onto 96-well half-area high-binding ELISA plates (Corning, 3690) at 5ug/mL in PBS overnight at 4°C.
    CHO
    suggested: None
    K18-hACE2 mice challenge: SARS-CoV-2 Wuhan (catalogue number: USA-WA1/2020) and B.1.617.2 delta (hCoV-19/USA/PHC658/2021 catalogue number NR-55611) were procured from BEI resources (https://www.beiresources.org/) and were expanded in Vero E6 cells to produce stocks required for the experiments.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Recombinant DNA
    SentencesResources
    Pseudovirus (PSV) neutralization assay: Pseudoviruses expressing complete SARS-CoV2 spike genes were prepared by transient transfection of HEK293T cells with three plasmids: SARS-CoV2 MLV-gag/pol and MLV-CMV-luciferase plasmids using Fugene 6 (Promega Inc.) as described earlier (Rogers et al., 2020).
    MLV-CMV-luciferase
    suggested: None
    Software and Algorithms
    SentencesResources
    Concentration of IgG was measured by NanoDrop spectrophotometer and IgG heavy and light chain bands were visualized with 12% SDS PAGE analysis.
    NanoDrop
    suggested: None
    50% neutralization values were calculated with four-parameter logistic regression using GraphPad Prism 7·0 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    K18-hACE2 mice challenge: SARS-CoV-2 Wuhan (catalogue number: USA-WA1/2020) and B.1.617.2 delta (hCoV-19/USA/PHC658/2021 catalogue number NR-55611) were procured from BEI resources (https://www.beiresources.org/) and were expanded in Vero E6 cells to produce stocks required for the experiments.
    https://www.beiresources.org/
    suggested: (BEI Resource Repository, RRID:SCR_013698)

    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.
    • 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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.12.25.474152v1 on bioRxiv