Chatikorn Boonkrai , Thomas S. Cotrone , Watchadaporn Chaisuriyong , Terapong Tantawichien, Usa Thisyakorn, Stefan Fernandez, Taweewun Hunsawong, Matthew Reed,Tossapon Wongtangprasert, Thittaya Audomsun, Tanapati Phakham, Chadaporn Attakitbancha, Pijitra Saelao, Dorota Focht, Raymond Kimbung, Martin Welin, Aijaz Ahmad Malik, Trairak Pisitkun , Nattachai Srisawat
Abstract
The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the biggest healthcare issue worldwide. This study aimed to develop a monoclonal antibody against SARS-CoV-2 from B cells of recovered COVID-19 patients, which might have beneficial therapeutic purposes for COVID-19 patients. We successfully generated human monoclonal antibodies (hmAbs) against the receptor binding domain (RBD) protein of SARS-CoV-2 using developed hybridoma technology. The isolated hmAbs against the RBD protein (wild-type) showed high binding activity and neutralized the interaction between the RBD and the cellular receptor angiotensin-converting enzyme 2 (ACE2) protein. Epitope binning and crystallography results displayed target epitopes of these antibodies in distinct regions beneficial in the mix as a cocktail. The 3D2 binds to conserved epitopes among multi-variants. Pseudovirion-based neutralization results revealed that the antibody cocktail, 1D1 and 3D2, showed high potency in multiple variants of SARS-CoV-2 infection.
Introduction
Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has rapidly spread causing a worldwide pandemic [1–4]. The SARS-CoV-2 is an RNA virus with the characteristic of multiple spike glycoproteins on its envelope [5–10]. The receptor-binding domain (RBD) on the spike proteins binds specifically with the cellular receptor angiotensin-converting enzyme 2 (ACE2) of its host cells, resulting in a fusion cascade and virus entry [11–14]. This virus can transmit efficiently through respiratory droplets and aerosols with a reproduction number of up to 8 [15–17]. It replicates in the upper airway during the incubation period before developing symptoms. As of 13 September 2022, there have been 606,459,140 confirmed cases of COVID-19 globally, including 6,495,110 deaths reported to WHO.
Materials and methods
Ethical statement
This project was approved by the Institutional Review Board on Human Research of the Faculty of Medicine, Chulalongkorn University with certificate of approval number 814/2020. Written informed consent was obtained from all subjects ≥ 18 years old who were informed of the risks and signed a consent form before enrolling in the study. The study was conducted according to the Helsinki Declaration and Good Clinical Practice guidelines. All experimental procedures involving animals were conducted under a protocol approved by the Institutional Animal Care and Use Committee of AFRIMS. This animal protocol was executed in compliance with Thai laws, the Guide for the Care and Use of Laboratory Animals, the Animals for Scientific Purposes Act (National Research Council of Thailand, 2015), the Animal Welfare Act, and all applicable U.S. Department of Agriculture, Office of Laboratory Animal Welfare, and U.S. Department of Defence guidelines.
Results
To develop a fully human monoclonal antibody against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human PBMCs were isolated from patients who had recovered from COVID-19. After generating human hybridoma cells using human hybridoma technology, these cells were cultured and screened for the clones that produce anti-RBD (wild-type) SARS-CoV-2. We were then able to successfully isolate the human hybridoma clones producing antibodies against RBD (wild-type) of SARS-CoV-2. The ELISA binding profile is shown in Fig 1A. Results demonstrated that all isolated antibodies exhibited high binding to the RBD (wild-type) of SARS-CoV-2. The 1A5 mAb displayed the highest binding activity. The EC50 values of binding activity of each antibody on RBD (wild type) are summarized in Table 1. The neutralization activity of isolated human antibodies on the SARS-CoV-2 surrogate (wild-type) infection was also evaluated by the cPass assay (Fig 1B). Results showed that the cocktail combination of 1D1 and 3D2 antibodies exhibited the highest neutralizing activity with the IC50 of 17.16 ng/ml. The IC50 values of each antibody on SARS-CoV-2 surrogate virus neutralizing activity are summarized in Table 1.
Discussion
In our present study, we successfully generated human monoclonal antibodies using hybridoma technology and successfully isolated the human hybridoma clones producing antibodies against the RBD protein (wild-type) of SARS-CoV-2. We characterized mAbs that were isolated from B cells of recovered COVID-19 patients. The isolated mAbs showed sufficient specificity and sensitivity to bind and neutralize the RBD protein (wild-type) of SARS-CoV-2 assessed by ELISA and cPass assay respectively. For the binding kinetics assay, data showed that all antibodies exhibited the equilibrium constant (KD) values in the subnanomolar range. Interestingly, two of these isolated mAbs, 1D1 and 3D2 targeted non-competing epitopes on RBD. 1D1 and 3D2 antibodies were combined to form mAb cocktail to develop a product that had an improved chance of maintaining efficacy against future variants of SARS-CoV-2. These antibodies showed high pseudo-virus neutralization of Wild-type, Alpha, Beta, Gamma, and Delta VOCs
Conclusion
In conclusion, this study demonstrated that this novel mAb cocktail exerts protective effects against two variants of SARS-CoV-2. Moreover, this protective effect was demonstrated in two different routes of administration and dosing regimens. The results of this study illustrate the potential of this mAb cocktail as a viable multi-variant intervention against SARS-CoV-2 infection. However, future studies need to consider adjusting dose, route of administration, frequency of dosing, testing with new/different strains and pathology for future clinical implementation. Moreover, for using the mAb cocktail as a nasal spray solution as a medical device, future studies may need to evaluate the preclinical studies following the ISO 10993 standards with good biocompatibility based on cytotoxicity, skin sensitization, and intracutaneous reactivity as well as satisfactory safety profiles by acute and subacute systemic toxicity before starting clinical trials.
Acknowledgments
We are grateful to the volunteer for participating in the study. We thank the Institute of Biological Products, Department of Medical Sciences, Ministry of Public Health for PRNT results. We also thank Praneet Opanasopit and the team from Department of Pharmaceutical Technology, Faculty of Pharmacy, Silpakorn University for the formulation of the intranasal administration cocktail antibody therapy. We are grateful to the MAX IV laboratory in Lund for providing beamtime at BioMAX. We also want to thank Dr. Ana Gonzales for excellent support during the beamtime.
Citation: Boonkrai C, Cotrone TS, Chaisuriyong W, Tantawichien T, Thisyakorn U, Fernandez S, et al. (2023) Efficacy of the combination of monoclonal antibodies against the SARS-CoV-2 Beta and Delta variants. PLoS ONE 18(5): e0284173. https://doi.org/10.1371/journal.pone.0284173
Editor: Faten Abdelaal Okda, St Jude Children’s Research Hospital, UNITED STATES
Received: October 4, 2022; Accepted: March 25, 2023; Published: May 4, 2023
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This study was supported by Ratchadapiseksompotch Fund, Faculty of Medicine, Chulalongkorn University, grant number RA(P0)001/64, Health Systems Research Institute Fund, grant number 64-112, Education and Research Management Committee, Thai Red Cross Society fund 2021, and Sirivadhanabhakdi foundation.
Competing interests: The authors have declared that no competing interests exist.
