PRODUCTION OF POTENT NEUTRALIZING POLYCLONAL ANTIBODIES AGAINST SEVERE ACUTE RESPIRATORY SYNDROME-CORONAVIRUS-2 (SARS-CoV-2) IN RABBITS, IMMUNIZED WITH RECEPTOR BINDING DOMAIN HEPATITIS B SURFACE ANTIGEN CONJUGATE PROTEIN AND WHOLE INACTIVATED SARS-CoV-2 (MT416726): A COMPARATIVE STUDY

DHAIRYASHEEL YADAV; NANDKUMAR KADAM; S. MOHAN KARUPPAYIL; MAYUR VIKHARANKAR; UMESH SHALIGRAM; ASHWINI K. JADHAV

doi:10.22159/ijap.2025v17i2.52098

Authors

DHAIRYASHEEL YADAV SERA Biological Pvt Ltd, Shirala, Sangli-415408, Maharashtra India. Department of Stem Cell and Regenerative Medicine and Medical Biotechnology, Centre for Interdisciplinary Research, DY Patil Education Society (Deemed to be University), Kolhapur, Kasaba Bawada-416006, Maharashtra, India
NANDKUMAR KADAM SERA Biological Pvt Ltd, Shirala, Sangli-415408, Maharashtra India. Department of Stem Cell and Regenerative Medicine and Medical Biotechnology, Centre for Interdisciplinary Research, DY Patil Education Society (Deemed to be University), Kolhapur, Kasaba Bawada-416006, Maharashtra, India
S. MOHAN KARUPPAYIL Department of Stem Cell and Regenerative Medicine and Medical Biotechnology, Centre for Interdisciplinary Research, DY Patil Education Society (Deemed to be University), Kolhapur, Kasaba Bawada-416006, Maharashtra, India
MAYUR VIKHARANKAR Serum Institute of India Pvt Ltd, 212/2, Hadapsar, Off Soli Poonawalla Road, Pune-411028 India
UMESH SHALIGRAM Serum Institute of India Pvt Ltd, 212/2, Hadapsar, Off Soli Poonawalla Road, Pune-411028 India
ASHWINI K. JADHAV Department of Stem Cell and Regenerative Medicine and Medical Biotechnology, Centre for Interdisciplinary Research, DY Patil Education Society (Deemed to be University), Kolhapur, Kasaba Bawada-416006, Maharashtra, India https://orcid.org/0000-0003-4204-301X

DOI:

https://doi.org/10.22159/ijap.2025v17i2.52098

Keywords:

ELISA, Immunization, Rabbit, RBD, Polyclonal antibodies, SARS-CoV-2

Abstract

Objective: The current study aims to produce potent neutralizing polyclonal antibodies against novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) by immunization of rabbits.

Methods: Whole inactivated SARS-CoV-2 and purified Receptor Binding Domain-Hepatitis B surface Antigen (RBD-HBsAg) conjugate protein were used as immunogens along with Freud’s incomplete adjuvant for systematic immunization in rabbits by following a protocol approved by the Committee for Control and Supervision of Experiments on Animals (CCSEA) approved Institutional Ethics Committee (IAEC). During the systematic immunization cycle, blood samples were collected periodically after some intervals and checked for in vitro efficacy against SARS-CoV-2 by using Enzyme-Linked Immunosorbent Assay (ELISA) and Plaque Reduction Neutralization Test (PRNT50) methods.

Results: The study revealed that 28, 35, and 42 d are required to generate high-neutralizing hyperimmune polyclonal antibodies in rabbits against immunogens. A combination of Freud’s incomplete adjuvant with whole inactivated SARS-CoV-2 and RBD-HBsAg conjugate protein has shown good response in the generation of potent highly specific polyclonal antibodies. RBD-HBsAg Conjugate protein has shown threefold more immunogenicity and neutralizing efficacy as compared to a whole inactivated SARS-CoV-2.

Conclusion: Rabbits immunized with RBD-HBsAg Conjugate protein immunogen generated high neutralizing and more specific polyclonal antibodies. After extensive preclinical and clinical studies, such purified polyclonal antibodies can be used as alternative therapeutic drugs against SARS-CoV-2 infection.

Author Biography

MAYUR VIKHARANKAR, Serum Institute of India Pvt Ltd, 212/2, Hadapsar, Off Soli Poonawalla Road, Pune-411028 India

^Scientist,

Serum Institute of India Pvt Ltd, 212/2, Hadapsar, Off Soli Poonawalla Road, Pune India.411028

References

Yang J, Vaghela S, Yarnoff B, DE Boisvilliers S, DI Fusco M, Wiemken TL. Estimated global public health and economic impact of COVID-19 vaccines in the pre-omicron era using real-world empirical data. Expert Rev Vaccines. 2023 Dec 31;22(1):54-65. doi: 10.1080/14760584.2023.2157817, PMID 36527724.

Karia R, Gupta I, Khandait H, Yadav A, Yadav A. COVID-19 and its modes of transmission. SN Compr Clin Med. 2020 Oct;2(10):1798-801. doi: 10.1007/s42399-020-00498-4, PMID 32904860.

Salian VS, Wright JA, Vedell PT, Nair S, LI C, Kandimalla M. COVID-19 transmission current treatment and future therapeutic strategies. Mol Pharm. 2021 Jan 19;18(3):754-71. doi: 10.1021/acs.molpharmaceut.0c00608, PMID 33464914.

Singh MS, Yellaboina S, Ansari MA. A comprehensive review on the multifaceted interactions between host immunity and viral pathogenesis in COVID-19. Int J App Pharm. 2024;16(4):37-45. doi: 10.22159/ijap.2024v16i4.50576.

Ansari MZ, Mehta SK, Priyadarshi K, Mohanty B, Sunder A. The impact of different variables on the three cell lines in cobalamin deficient patients in a tertiary care teaching hospital. Int J Pharm Clin Res. 2024;16(7):905-11.

Folegatti PM, Ewer KJ, Aley PK, Angus B, Becker S, Belij Rammerstorfer S. Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind randomised controlled trial. Lancet. 2020;396(10249):467-78. doi: 10.1016/S0140-6736(20)31604-4, PMID 32702298.

Izda V, Jeffries MA, Sawalha AH. COVID-19: a review of therapeutic strategies and vaccine candidates. Clin Immunol. 2021 Jan 1;222:108634. doi: 10.1016/j.clim.2020.108634, PMID 33217545.

Kaznadzey A, Tutukina M, Bessonova T, Kireeva M, Mazo I. BNT162b2 mRNA-1273 and sputnik V vaccines induce comparable immune responses on a par with severe course of COVID-19. Front Immunol. 2022 Apr 13;13:797918. doi: 10.3389/fimmu.2022.797918, PMID 35493476.

Nicola M, Alsafi Z, Sohrabi C, Kerwan A, Al Jabir A, Iosifidis C. The socio-economic implications of the coronavirus pandemic (COVID-19): a review. Int J Surg. 2020 Jun 1;78:185-93. doi: 10.1016/j.ijsu.2020.04.018, PMID 32305533.

Behnood SA, Shafran R, Bennett SD, Zhang AX, O Mahoney LL, Stephenson TJ. Persistent symptoms following SARS-CoV-2 infection amongst children and young people: a meta-analysis of controlled and uncontrolled studies. J Infect. 2022 Feb 1;84(2):158-70. doi: 10.1016/j.jinf.2021.11.011, PMID 34813820.

Castro Dopico X, Ols S, Lore K, Karlsson Hedestam GB. Immunity to SARS‐CoV‐2 induced by infection or vaccination. J Intern Med. 2022 Jan;291(1):32-50. doi: 10.1111/joim.13372, PMID 34352148.

Jahanshahlu L, Rezaei N. Monoclonal antibody as a potential anti-COVID-19. Biomed Pharmacother. 2020 Sep 1;129:110337. doi: 10.1016/j.biopha.2020.110337, PMID 32534226.

Rosenstein S, Vaisman Mentesh A, Levy L, Kigel A, Dror Y, Wine Y. Production of F (ab) 2 from monoclonal and polyclonal antibodies. Curr Protoc Mol Biol. 2020 Jun;131(1):e119. doi: 10.1002/cpmb.119, PMID 32319727.

Zheng X, Wong G, Zhao Y, Wang H, HE S, BI Y. Treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from Ebola virus infection. Sci Rep. 2016 Apr 12;6(1):24179. doi: 10.1038/srep24179, PMID 27067649.

Graham BS, Ambrosino DM. History of passive antibody administration for prevention and treatment of infectious diseases. Curr Opin HIV AIDS. 2015 May 1;10(3):129-34. doi: 10.1097/COH.0000000000000154, PMID 25760933.

Habarugira G, Suen WW, Hobson Peters J, Hall RA, Bielefeldt Ohmann H. West nile virus: an update on pathobiology epidemiology diagnostics control and one health implications. Pathogens. 2020 Jul 19;9(7):589. doi: 10.3390/pathogens9070589, PMID 32707644.

HU T, Liu Y, Zhao M, Zhuang Q, XU L, HE Q. A comparison of COVID-19 sars and mers. Peer J. 2020 Aug 19;8:e9725. doi: 10.7717/peerj.9725, PMID 32879801.

Kulkarni R, Patil HP, Palkar S, Lalwani S, Mishra AC, Arankalle V. Anti SARS-CoV-2 IgG antibody response among Indian COVID-19 patients using β-propiolactone inactivated whole virus-based indirect ELISA. J Virol Methods. 2021 Jan 1;287:113996. doi: 10.1016/j.jviromet.2020.113996, PMID 33126149.

Barbier M, Lee KS, Vikharankar MS, Rajpathak SN, Kadam N, Wong TY. Passive immunization with equine rbd-specific fab protects k18-hace2-mice against alpha or beta variants of SARS-COV-2. Front Immunol. 2022 Aug 15;13:948431. doi: 10.3389/fimmu.2022.948431, PMID 36091051.

Muruato AE, Fontes Garfias CR, Ren P, Garcia Blanco MA, Menachery VD, Xie X. A high throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation. Nat Commun. 2020 Aug 13;11(1):4059. doi: 10.1038/s41467-020-17892-0, PMID 32792628.

Perera RA, Mok CK, Tsang OT, LV H, KO RL, WU NC. Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) March 2020. Euro Surveill. 2020 Apr 23;25(16):2000421. doi: 10.2807/1560-7917.ES.2020.25.16.2000421, PMID 32347204.

Deshpande GR, Sapkal GN, Tilekar BN, Yadav PD, Gurav Y, Gaikwad S. Neutralizing antibody responses to SARS-CoV-2 COVID-19 patients. Ind J Med Res. 2020 Jul 1;152(1):82-7.

Kanne JP, Little BP, Schulte JJ, Haramati A, Haramati LB. Long term lung abnormalities associated with COVID-19 pneumonia. Radiology. 2023;306(2):e221806. doi: 10.1148/radiol.221806, PMID 36040336.

Assefa N, Hassen JY, Admassu D, Brhane M, Deressa M, Marami D. COVID-19 testing experience in a resource-limited setting: the use of existing facilities in public health emergency management. Front Public Health. 2021 Jun 14;9:675553. doi: 10.3389/fpubh.2021.675553, PMID 34195170.

Hebbani AV, Pulakuntla S, Pannuru P, Aramgam S, Badri KR, Reddy VD. COVID-19: comprehensive review on mutations and current vaccines. Arch Microbiol. 2021;204(1):8. doi: 10.1007/s00203-021-02606-x, PMID 34873656.

Takashita E, Kinoshita N, Yamayoshi S, Sakai Tagawa Y, Fujisaki S, Ito M. Efficacy of antibodies and antiviral drugs against COVID-19 omicron variant. N Engl J Med. 2022 Mar 10;386(10):995-8. doi: 10.1056/NEJMc2119407, PMID 35081300.

Omolo CA, Soni N, Fasiku VO, Mackraj I, Govender T. Update on therapeutic approaches and emerging therapies for SARS-CoV-2 virus. Eur J Pharmacol. 2020 Sep 15;883:173348. doi: 10.1016/j.ejphar.2020.173348, PMID 32634438.

Gupta A, Pradhan A, Maurya VK, Kumar S, Theengh A, Puri B. Therapeutic approaches for SARS-CoV-2 infection. Methods. 2021 Nov 1;195:29-43. doi: 10.1016/j.ymeth.2021.04.026, PMID 33962011.

Kumar S, Chandele A, Sharma A. Current status of therapeutic monoclonal antibodies against SARS-CoV-2. PLOS Pathog. 2021 Sep 3;17(9):e1009885. doi: 10.1371/journal.ppat.1009885, PMID 34478455.