Original Article 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 1,2 , NANDKUMAR KADAM 1,2 , S. MOHAN KARUPPAYIL 2 , MAYUR VIKHARANKAR 3 , UMESH SHALIGRAM 3 , ASHWINI K. JADHAV 2* 1 iSERA Biological Pvt Ltd, Shirala, Sangli-415408, Maharashtra India. 2 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. 3 Serum Institute of India Pvt Ltd, 212/2, Hadapsar, Off Soli Poonawalla Road, Pune-411028 India * Corresponding author: Ashwini K. Jadhav; * Email: ashujadhav09@gmail.com Received: 19 Jul 2024, Revised and Accepted: 04 Feb 2025 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. Keywords: ELISA, Immunization, Rabbit, RBD, Polyclonal antibodies, SARS-CoV-2 © 2025 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/) DOI: https://dx.doi.org/10.22159/ijap.2025v17i2.52098 Journal homepage: https://innovareacademics.in/journals/index.php/ijap INTRODUCTION COVID-19 is an infectious disease caused by the novel Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2), originated in Wuhan, China, in December 2019. It rapidly proliferated globally, gaining pandemic status by March 11, 2020. Statistics from October 2022, show that the virus affected over 618 million confirmed cases, and 6.5 million fatalities were reported globally as of October 9 th , 2021 [1]. SARS-CoV-2 has single-stranded RNA as the genetic material of size ~30000 nucleotides with only 15 coding genes [2]. Among all the potential targets of SARS-CoV-2, the spike glycoprotein (S) has been well studied due to its critical role in mediating viral entry and in inducing a protective antibody response in infected individuals [3]. The initial stage of viral entry involves the binding of the S protein of SARS-CoV-2 to the ACE2 receptor located on the surface of host cells of vital organs. This binding event induces conformational changes in the S protein, exposing the fusion peptide located within the S2 subunit [4]. Hence, S protein is one of the best target for development of vaccines. Currently, an appreciative response is seen among people with a vaccine acceptance [5] there are few drugs approved for the treatment of SARS-CoV-2 infection, and many are under different phases of the trial, finding a vaccine for this virus, therefore, remains a high priority [6]. Various areas explored in the search for an ideal vaccine against SARS-CoV-2 include inactivated virus vaccines, recombinant viral vaccines, subunit vaccines, DNA vaccines, and attenuated vaccines. More than 20 vaccines have been approved for human use in different countries for COVID-19 [7]. Some of those are BNT162b2, mRNA-1273, and Sputnik V after two doses had the highest efficacy (>90%) in preventing symptomatic cases in phase III trials [8]. mRNA vaccines, AZD1222, and CoronaVac were effective in preventing symptomatic COVID-19, developed by Pfizer/BioNTech, Moderna, and Oxford University [9]. It is believed that symptoms of SARS-CoV-2 may appear in 2 to 14 d [10]. A strategy for treating SARS-CoV-2 is directly attacking the virus. Blocking a virus’s ability to recognize, attach to, or penetrate host cells will prevent infection altogether. In many cases, the human body naturally produces antibodies against the SARS-CoV-2 virus [11]. In SARS-CoV-2 infection, in humans, both humoral and cellular immune responses are crucial for the clearance of infections. The immune response can be enhanced by Active Immunization or Passive Immunization [10]. Vaccination introduces a dead or weakened version of the pathogen, resulting in the development of vaccine-induced immunity. In either case, if an immune person later comes into contact with that virus, their immune system will instantly recognize it and develop the antibodies required to attack it. Active immunity can last for a very long time, even a lifetime. When a person receives antibodies to a disease rather than creating them through his or her immune system, passive immunity is supplied [10]. The use of monoclonal antibodies is a new outlook in the prevention of infectious diseases [14]. Monoclonal antibodies are utilized to bind to one specific substance in the body. This binding is very versatile and can mimic, block, or cause changes to enact precise mechanisms and provide an effective therapeutic intervention with a very specific treatment for diseases [14]. Many monoclonal antibodies have been recognized to identify the S1 fragment of International Journal of Applied Pharmaceutics ISSN- 0975-7058 Vol 17, Issue 2, 2025