Evaluation of the efficacy of different adjuvants in producing murine monoclonal antibodies to the receptor-binding domain of the S-protein of SARS-CoV-2
AbstractIntroduction. The SARS-CoV-2-infection/COVID-19 pandemic began at the end of December 2019. In May 2023, the WHO leadership declared the end of the pandemic. The reason for this conclusion was the reduction of SARS-CoV-2 infection/COVID-19-caused mortality. However, the circulation of the virus continues, new mutations appear, new epidemiologically significant strains arise. Monoclonal antibodies (MAbs) are considered as potential drugs for the treatment or prevention of SARS-CoV-2 infection. To date, most potent neutralizing MAb aim to neutralize the receptor-binding domain (RBD) of S-protein of the SARS-CoV-2. There are many approaches to obtain MAb, but they are still a challenge. The correct choice of antigen and adjuvant plays a key role in the formation of an intense immune response. We performed a comparative evaluation of the efficacy of recombinant RBD and RBD fused to Fc fragment of immunoglobulin (RBD-Fc) emulsified with different adjuvants to achieve a high immune response in BALB/c mice.
The purpose of this study was to select the most effective antigen and adjuvant to increase the immune response in animals in order to obtain MAb.
Material and methods. Recombinant RBD and RBD fused to Fc-fragment of human immunoglobulin G1 were obtained for this work. In the next step, BALB/c mice were immunized according to nine schemes to obtain MAb specific to the RBD domain of the S-protein of SARS-CoV-2. On the third day after the last immunization blood was collected from each group of animals to determine the antibody specificity titer. Spleens were extracted from the group of hyperimmune mice to obtain a suspension of splenocytes for further fusion with Sp2/0-Ag14 partner cells. After fusion, cells were cultured and screened, cloned, and expanded upon monolayer formation.
Results. Comparative analysis of specific antibody titers to RBD in the blood of mice showed that the adjuvants aluminum hydroxide, complete Freund's adjuvant (CFA), aluminum hydroxide in combination with PAF and MagicTM Mouse have different abilities to induce an immune response when combined with recombinant RBD or RBD-Fc. The most effective immunogen was RBD-Fc emulsified with aluminum hydroxide and CFA, the antibody titer was 1 : 256,000. Seventeen hybrids were obtained by electrofusion, three of which were stable.
Conclusion. Adjuvant composition of aluminum hydroxide and IFA is the most effective for stimulation of immune response to obtain murine MAb to RBD domain of the S-protein of SARS-CoV-2. It is possible that this effect in enhancing immunogenicity was due to the mechanisms of action of the two adjuvants. IFA acted as an antigen depot due to the mineral oil in its composition, and aluminum hydroxide acted as antigen delivery. Since the molecular weight of the recombinant RBD protein was doubled, this allowed the aluminum hydroxide-based adjuvant to retain the antigen on its surface and provide it to the immune cells for a long time, stimulating the humoral immune response.
Keywords:SARS-CoV-2; receptor-binding domain; angiotensin-converting enzyme 2; immunization; adjuvant; monoclonal antibodies
Funding. The work was supported by the Sectoral Scientific Program of the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing.
Conflict of interests. The authors declare no conflict of interests.
Authors’ contribution. Design and conduct of the study – Ivashchenko T.A.; collection and processing of material – Marin M.A., Silkina M.V., Zeninskaya N.A.; statistical processing – Kartseva A.S.; design and text editing – Ivashchenko T.A., Romanenko Ya.O., Kartseva A.S.; responsibility for the integrity of all parts of the article – Shemyakin I.G.; research concept and final editing – Fyrstova V.V.
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