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. 2024 May 2;12(5):1007.
doi: 10.3390/biomedicines12051007.

Natural Antibodies Produced in Vaccinated Patients and COVID-19 Convalescents Hydrolyze Recombinant RBD and Nucleocapsid (N) Proteins

Anna M Timofeeva  1   2 , Liliya Sh Shayakhmetova  2 , Artem O Nikitin  1   2 , Tatyana A Sedykh  1 , Andrey L Matveev  1 , Daniil V Shanshin  3 , Ekaterina A Volosnikova  3 , Iuliia A Merkuleva  3 , Dmitriy N Shcherbakov  3   4 , Nina V Tikunova  1   2 , Sergey E Sedykh  1   2 , Georgy A Nevinsky  1   2
Affiliations

Natural Antibodies Produced in Vaccinated Patients and COVID-19 Convalescents Hydrolyze Recombinant RBD and Nucleocapsid (N) Proteins

Anna M Timofeeva et al. Biomedicines. .

Abstract

Antibodies are protein molecules whose primary function is to recognize antigens. However, recent studies have demonstrated their ability to hydrolyze specific substrates, such as proteins, oligopeptides, and nucleic acids. In 2023, two separate teams of researchers demonstrated the proteolytic activity of natural plasma antibodies from COVID-19 convalescents. These antibodies were found to hydrolyze the S-protein and corresponding oligopeptides. Our study shows that for antibodies with affinity to recombinant structural proteins of the SARS-CoV-2: S-protein, its fragment RBD and N-protein can only hydrolyze the corresponding protein substrates and are not cross-reactive. By using strict criteria, we have confirmed that this proteolytic activity is an intrinsic property of antibodies and is not caused by impurities co-eluting with them. This discovery suggests that natural proteolytic antibodies that hydrolyze proteins of the SARS-CoV-2 virus may have a positive impact on disease pathogenesis. It is also possible for these antibodies to work in combination with other antibodies that bind specific epitopes to enhance the process of virus neutralization.

Keywords: COVID-19; IgG; N-protein; RBD; SARS-CoV-2; autoimmunity; catalytic antibodies; coronavirus; proteolytic antibodies.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analysis, or interpretation of the data, in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Analysis of N-protein hydrolysis by IgG isolated from the blood of patients who have recovered from COVID-19. The green rectangle shows a fragment of the gel corresponding to the original protein, which was taken into account for calculations (A). SDS-PAGE analysis of hydrolysis of the RBD (B,C) and N-protein (D,E) with antibodies isolated from the blood of patients who have recovered from COVID-19 (B,D)—lanes 1–5; depleted antibody subfractions after chromatography on RBD- and N-Sepharose (C,E)—lanes 6–9. c—reaction mixture which did not contain antibodies, incubated under the same conditions (control). m—protein mass markers RAV11 (Biolabmix, Novosibirsk, Russia).
Figure 2
Figure 2
Analysis of RBD (A) and N-protein (B) hydrolysis by RBD-IgG and S-IgG preparations from COVID-19 convalescents who were subsequently vaccinated with Sputnik V (Con+Vac), COVID-19 convalescents (Con), and patients vaccinated with Sputnik V (Vac). Presented in the figure are the results of a series of three experiments.
Figure 3
Figure 3
(A) 1 indicates the plasma sample of patient 1176, 2 indicates the intact IgG preparation, 3 indicates IgG incubated with 40 mM DTT at 100 °C, and m indicates protein molecular weight markers (cat. #00569095, Thermo Fisher Scientific, Waltham, MA, USA). (B) Gel filtration profile of RBD-IgG preparation from COVID-19 convalescents on a Superdex-200 column at pH 2.6. The solid line indicates the optical absorbance of the eluate at λ = 280 nm. The line with squares (□) reflects the relative activity of RBD hydrolysis by RBD-IgG preparation after gel filtration. (C) The relative activity of RBD-IgG in RBD hydrolysis after SDS electrophoresis in 4–18% gradient PAGE. (D) Electrophoretic analysis of RBD-IgG preparation (Coomassie blue staining of the control lane), where m indicates the protein mass marker and 1 indicates the RBD-IgG sample.
Figure 4
Figure 4
Hydrolysis activity of (A) RBD and (B) N-protein hydrolysis by the IgG preparations of the selected patient groups: COVID-19 convalescents vaccinated with Sputnik V (Con+Vac, black); COVID-19 convalescents (Con, red); and patients vaccinated with Sputnik V (Vac, green). The data from non-diseased and unvaccinated individuals (Neg, blue) are shown as controls. Presented are the mean values derived from a series of three independent experiments.
Figure 5
Figure 5
Analysis of RBD hydrolysis by individual antibody preparations in the presence of divalent metal ions. The data are presented for the donors of three groups under study, with (A) for COVID-19 convalescents who were then vaccinated (Con+Vac), (B) for COVID-19 convalescents (Con), and (C) for vaccinated donors (Vac). The preparations incubated without metal ions were used as the control. * p-value < 0.05.
Figure 6
Figure 6
Analysis of N-protein hydrolysis by individual antibody preparations in the presence of divalent metal ions. The data provided include information on two groups under study: (A) is for COVID-19 convalescents who were subsequently vaccinated, and (B) is for COVID-19 convalescents only. Preparations for the control group were incubated without including metal ions. * p-value < 0.05.
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