| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1196-1 | ||||
Resumo:As the pandemic of COVID-19 has progressed, diagnostic, therapeutic, and vaccine approaches have emerged as priorities for infection management and control. Today, even with the low numbers of cases and deaths, the risk of the emergence of a new highly pathogenic variant supports the need for strategies for the rapid development of therapies. Therefore, this project aimed to generate neutralizing recombinant antibody fragments (scFv) against SARS-CoV-2, using the Phage Display technology. Using B cell extracted from 20 convalescent COVID-19 patients, we constructed a scFv antibody fragment library. The library was transformed into electrocompetent XL1-BLUE MRF' bacteria and recovered by the VCSM13 phage. Antibody epitope-specific fragment selection was performed using the scFv phage library generated by Biopanning on 96-well microplates coated with the S-RBD protein in TBS solution. Unbound phages were removed by TBST solution (TBS with 0.05% Tween-20), while bound phages were eluted in Glycine-HCl solution 100 mM pH 2.2, and then neutralized with 3 µL of Tris-base 2 M. The eluted phages were used to infect new bacteria for a new selection, and this procedure was repeated for 5 cycles (R1, R2, R3, R4 and R5). The reactivity of the generated phages against S-RBD protein were evaluated by Phage ELISA. Using the Single-Cell methodology, E. coli Top-10F' bacteria were transformed with plasmids extracted from the R3. Briefly, selected bacterial colonies were incubated in SB medium, followed by IPTG induction to evaluate the expression of specific antibodies. With this methodology, we observed the selection of 4 clones (A6, B6, D5, and D7) capable of recognizing the S-RBD protein. The selected clones were expressed in larger volumes (200 mL), and their culture supernatants were purified by affinity HPLC using nickel columns, which recognize the Histidine tag (His-tag) inserted into our plasmid to facilitate the purification process. The antibody fragments were purified into fractions corresponding to their expected molecular weight (~25kDa), at concentrations ranging from 0.12 to 0.37 mg/mL. The mass of antibodies obtained was sufficient for performing the neutralization assay. The four expressed recombinant antibody fragments capable of recognizing the Spike protein of SARS-CoV-2 were used in a Plaque Reduction Neutralization Tests (PRNT) to neutralize the Wuhan SARS-CoV-2 invasion in VERO cell culture. The number of plaques produced when the virus is pre-incubated with a negative control is used to determine the percentage reduction resulting from incubation with each antibody, while a sample from a patient with high levels of neutralizing antibodies was used as a positive control for the assay. Among generated antibodies the A6, B6, and D5 scFv reduced the number of observed plaques by 18% to 38%, while the D7 antibody was unable to neutralize invasion. This data demonstrates the neutralizing potential of the A6, B6, and D5 antibodies, supporting them as candidates for use in immunotherapeutic formulations for the treatment of COVID-19. This project resulted in the implementation of the Phage Display technology in the laboratory. We anticipate that we will continue to improve the process to achieve a greater yield of antibodies with a larger mass of it in the step of expression.
Keywords: SARS-CoV-2, scFv, Monoclonal Antibodies, Phage Display, Immunotherapy
Development Agency: Bio-Manguinhos, Fundação Oswaldo Cruz
Palavras-chave: Phage Display, Monoclonal Antibodies, scFv, Immunotherapy, SARS-COV-2 Agência de fomento:This research was funded by Bio-Manguinhos,Fiocruz/Fundação Oswaldo Cruz | |||||