Production and functional activity of scFv fragments obtained from recombinant antibodies against influenza virus

INTRODUCTION. Passive immunotherapy using broad-spectrum antibodies is a promising development vector of new drugs against influenza. However, production process, purification, and storage of clinically suitable recombinant antibodies still face significant challenges. Antibody scFv (single-chain variable fragments) represent a more reliable, flexible, and simpler alternative to full-length antibody analogues.

AIM. This study aimed to develop expression constructs used to synthesize scFv fragments of recombinant antibodies against influenza A and B viruses, produce scFv protein preparations, and evaluate their in vitro functional activity.

MATERIALS AND METHODS. Expression constructs encoding antibody scFv fragments were obtained by PCR using overlapping primers and genetic engineering methods. 3D models of the developed scFv fragments were constructed from the primary amino acid sequence using AlfaFold Server. Antibodies were produced in a HEK293 cell line via transient expression. Antibody preparations were purified from the culture fluid by metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) was used to study the virus-specific activity of the antibodies. Virus neutralising activity was studied in Madine-Darbi canine kidney (MDCK) cell monolayer culture based on cytopathic effect and recorded in a haemagglutination assay.

RESULTS. Based on recombinant antibodies specific to influenza A and B viruses, configuration of scFv fragments was designed and predicted, and three genetic constructs were obtained for expression of scFv proteins in a eukaryotic cell culture. The scFv fragments were produced and purified by affinity chromatography using Ni sorbent (no less than 0.5 mg) at a concentration of about 1 mg/mL of each fragment. Protein polyacrylamide gel electrophoresis confirmed that the isolated scFv fragments matched the expected size of approximately 28 kDa. ELISA demonstrated specific binding of scFv fragments to various influenza A and B strains. It was established that a 50% virus neutralising dose of the scFv antibody fragment against the influenza virus surface haemagglutinin (170 ng/mL) is comparable to that of the original full-length antibody (179 ng/mL).

CONCLUSIONS. The scFv fragments have been designed and obtained for two antibodies; the one has broad neutralising activity against influenza A virus, the other is specific for influenza B virus. Due to their small size, scFv fragments can effectively penetrate mucous membranes upon intranasal administration. This makes scFv fragments potentially useful in the emergency prophylaxis and early therapy of acute respiratory viral infections. A promising decision for enhanced neutralising activity is to create bispecific scFv fragments capable of simultaneously targeting two viral epitopes.

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