Evaluation of an F Protein-Based Recombinant Protein for Immunization Against Respiratory Syncytial Virus.

Abstract

Respiratory syncytial virus (RSV) is one of the most important etiologies of acute respiratory infections that cause bronchiolitis in children under 5 years of age. Treatments are expensive, no vaccine is available, and this is an important cause of hospitalization. Costimulatory molecules have been reported to be good inducers of antiviral type 1 immune response. This study aimed to generate a recombinant vaccine with the RSV F protein fused to 4-1BBL to evaluate the activation of an antiviral response in vitro and the production of neutralizing antibodies in vivo. The codon-optimized F gene was subcloned into an expression vector as follows: streptavidin core, gene F, and costimulatorytumor necrosis factor receptor superfamily member 9 -TNFRS9- ligand (4-1BBL). After the induction of expression in Escherichia coli C43, the recombinant protein (SA-F3x-4-1BBL, denominated SF4) was purified and verified by western blotting. Cultured RAW264.7 macrophages were stimulated with SF4 protein, then tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), p38, and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) were analyzed by western blot, and mRNA cytokines were analyzed by RT-qPCR. Finally, male C57BL/6 mice were inoculated with SF4, and the generation of anti-RSV neutralizing antibodies and serum cytokines was examined. SF4 had a size of 84.4 kDa with a 5.6% yield. SA-F-4-1BBL upregulated TRAF2, TNF-α, and interferon (IFN)-γ expression levels and activated p38 mitogen-activated protein kinase and NF-κΒ pathways in RAW264.7 cells. Importantly, antibodies capable of neutralizing RSV infection and producing type 1 cytokines were detected in the sera of immunized animals. These results suggest that the fusion protein SF4 activates the 4-1BBL signaling pathway, resulting in an effective antiviral response mediated by neutralizing antibodies and antiviral cytokines.