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Published on: May 2, 2011
Evaluation of an F Protein-Based Recombinant Protein for Immunization Against Respiratory Syncytial Virus
Alicia Hernández-Mercado1, Claudia Berenice Barrón-García1, Jayline Romo-Amador1
1Department of Microbiology, Basic Science Center, Autonomous University of Aguascalientes, Aguascalientes, Mexico.
Insights
A novel recombinant fusion protein, SF4, combining Respiratory Syncytial Virus (RSV) F protein and 4-1BBL, effectively activates antiviral immune responses and generates neutralizing antibodies against RSV.
Area of Science:
- Immunology
- Vaccinology
- Molecular Biology
Background:
- Respiratory Syncytial Virus (RSV) is a major cause of severe respiratory infections in young children, with no available vaccine and costly treatments.
- Costimulatory molecules can enhance antiviral type 1 immune responses, suggesting their potential in vaccine development.
Purpose of the Study:
- To create a recombinant vaccine candidate (SF4) fusing the RSV F protein with 4-1BBL.
- To evaluate SF4's ability to induce antiviral responses in vitro and generate neutralizing antibodies in vivo.
Main Methods:
- Constructed and purified a fusion protein (SA-F3x-4-1BBL, SF4) in E. coli.
- Stimulated RAW264.7 macrophages with SF4 to analyze signaling pathways (TRAF2, p38, NF-κB) and cytokine production (TNF-α, IFN-γ).
- Immunized mice with SF4 and assessed anti-RSV neutralizing antibody and serum cytokine levels.
Main Results:
- SF4 protein successfully expressed and purified.
- SF4 activated key signaling pathways (p38 MAPK, NF-κB) and upregulated inflammatory cytokines (TNF-α, IFN-γ) in macrophages.
- Immunized mice developed RSV-neutralizing antibodies and type 1 cytokines.
Conclusions:
- The SF4 fusion protein effectively activates the 4-1BBL pathway.
- SF4 demonstrates potential as a vaccine candidate for inducing protective immunity against RSV through neutralizing antibodies and antiviral cytokines.
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.
