Cell-penetrating peptide-mediated subunit vaccine generates a potent immune response and protection against Streptococcus iniae in Japanese flounder (Paralichthys olivaceus)

The efficiency of antigen capture, processing, and presentation by antigen-presenting cells is the key to induce an effective immune response. Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular uptake of various molecular cargoes and have an attractive potential for vaccine delivery. In this study, the Drosophila Antennapedia homeoprotein (Antp) and the human immunodeficiency virus-1 transactivator of transcription (TAT) peptides were fused to the N- or C-terminus of Sia10, a protective antigen of Streptococcus iniae, resulting in four recombinant fusion proteins, i.e., rAntp-Sia10, rSia10-Antp, rTAT-Sia10, and rSia10-TAT. All fusion proteins were expressed and purified, and their ability to penetrate into cells was examined. The results showed that rTAT-Sia10 had the strongest ability to translocate through the cellular membrane into cells. Immunofluorescence microscopy and Western blot assay confirmed that rTAT-Sia10 could penetrate into the head kidney lymphocytes and gill cells of Japanese flounder (Paralichthys olivaceus). Immunological analysis showed that rTAT-Sia10 significantly enhanced macrophage activation and peripheral blood leukocyte proliferation, and induced production of specific serum antibodies at 2–8 weeks post-vaccination. Transcriptional analysis showed that vaccination with rTAT-Sia10 up-regulated the expression of the genes encoding IL-1β, IL-8, NKEF, Mx, IgD, IgM, TNFα, MHC I α, MHC IIα, and CD8α. Fish vaccinated with rTAT-Sia10 exhibited significantly higher levels of survival rates (98% at 1 month and 92% at 2 months) compared to fish vaccinated with rSia10 (57% at 1 month and 53% at 2 months). Taken together, these results indicate that TAT-derived peptide has a great potential in the application of bacterial vaccines.