Three plasmids were used to produce AAV9-SFTSV Vaccine: pAAV2/9n (pRC9), pAdDeltaF6 (pHelper), and pAAV-CAG-GFP (pAAV). They edited pAAV to obtain two plasmids: pAAV-CAG-Gn and pAAV-CAG-Gc. They used restriction enzyme (5' side: BamHI, 3' side: EcoRV) to cut eGFP genome of pAAV to replace with Gn and Gc sequences amplified by PCR from the whole genome of SFTSV YG1 strain. Adherent HEK293T cells was cultured in DMEM WITH 5% FBS and seeded at 1.0 x 10^5 cells/cm2 one-day before transfection. Three plasmids (pAAV-CAG-Gn or pAAV-CAG-Gc, pRC9, pHelper at a weight ratio of 1:1:1, 0.5 µg/10^5 cells) mixed with the same weight of PEIpro in PBS solution was added to the medium for transfection and incubated in 5% CO2 incubator at 37 °C for 96 hr. One-tenth volume of Lysis Buffer (1% Triton-X, 200 mM Tris, 20 mM MgCl2, pH 7.2) and 50 u/mL of Benzonase were added to the culture medium. The mixture was incubated at 37 °C with shaking for 1 hour. After removing the debris by centrifugation (10,000 × g/10 min), the culture supernatant containing AAV9-SFTSV Gn and AAV9-SFTSV Gc was concentrated using Amicon (MWCO 100 kDa). The solution was purified by ultracentrifugation using an OptiPrep. A 40% OptiPrep fraction containing full capsid AAV9-SFTSV vaccine was collected. Finally, the solutions were concentrated and diluted in Formulation Buffer (20mM Tris,150mM NaCl, 2mM MgCl2, 5% sucrose, 0.05% Pliuronic F-68, pH 8.0) using Amicon (MWCO 100kDa). The resulting AAV9-SFTSV vaccine solutions were aliquoted and stored at –80 °.

Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by a tick-borne virus called severe fever with thrombocytopenia syndrome virus (SFTSV). In recent years, human infections through contact with ticks and through contact with the bodily fluids of infected dogs and cats have been reported; however, no vaccine is currently available. SFTSV has two glycoproteins (Gn and Gc) on its envelope, which are vaccine-target antigens involved in immunogenicity. In the present study, we constructed novel SFTS vaccine candidates using an adeno-associated virus (AAV) vector to transport the SFTSV glycoprotein genome. AAV vectors are widely used in gene therapy and their safety has been confirmed in clinical trials. Recently, AAV vectors have been used to develop influenza and SARS-CoV-2 vaccines. Two types of vaccines (AAV9-SFTSV Gn and AAV9-SFTSV Gc) carrying SFTSV Gn and Gc genes were produced. The expression of Gn and Gc proteins in HEK293T cells was confirmed by infection with vaccines. These vaccines were inoculated into mice, and the collected sera produced anti-SFTS antibodies. Furthermore, sera from AAV9-SFTSV Gn infected mice showed a potent neutralizing ability, similar to previously reported SFTS vaccine candidates that protected animals from SFTSV infection. These findings suggest that this vaccine is a promising candidate for a new SFTS vaccine.