Fig 1.
Production and identification of hmAbs to SFTSV Gn.
(A) The amino acid sequences of VL and VH domains of hmAbs. The nucleotides in the red boxes are conserved among the 3 hmAbs and dots represent gaps. Black boxes indicate complementary determining regions (CDR) of each variable region defined by the International Immunogenetics Information System (IMGT). (B) ELISA analysis of the binding ability of hmAbs (0.1 µg/mL) to recombinant protein Gn, Gc, or bovine serum albumin (BSA) pre-coated ELISA plates. Strep was used as a positive control. (C) Vero cells were infected with 1 MOI of SFTSV and the protein levels of SFTSV Gn and NP were analyzed with Western blot. TF2, an anti-SFTSV NP-specific mAb.
Fig 2.
hmAbs derived from Gn neutralizing SFTSV infection in vitro.
(A-C) Different doses of each hmAb were premixed with SFTSV (1 MOI) at 37°C for 1 h, and the mixture was incubated with cells for 2 h, and then the supernatant was replaced with 2% maintenance medium and cultured at 37°C for 24 h. Western blot analysis was used to determine the protein level of SFTSV NP. PBS was used as negative control. (B) SFTSV titer in the supernatant of infected cells was measured with TCID50 assays at 24 h post-infection. (C) Different doses of each hmAb were premixed with 100 TCID50 SFTSV at 37°C for 1 h, and the mixture was incubated with cells for 2 h, and then the supernatant was replaced with 2% maintenance medium and cultured at 37°C for 24 h. RT-qPCR was used to determine the viral RNA level of SFTSV L/M/S segments. (D) Different doses of each hmAb (6.25, 12.5, 25, 50, and 100 µg/mL) were premixed with 100 TCID50 SFTSV at 37°C for 1 h, and the mixture was incubated with cells for 2 h, and then the supernatant was replaced with 2% maintenance medium and cultured at 37°C for 24 h. RT-qPCR was used to detect the viral RNA level of SFTSV. IC50: 50% inhibitory concentration. Data were obtained from three independent experiments (n = 3) and were analyzed with a two-tailed Student’s t-test. Data are presented as mean ± standard deviation (SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance.
Fig 3.
Potency of a single human monoclonal antibody to neutralize SFTSV infection.
(A-D) Effects of different antibodies on SFTSV infection at the concentration of 10 µg/mL or 100 µg/mL, respectively. (A-B) The hmAbs were premixed with SFTSV (1 MOI) at 37°C for 1 h, the mixture was incubated with cells for 2 h, and then the supernatant was replaced with 2% maintenance medium and cultured at 37°C for 24 h. Western blot was used to determine the protein level of SFTSV NP. (C-D) The hmAbs were premixed with 100 TCID50 SFTSV at 37°C for 1 h, the mixture was incubated with cells for 2 h, and then the supernatant was replaced with 2% maintenance medium and cultured at 37°C for 24 h. RT-qPCR was used to analyze the viral RNA level of SFTSV. PBS was used as negative control. Data were obtained from three independent experiments (n = 3) and were analyzed with a two-tailed Student’s t-test. Data are presented as mean ± standard deviation (SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance.
Fig 4.
hmAbs neutralize SFTSV binding and internalization.
(A) Effect of hmAbs on the binding and internalization of SFTSV. For the binding assay: viruses (1 MOI) were first premixed with hmAbs (100 µg/mL) or PBS at 37°C for 1 h. This mixture was then added to Vero cells at 4°C. After 1 h, the mixture was removed and the cells were washed, and the relative viral RNA level of SFTSV was measured to assess the binding efficiency. For internalization assay, SFTSV (1 MOI) was incubated with cells at 4 °C for 1 h. Then, 100 µg/mL of hmAbs were added to cells, and cells were incubated at 37 °C for 2 h to allow viral entry into cells. Cell surface-bound virions were then removed by trypsin treatment, and relative viral RNA level of internalized SFTSV was measured. (B) Each hmAb (25 µg/mL) or PBS control was premixed with 100 TCID50 SFTSV at 37°C for 1 h, and the mixture was incubated with cells for 2 h, and then the supernatant was replaced with replacement medium with hmAb (25 µg/mL) (hmAb/hmAb) or without hmAb (hmAb/PBS) and was cultured at 37°C for 48–72 h. RT-qPCR was used to determine the viral RNA level of SFTSV. (C) Schematic timeline of hmAb treatment on SFTSV. hmAbs or NH4Cl was added to cells 12 h before and 0, 2, 6, or 12 h after SFTSV (1 MOI) infection. (D) 100 µg/mL of hmAbs were added to cells before SFTSV infection (−12 h), at the same time as SFTSV infection (0 h), or after SFTSV infection (2, 6, and 12 h) at different time points. The cells were harvested 24 h after SFTSV infection and the protein level of SFTSV NP were analyzed with Western blot. (E) Total cell RNA was extracted 24 h after SFTSV infection. RT-qPCR was used to determine the viral RNA level of SFTSV. Data were obtained from three independent experiments (n = 3) and were analyzed with a two-tailed Student’s t-test. Data are presented as mean ± standard deviation (SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance.
Table 1.
Survival rates of IFNAR1-/- A129 mice treated with hmAbs against lethal SFTSV challenge.
Fig 5.
hmAbs protect mice against a lethal SFTSV challenge.
(A) Six to eight weeks old IFNAR1-/- A129 mice (n = 6 per group) were infected with 10 lethal doses (LD50) of SFTSV intraperitoneally. At 1, 24, 48, and 72 h post-infection, IFNAR1-/- A129 mice were treated with 100 μg or 600 μg hmAb, human IgG control or PBS control intraperitoneally. The figure was created with BioRender.com. (B) The survival of IFNAR1-/- A129 mice was monitored for 10 days after SFTSV infection. Kaplan–Meier survival curves were obtained using GraphPad Prism 8. (C) Body weight of mice was monitored daily for 10 days post-infection. Relative body weight values are presented as the mean with standard deviation of surviving mice in each group. (D) The SFTSV RNA copies in the mouse spleens were determined with RT-qPCR and were normalized by mouse β-actin. Two-tailed Student’s t-test was used to determine the level of statistical significance. The calculated P-values are shown above the groups that were compared. U.D., under the detection limit. Data are presented as mean ± standard deviation (SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance.
Fig 6.
Pair combinations of hmAb fully protected mice from lethal SFTSV challenge.
(A) Biotin-labeled (HRP-conjugated) hmAbs were detected with ELISA pre-coated with recombinant protein Gn. (B) Competitive ELISA was performed with 4 anti-Gn hmAbs. Biotinylated hmAbs were incubated with antigens (SFTSV Gn) in the presence of unlabeled competitor hmAbs, followed by detection. All experiments were performed in triplicate, and the data represented mean ± standard deviation. (C) Sandwich ELISA was performed using 4 unlabeled hmAbs as capture antibodies to coat 96-well EIA/RIA plates, followed by incubation with SFTSV Gn and biotin-labeled hmAbs. All experiments were performed in triplicate, and the data represented mean ± standard deviation. (D) Six to eight weeks old IFNAR1-/- A129 mice (n = 6 per group) were infected with 10 LD50 of SFTSV intraperitoneally. At 1, 24, 48, and 72 h post-infection, IFNAR1-/- A129 mice were injected intraperitoneally with individual antibodies or paired combinations of hmAbs 1F6, 1B2, and 4-5 at a total dose of 600 μg. Human IgG was used as control. The figure was created with BioRender.com. (E) Survival of IFNAR1-/- A129 mice was monitored for 10 days. Kaplan–Meier survival curves were obtained using GraphPad Prism 8. (F) Body weight of mice was monitored daily for 10 days after SFTSV infection. Relative body weight values are presented as the mean with standard deviation of surviving mice in each group. (G) The SFTSV RNA copies in the mouse spleens were determined with RT-qPCR and were normalized by mouse β-actin. Two-tailed Student’s t-test was used to determine the level of statistical significance. The calculated P-values are shown above the groups that were compared. U.D., under the detection limit. Data are presented as mean ± standard deviation (SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance.
Table 2.
Survival rates of IFNAR1-/- A129 mice treated with hmAb cocktails against lethal SFTSV challenge.
Fig 7.
Schematic model illustrating the process of antibody neutralizing SFTSV in vivo.
Neutralizing antibodies can bind to virus particles, prevent their attachment to host cell surface receptors, and thereby block the virus from entering host cells. Following viral neutralization, they stimulate macrophages to engulf and degrade the virus. Non/low-neutralizing antibodies binding to Gn can induce the conformational change, making the neutralization epitope of medium/high-neutralizing antibodies being more accessible, thus enhancing the neutralizing activity. In addition, the antibody cocktails achieved more efficient clearance by targeting different neutralizing epitopes on the viral Gn protein. The model was created with BioRender.com.
Table 3.
Primers used for RT-qPCR.