https://orcid.org/0000-0002-0855-5815
Figures & data
Figure 1. The eukaryotic expression vector pCAG-scFv4F5-GFP was constructed and then transfected into A549 cells.
The recombinant vector pCAG-scFv4F5-GFP was amplified by PCR with vector primer, and the PCR products showed that scFv4F5 with the tag sequence was ~850 bp, pCAG-GFP had an ~ 750 bp GFP band and pCAG-scFv4F5-GFP had an ~1600 bp band (a). A549 cells were plated in 6-well plates at a density of 5 × 105 cells/well in 2 mL and grown overnight. Cells were transfected with 3 μg pCAG-scFv4F5-GFP vector plus different ratios of Lipofectamine™ 2000 (1:1,1:2,1:3). GFP green fluorescence was detected and fluorescence intensity of cells transfected with 1:3 of pCAGsc-Fv4F5-GFP:Lipofectamine™ was obviously higher than that of others at the same time (b). Cells transfected at 1:3 pCAG-scFv4F5-GFP:Lipofectamine™ 2000 were collected and lysed in RIPA buffer at 24, 48, and 72 hours. The supernatants of the lysed cells were analyzed by Western blot for expression of scFv4F5 antibody. A 55 kD fragment, which is the size of the antibody fusion with GFP, was detected for all treated cells, and the highest production occurred at 48 hours (c).
Figure 2. The expressed intracellular antibody can inhibit H5N1 virus propagation in A549 cells.
pCAG-scFv4F5-GFP was transfected into A549 cells as above. After 24 hours, the cells were infected with H5N1 virus and the inhibition of H5N1 virus propagation by the intrabody was measured at 48 hours after infection. The treated cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 and then consecutively incubated with mouse anti-influenza A virus NP antibody and R-PE-conjugated goat anti-mouse IgG. The cells were observed using a Carl Zeiss immunofluorescence microscope, virus quantity decreased with increasing dose of pCAG-scFv4F5-GFP transfected from 1 to 5 μg (a). The virus titer of each supernatant was decreasing with the increasing dose of pCAG-scFv4F5-GFP (b).
Figure 3. Full-length human anti-H5N1 virus IgG1 antibody-expressing vectors were constructed and then stably transfected into CHO-DG44 cells.
The sizes of genes Vλ, VH, Cλ, CH1 were all ~350 bp, VH2-3 and λ were ~750 bp, H was ~ 1500 bp, as tested by PCR (a). Transfected cells were selected with G418 added 2 days later, and obvious cell colonies were seen at 5 and 10 days, the cells were marked with red circles and black arrows (b). The expression of IgG1 was tested by Western blot and antibody H and λ were both expressed in cells and supernatants. The size of λ was 25 kD, and H was 55 kD (c). The purified IgG1 was identified by SDS-PAGE, and the size was correct. The purity of the purified IgG1 reached 95% as analyzed by ImageJ software, with the mean gray value of the protein band areas methods (d).
Figure 4. The purified full-length human IgG1 antibody has high specificity, sensitivity and affinity against H5N1 virus.
ELISA showed that the OD450 of IgG1 and scFv4F5 were almost the same at the same concentrations against the H5N1 virus (a). The binding reactivity of IgG1 was detected by flow cytometry, and IgG1 and negative commercial human IgG1 antibody were added 48 hours after MDCK cells were infected by A/Jiangsu/1/2007(H5N1) virus. After incubation for 1 hour at 4°C, R-PE-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG was added. After incubation in the dark for 30 minutes at 4°C, the cells were resuspended in 100 µl 4% paraformaldehyde and analyzed by flow cytometry. The results showed that IgG1 could bind 95% of virus-infected cells, while the negative IgG1 did not bind with the cells (b). Biacore SPR results showed that the affinity constant kD of IgG1 against the virus was 4.479 × 10−12, while the kD of scFv4F5 was only 9.740 × 10−9 (c). The microneutralization assay was used to detect the IC50 of the antibodies against virus in vitro. The IC50 of IgG1 was 0.01521 μg/ml, lower than 0.3451 μg/ml of scFv4F5. The inhibitory effects of antibodies against H5N1 virus proliferation in MDCK cells are shown (d).
Figure 5. The combination of intracellular and extracellular antibodies has a better protective effect in a mouse model challenged with H5N1 virus.
The procedures of the combination of intracellular and extracellular antibodies used in this experiment was illustrated (a). Survival of 100 TCID50 A/Jiangsu/1/2007(H5N1)-challenged mice treated with IgG1. Mice in the PBS group and the 2 mg/kg group all died and the survival rates of the 5 mg/kg 10 mg/kg, and 15 mg/kg groups were 16.67%, 66.67%, and 100%, respectively. Differences between groups were considered significant when the P was < 0.05 and its value was shown in the table below (b). The weight loss of the mice was also shown (c). Survival of mice treated with antibody combinations and challenged with 500 TCID50 A/Jiangsu/1/2007(H5N1). IgG1 was used as 15 mg/kg invariantly, and when the dose of pCAG-scFv4F5-GFP was 0, 5 μg, 10 μg, 20 μg, or 30 μg, the survival rates were 16.67%, 33.33%, 66.67%, 66.67% or 83.33%, respectively, the group only treated with PBS was 0. Differences between groups were considered significant when the P was < 0.05 and its value was shown in the table below (d). The weight loss of the mice was also shown (e).
Figure 6. The secretion of cytokines and the apoptosis-related proteins increased after administration of the antibody combination.
ELISA was used to detect the levels of cytokines IFN-α, IFN-β, IFN-γ, IL-6, and IL-10 (a). Real-time PCR was used to detect the transcriptional level of the cytokines, data generated in triplicate and fold changes are shown as the mean ± SD compared to day 0 (b). Western blot was used to detect the expression of Bim and cleaved PARP (c). The relative gray intensity ratio analyses of the bands (d). Virus titers (TCID50) of each lung at different times were determined by the Reed and Muench method (e).
