The physical structure of the EV71-VLPs was determined using transmission electron microscope (TEM) analysis at the instrument center of the College of Life Sciences, Wuhan University. TEM images of negatively stained samples showed the presence of intact VLPs (Figure 1A), which had an icosahedral structure and which were morphologically similar to EV71 virus par-ticles (Wang et al., 2012). The SDS-PAGE assay showed the presence of three major structural proteins, VP0 (38 kDa), VP1 (36 kDa), and VP3 (27 kDa), of EV71 in the VLPs (Figure 1B). Thus, VLPs produced in yeast are of good quality and can be used as immunogens.
Figure 1. Virus-like particles were characterized using TEM examination (A) and SDS-PAGE (B). (A) Images of VLPs were analyzed by TEM at 160, 000× magnifcation. The VLPs were approximately 30 nm in diameter and were morphologically similar to empty particles of EV71. (B) SDS-PAGE assay demonstrated the presence of three EV71 capsid proteins (5 μg VLPs): VP0 (38 kDa), VP1 (36 kDa), and VP3 (27 kDa) in the VLPs.
The supernatant of the hybridoma cell culture was assayed by ELISA to evaluate its ability to bind to inactivated EV71 virus. The neutralization capacity of the positive supernatants was determined by an in vitro neutralization assay. Following four rounds of ELISA and neutralization assay screening, two hybridomas (D4 and G12) that could produce anti-EV71 antibodies that showed a strong reaction with the above antigens and that could effectively inhibit EV71 infection were identifed.
The purity and integrity of the antibodies that were purifed from the supernatant of the hybridoma D4 or G12 cell cultures with protein A+G SepharoseTM were analyzed by SDS-PAGE. The heavy chain (55 kDa) and light chain (25 kDa) molecules of both antibodies (mAb D4 and G12) were as expected (Figure 2A, lanes 1 and 2).
Figure 2. Relative titer of mAbs reacted with different antigens and specifcity testing of mAbs. (A) The purifed antiEV71 antibodies (10 μg per lane) were analyzed by SDS-PAGE. Lane M, protein size standards; Lane 1, mAb from G12 hybridoma cells (mAb G12); lane 2, antibodies from D4 hybridoma cells (mAb D4). (B) Triplicate samples (20 ng/well of VP1 protein, inactivated EV71 virus, or PBS) were added to a 96-well ELISA plate. Purifed antibody (0.5 μg/well) was applied and incubated at 37 ℃ for 1 h, followed by an anti-mouse IgG secondary antibody. Anti-HBsAg mAb and PBS were used as the negative control and the blank control, respectively. The average OD450 ± standard showed the relative titer. (C) Five micrograms of inactivated EV71 virus was resolved using a 10% SDS-PAGE gel, followed transfer to a PVDF membrane, probing with mAb D4, G12, HBsAg mAb, or VP1-specifc antiserum and incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibodies. Uninfected RD cells were used as the negative control.
The D4 and G12 mAbs were then analyzed with ELISA to determine their relative titers with different antigens (VP1 protein, inactivated EV71 virus, or PBS). As shown in Figure 2B, both purifed mAbs reacted with the VP1 protein and the inactivated EV71 virus, but the anti-HBsAg negative control and the PBS blank control showed no signifcant reactivity with the antigens.
The specifcity of the D4 and G12 mAbs were evaluated by Western blot analysis. Comparison of VP1-specifc antiserum with control anti-HBsAg showed that both purifed mAbs could react with VP1 of EV71 positively and specifcally (Figure 2C).
The neutralizing effect of the purifed antibodies was tested using an in vitro neutralization assay. EV71 (100 TCID50) was incubated with serially diluted antibodies, followed by infection of RD cells. Uninfected RD cells were used as negative controls and infected RD cells with PBS were used as positive controls. As shown in Table 1, the lowest mAb concentration that could protect > 95% cells from CPE was 25 μg/mL. In contrast, RD cells infected with the virus alone showed severe CPE that resulted in cell rounding, aggregation, and flotation (Figure 3A).
Table 1. Neutralization capacity of purifed mAbs to inhibit CPE
Figure 3. Effect of mAbs treatment on EV71-induced CPE. Cells were photographed at 6 days post-infection. (A) Infected RD cells incubated with PBS. (B) Uninfected RD cells. (C) pretreated EV71 with 25 μg/mL of mAb G12. (D) Pretreated EV71 with 25 μg/mL of mAb D4.
Uninfected RD cells grew normally (Figure 3B). As shown by the plaque reduction and TCID50 assays, pretreatment of the virus with the lowest concentration anti-EV71 antibodies (25 μg/mL of G12 or D4) protected > 95% of cells from CPE development (Figure 3C, mAb G12; Figure 3D, mAb D4). Thus, the two purifed antibodies have EV71-neutralizing effects.