Enterovirus D68 virus-like particles expressed in Pichia pastoris potently induce neutralizing antibody responses and confer protection against lethal viral infection in mice

Fig. 1. Co-expression of P1 and 3CD of EV-D68 in P. pastoris a Schematic diagrams of the constructs made in this study. TRP2-L and TRP2-R, the up-stream and down-stream parts of the TRP region, used for homologous recombination; PAOX1, AOX1 promoter; CYC1 TT, CYC1 transcription termination region; ADE2, expression cassette encoding phosphoribosylaminoimidazole carboxylase, used as the selection marker. b ELISA analysis of cell lysates from pEV-D68-003-transformed yeast clones. A polyclonal antibody against inactivated EV-D68 was used for detection. Cell lysate from a yeast clone transformed with the empty vector pPink-HC was used as the negative control (ctr). Data are mean ± SD of triplicate wells. c Western blotting analysis with an anti-VP3 polyclonal antibody. Lane M, marker; lane 1, empty vector-transformed yeast; lane 2, pEV-D68-003-transformed yeast; lane 3, inactivated EV-D68 virus

Fig. 2. Characterization of EV-D68 VLP assembly a–b Sucrose gradient analyses. Lysates from pEV-D68-003-transformed yeast were subjected to 10–50% sucrose gradient sedimentation. Twelve fractions were collected from top to the bottom and analyzed by (a) Western blotting with anti-VP0, anti-VP1, or anti-VP3 polyclonal antibodies, respectively, and (b) ELISA with a polyclonal antibody against inactivated EV-D68. c Electron microscopy of EV-D68 VLPs. Bar = 100 nm

Fig. 3. EV-D68 VLPs-induced potent neutralizing antibody responses in mice a SDS-PAGE and Western blotting analysis of the purified antigens. Lane M, protein marker; ctr, the control antigen prepared from empty vector-transformed yeast; VLP, purified EV-D68 VLPs. b ELISA reactivity of antisera with EV-D68 virus coated on the plate. Mouse antisera taken at 2 weeks after the last immunization were diluted 1:1000 and then used in this assay. c Neutralizing antibody titers against US/MO/14-18947 measured by the micro-neutralization assay. The antisera from mice immunized with control antigen did not exhibit any neutralization effect at 1:16 dilution (the lowest dilution tested) and were therefore denoted as a titer of eight for geometric mean titer (GMT) computation. Each symbol represents an individual mouse, and the solid line indicates the GMT of the group

Fig. 4. Maternal immunization with EV-D68 VLPs provided complete protection for neonatal mice against lethal challenge One-day-old ICR mice born to dams immunized with EV-D68 VLPs or the control antigen were i.p. injected with 1.2 × 105 TCID50 of US/MO/14-18947. All infected mice were observed daily for a survival and b clinical score for 14 days. Clinical scores were graded as follows: 0, healthy; 1, lethargy and reduced mobility; 2, limb weakness; 3, limb paralysis; 4, death. The numbers of mice in each group were shown in brackets

Fig. 5. Passive transfer of anti-VLP sera fully protected newborn mice against lethal infection Groups of naive ICR mice (age <24 h) were i.p. injected with 20 µl of anti-VLP or control sera, and 1 day later i.p. inoculated with 1.2 × 105 TCID50 of US/MO/14-18947. Following challenge, mice were monitored daily for a survival and b clinical score for 14 days. Clinical scores were graded as described in the legend of Fig. 4. The numbers of mice in each group were shown in brackets