Cells and Viruses
RD and Vero cells were grown as described previously 
. EV71 strain G082 (genogroup C4) was propagated in RD cells. Virus titers were determined using RD cells by the microtitration method and expressed as the 50% tissue culture infective dose (TCID50
) according to the Reed–Muench method 
. Purified, inactivated EV71 was obtained from Hualan Inc. (Henan, China).
Capsid Subunit Protein-specific Polyclonal Antibodies
The anti-VP0 guinea pig polyclonal antibody has been described previously 
. The anti-VP3 guinea pig polyclonal antibody was generated by immunization of guinea pigs with the recombinant VP3 protein of coxsackievirus A16 (CVA16) 
, which was found to cross-react strongly with EV71 (data not show) and hence was used in this study to detect the VP3 protein of EV71. The anti-VP1 polyclonal antibody was generated by immunization of rabbits with recombinant EV71 VP1 protein produced from Escherichia coli
RNA was extracted from RD cells infected with the EV71 strain G082 and subsequently reverse transcribed using oligo(dT) primers and M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The resultant cDNA was used as a template for the amplification of gene fragments coding for P1 and 3CD, respectively. The P1 fragment was amplified with primers (forward 5′–AATCCATGGGTTCGCAGGTGTCT-3′ and reverse 5′- GACAAGCTTTCAAAGAGTAGTGATCGC-3′), digested with NcoI and HindIII, and then inserted into the plasmid pIExBac-1 (Novagen, Merck KGaA, Darmstadt, Germany) from the same sites, to make pIExBac-P1. The 3CD fragment was amplified with primers (forward 5′-AATCCATGGGCCCGAGCCTTGATTTT-3′ and reverse 5′-GACAAGCTTTCAAAATAACTCGAGCC-3′), digested with NcoI and HindIII, and inserted into pIExBac-1 from the same site, to make pIExBac-3CD.
Generation of Recombinant Baculoviruses
pIExBac-P1 and pIExBac-3CD were used to generate the corresponding baculoviruses. The procedures, including vector transfection, and generation, selection and propagation of the recombinant viruses, were carried out according to the manufacturer's instructions (Novagen). For expression of proteins of interest, Sf9 insect cells were infected with recombinant baculoviruses. Infected cells and supernatants were harvested at 72 hours post-infection and subjected to biochemical analyses.
ELISA and Western Blot Analyses of Protein Expression
Protein lysates from baculovirus-infected Sf9 cells were obtained by treating cells with a Tris–NaCl buffer containing 1% NP-40, followed by centrifugation at 12,000 rpm for 5 minutes. For indirect ELISA, wells of 96-well microtiter plates were coated with 5 µl lysate diluted in 50 µl PBS and incubated at 4 °C for 12 hours; then and after each of the following steps, the plates were washed three times with PBST buffer (1× PBS with 0.05% Tween 20). Consecutively, 200 µl/well of 5% milk in PBST was added for blocking and incubated at 37 °C for 1 hour; 50 µl/well of the primary antibodies (guinea pig anti-VP0 antiserum, rabbit anti-VP1 antiserum or guinea pig anti-VP3 antiserum) diluted 1:1000 in PBST plus 1% milk was added and incubated at 37 °C for 2 hours; then 50 µl/well of the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000 diluted in PBST plus 1% milk) was added and incubated at 37 °C for 1 hour. For color development, 50 µl/well TMB mixture was added and incubated for 5–10 minutes; then 50 µl/well 1 N H3PO4 was added to stop the reaction. Absorbance was measured at 450 nm in a 96-well plate reader.
For Western blotting, protein samples were separated on 12% polyacrylamide gels and then transferred onto PVDF membranes. The membranes were probed with an antigen-specific primary antibody, followed by a corresponding HRP-conjugated secondary antibody. Positive signals on the membranes were developed by chemiluminescence using the BeyoECL Plus kit (Beyotime, Shanghai, China) and recorded using a LAS-4000 Luminescent Image Analyzer (Fujifilm Life Science USA, Stamford, CT, USA).
Preparation of VLPs and Control Antigens for Immunization
Lysate from baculovirus-infected Sf9 cells was subjected to ultracentrifugation at 25,000 rpm for 6 hours on a 20% sucrose cushion. The resultant pellets were resuspended in PBS and then layered onto 10–50% sucrose gradients for ultracentrifugation at 39,000 rpm for 3 hours. Ten fractions were taken from top to bottom and assayed. Based on the SDS-PAGE and Western blotting results, VLP-rich fractions were pooled, dialyzed against PBS, and then pelleted by ultracentrifuge through a 20% sucrose cushion. The pelleted VLPs were resuspended in PBS and subsequently used for animal immunization. The VLP preparations were quantitated for VP0 content by Western blotting using the recombinant VP0 as a reference standard and anti-VP0 as the detection antibody. The uninfected Sf9 cell lysate was subjected to the same purification processes as the VLP, and served as the negative control antigen for immunization.
VLP samples were subjected to negative staining with 0.5% aqueous uranyl acetate, and transmission electron microscopy was performed with a Philips CM-12S microscope.
Prior to immunization, EV71 VLPs or similarly prepared negative control (Sf9) antigens were mixed with Imject alum (Pierce, Rockford, IL, USA) at a volumetric ratio of 1:1 according to manufacturer's instructions. Groups of five female Balb/c mice (6–8 weeks old) were injected intraperitoneally (i.p.) with the antigen/alum mixtures containing the VLPs (equivalent to 5 µg VP0) or the Sf9 lysate, respectively, at weeks 0, 2 and 5. Blood samples were collected before immunization and at week 7 when mice were terminated. Sera were kept at −80 °C until use. The animal studies were approved by the Institutional Animal Care and Use Committee at the Institut Pasteur of Shanghai.
To measure EV71-specific antibody responses in serum samples, 96-well ELISA plates were coated with inactivated EV71 (10 ng/well) and incubated for 3 hours at 37 °C. Plates were blocked with 5% milk in PBST for 1 hour at 37 °C. Subsequently, the plates were incubated with the serum samples diluted with 1% milk in PBST for 2 hours at 37 °C, and then with a HRP-conjugated anti-mouse IgG antibody (Sigma, St. Louis, MO, USA) for 1 hour at 37 °C. After color development, absorbance was determined at 450 nm in a 96-well plate reader. Endpoint titer was reported as the reciprocal of the highest serum dilution that had an absorbance ≥0.1 OD unit above the blank (absorbance of the preimmune samples).
For B cell ELISPOT assay, spleens and bone marrow from three immunized mice were collected for each group at 3 weeks after the last immunization. The splenocytes and bone marrow cells were isolated, pooled, and counted. Ninety-six-well PVDF plates (Millipore, Billerica, MA, USA) were precoated with 100 ng/well of the purified inactivated EV71 and incubated overnight at 4 °C. Plates were blocked with complete RPMI 1640 medium for 2 hours at 37 °C. Freshly isolated spleen cells and bone marrow cells (1×105/well) were added to the plates and incubated for 40 hours at 37 °C with 5% CO2. Subsequently, the plates were incubated with biotinylated goat anti-mouse IgG (0.1 µg/well) diluted in PBS buffer containing 1% FBS and 0.05% Tween-20 for 2 hours at 37 °C, and then with alkaline-phosphatase-conjugated streptavidin diluted 1:1000 in PBS for 1 hour at 37 °C. After washing the plate six times with water, 100 µl/well of NBT/BCIP substrate was added to the plates and incubated for 20 minutes for color development. The plates were washed with water to stop the reaction and then air dried. The antibody-secreting cell spots were imaged and counted using a CTL Immunospot reader (Cellular Technology Ltd., Shaker Heights, OH, USA).
Serum samples were diluted serially twofold using Dulbecco's Modified Eagle's Medium (DMEM) containing 2% FBS. The EV71 stock was diluted to a working concentration of 2 TCID50/µl. The neutralization assay was conducted using 96-well plates. In each well, 50 µl diluted antiserum was mixed with 50 µl EV71 containing 100 TCID50 and incubated for 1 hour at 37°C. Next, 100 µl cell suspension containing 15,000 RD cells was added to wells containing the virus/antiserum mixtures and incubated at 37°C with 5% CO2. After 3 days, the cells were observed to evaluate the appearance of cytopathic effects (CPEs). Neutralization titers were determined as the highest serum dilutions that could completely protect cells from CPE.
A series of 58 peptides spanning the entire amino acid sequence of VP1 of the EV71-G082 were synthesized by GL Biochem Ltd. (Shanghai, China). Each peptide consisted of 15 amino acid residues with 10 residues overlapping with the adjacent peptides. Wells of 96-well ELISA plates were coated with 400 ng of individual peptide in PBS buffer overnight at 4 °C. Then, the wells were blocked with 5% milk in PBST for 2 hours at 37 °C, incubated with the antiserum diluted in PBST plus 1% milk for 2 hours at 37 °C, followed by incubation with an HRP-conjugated secondary antibody for 1 hour at 37 °C. After color development, the absorbance was determined at 450 nm using a 96-well plate reader.
Different concentrations of synthetic peptides were added to the anti-VLP sera diluted 1:5,000 in DMEM plus 2% FBS, and incubated for 1 hour at 37 °C. The peptide/antiserum mixtures were subjected to neutralization assay as described above. After 3 days, neutralization-inhibition was evaluated by analyzing cell viability using an MTT-based method. The medium in the 96-well plates was decanted gently. Then, 100 µl/well of 0.1%(w/v) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) diluted in DMEM containing 2% FBS was added and incubated for 4 hours at 37 °C. The formazan crystals were solubilized with 100 µl DMSO. The absorbance was determined at 490 nm using a microtiter plate reader.
Antisera were diluted serially with DMEM plus 2% FBS. Then, 8 µl of the diluted antiserum or the control medium was added to 400 µl of the virus suspension containing 9×106 TCID50 of EV71-G082, and incubated for 1 hour at 37 °C. The mixtures were then added to RD or Vero cells (3.5×105 cells/well in 12-well plates) and incubated for 1 hour at 4 °C to allow virus attachment. After incubation, the cells were washed with cold PBS three times, collected, and subjected to Western blotting with anti-EV71 VP1 or anti-actin polyclonal antibodies.
EV71 virus stock was diluted with DMEM plus 2% FBS to a working concentration of 2 TCID50/µl. Then, 50 µl/well of the diluted virus or the control medium was added to 96-well microplates containing 1.5×106/well of pre-seeded RD cells, and incubated for 1 hour at 4 °C to allow virus attachment. The cells were gently washed three times with DMEM to remove unbound virus. Subsequently, 50 µl/well of serially diluted antiserum was added and incubated with the cells for 1 hour at 37 °C. The cells were washed with DMEM three times before adding 100 µl DMEM. After 3 days, the cells were examined for CPE development.