Purification of EV71 Virions
Vero cells (ATCC CCL81) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) with 10% heat-inactivated fetal bovine serum (FBS), penicillin and streptomycin (PS; 100U/ml) at 37°C with 5% CO2. EV71 strain (HN08) (Genebank No. HM038013) used in this study was isolated in Henan province and was propagated in Vero cells cultured in DMEM containing 2% FBS and PS (100 U/ml). When 90% of cells exhibited cytopathic effect (CPE), supernatants were harvested and inactivated with β-propiolactone (1:2000). Crude supernatants were centrifuged at 12,000 × g for 30 min to remove cellular debris and filtered through a 0.22-μm pore-size membrane (Millipore, MA, USA). Then the supernatants were concentrated by Vivaflow membrane (Sartorius stedim, Germany) according to the manufacture instruction. The concentration product was purified using gel filtration chromatography by sepharose 4FF and then ion exchange exclusion chromatography by DEAE-sepharose Fast Flow column (GE Healthcare, England). The purity of the EV71 virions was detected by high performance liquid chromatography (HPLC) (SHIMADZU, Japan) and quantified with the BCA protein assay kit (ThermoFisher, USA). EV71 particles were stored at −80°C.
Construction of EV71 VP1 protein expression plasmid
QIAamp viral RNA mini Kit (Qiagen, Germantown, MD, USA) was used to extract EV71 RNA from infected Vero cell supernatants. Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Indianapolis, IN, USA) was used for cDNA synthesis. The total 891 basepairs fragment of VP1 gene (Genebank No.HM038013) was amplified by polymerase chain reaction (PCR) using forward primer VP1(1–19)-F:CGCCATATGGGAGATAGGGTGGCAGATG and reverse primer VP1(874–891)-R:CCGCTCGAGAAGAGTGGTGATCGCTGT, the PCR product was digested with NdeI and XhoI and then cloned into pET30a vector (Novagen, Billerica, MA, USA) and named pET30a-VP1, the encoded fusion protein had a His-tag at the C-terminus.
Purification and identification of recombninat VP1 protein and EV71 virions
BL21 (DE3) was transformed with pET30a-VP1 by the Ca2+ phosphate method . Single clone was selected and inoculated into Luria Broth (LB) culture medium containing kanamycin (50 mg/ml). The culture medium was rotated at 37°C until the OD600 reached 0.6–0.7, culture medium was transplanted (1:1000 ratio) into 1.5-L Erlenmeyer flasks. Induction and purification of protein was according to the pET System Manual (11th edition, Novagen) using the Ni2+ affinity method. Further, potential endotoxin Lipopolysaccharide (LPS) in purified rVP1 protien solution was removed using ion exchange exclusion chromatography by Q Sepharose Fast Flow column (GE Healthcare, England), then the LPS levels were determined using the Limulus amebocyte lysate assay kit (ACC, USA ) according to manufacture instruction. Purified EV71 virions obtained from Beijing Institute of Biological Products Company Limited was analyzed by SDS-PAGE with 4-20% Mini-Protein TGX™ Gel (Bio-Red Cat No 4561093, USA) followed by staining with Coomassie brilliant blue. For western blot, proteins were transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked for 1 h in 5% skim milk at room temperature followed by incubation with purified human recombinant monoclonal antibody to EV71 VP1, which was derived from a phage display liberary and converted to IgG form in bacuovirus/SF9 cell system . Goat anti-human IgG conjugated to alkaline phosphatase was used as the second antibody. Signals were visualized with NBT/BCIP substrate (Roche, Germany).
Preparation of immunogen EV71 rVP1-Chi and mice immunization
Immunogen rVP1-Chi was obtained by adding purified rVP1 40 μg or 160 μg to 1 ml 0.02% chitosan-phosphate buffer solution (PBS) (pH5.7) (Sigma, St. Louis, MO, USA). And high speed vortexing was used until the solution was homogenated. EV71 virions formulation was prepared as the rVP1. Female ICR mice (8 weeks old) were divided randomly into eight groups: (1) 40 μg rVP1 antigen (rVP1-40); (2) 160 μg rVP1 antigen (rVP1-160); (3) 40 μg rVP1 antigen with chitosan adjuvant (rVP1-40-Chi); (4) 160 μg rVP1 antigen with chitosan adjuvant (rVP1-160-Chi); (5) 40 μg purified EV71 virions antigen (Virion-40); (6) 40 μg purified EV71 virions antigen with chitosan adjuvant (Virion-40-Chi); (7) PBS (PBS); (8) PBS mixed with chitosan solution (PBS-Chi). Before oral immunization, mice were starved overnight and fed with sodium bicarbonate buffer to neutralize gastric acid. Eight mice per-group were immunized intragastrically on day 0, 7, 14 and 28. Blood samples were collected and centrifuged at 5000 × g, 4°C. Serum was inactivated at 56°C for 30 min and stored at −80°C until used for neutralization assays and measurement of serum IgG. Vaginal, respiratory tract, and small intestine secretions were collected by cold PBS washes (containing 0.5% saponin (w/v); 50 mM EDTA; and 0.1 mg/ml trypsin inhibitor) . Fresh fecal pellets were collected on day 42, and immediately frozen at −80°C. Before analysis, 0.2 g feces was dissolved in 400 μl of cold PBS (containing 0.01% sodium azide and 1 mM EDTA), and the suspensions stirred and centrifuged at 9000 × g for 10 min. IgA in the supernatants was assayed by enzyme linked immunosorbent assay (ELISA) .
Measurement of specific antibodies
VP1-specific IgG antibodies were measured by indirect ELISA by coating 96-well plates with EV71 virions (100 ng/well), overnight at 4°C. After blocking with 5% skim milk to avoid nonspecific binding, Serum diluted in 5% skim milk was added to each well and incubated for 1 h at 37°C. After washing with PBS contain 0.05% tween-20 (PBST, V/V), horse radish peroxidase (HRP) -conjugated goat anti-mouse IgG or anti-mouse IgA (Sigma, St. Louis, MO, USA) was added and incubated for 1 h at 37°C. 3,3’,3,5’-tetramethylbenzidine (TMB) was added as substrate, and absorbance was measured at 450 nm. The positive cut-off value was an optical density (OD) value 2.1-fold above normal negative control sample.
Serum neutralization assay
The mice serum neutralizing antibodies against EV71 were analyzed using the cytopathic effect (CPE)-determination assay based on Vero cells. Briefly, mice sera from all groups were heat-inactivated at 56°C for 30 min, then were two-fold serially diluted from 1:4 to 1:256 in DMEM (2% FBS, 1% PS) and mixed with the same volume of 200 tissue culture infective dose 50 (TCID50) EV71 virus. After 2 h incubation at 37°C, 100 μl of virus-serum mixture was added to each well of the 96-well plates and followed by 100 μl of 2 × 105/ml Vero cells. Every dilution of each serum sample was performed in quadruplicate. The plates were then incubated in a CO2 incubator at 37°C for 7 days. The neutralizing antibody titer was expressed as the maximum serum dilution at which CPE was not observed in all four wells. The serum dilution titer at start point of 1:4 was considered as the cut off value for sero-conversion determination.
Splenocyte proliferation assay and cytokine production
Splenocytes were isolated from ICR mice from each vaccine group, which were sacrificed 6 weeks after primary inoculation, and cultured in 96-well plates (1 × 106 cells per well) in 200 μl RPMI medium 1640 (10% FBS, 1% PS). 1 μg purified rVP1 protein was used as antigenic stimulant per well and concanavalin A (Con A) was served as positive control. Furthermore, to eliminate the cell activation due to the LPS contamination, splenocytes were also stimulated by nucleocapsid protein (NP) of Hantaan virus, which was expressed and purified the same way as rVP1. Plates were incubated at 37°C for 72 h. Culture supernatants from proliferating splenocytes were collected and the level of IFN-γ, IL-4, IL-5 and TGF-β were detected using mouse ELISA kits (R&D, Minneapolis, MN, USA). The concentration values of cytokines stimulated by rVP1 were all subtracted those stimulated by hantaan virus NP and were expressed as mean ± SD.
Lethal EV71 challenge suckling mice
Because EV71 infection caused no apparent clinical symptoms in adult mice, viral challenge was performed using newborn mice. The 8-week-old female ICR mice received vaccination of rVP1-40, rVP1-40-Chi, rVP1-160, rVP1-160-Chi, Virion-40, Virion-40-Chi, PBS, and PBS-Chi as described above at day 0, 7, 14 and were allowed to mate. The mice received a booster 2 weeks later and neonatal mice were born at week 5 to week 6, then challenged intracerebrally with 10 μl (105 PFU/ml) EV71 48–72 h after birth. Meanwhile, a challenge-free group was also included in this experiment in order to provide a basic survival background. The clinical symptoms and body weight changes of challenged suckling mice were recorded daily and the scores were graded as: health (0), inactivity and/or wasting (1), limb-shake weakness (2), hind limb paralysis (3), moribund and death (4).
Results are expressed as mean ± SD. Significant differences between groups were analyzed using SPSS 11.5 (IBM, Chicago, IL, USA). P < 0.05 was considered statically significant. Significant differences between two means are presented as P < 0.05 (*) and P < 0.01 (**).