The composition of the designed vaccines is shown in Table 1.
Vaccine group gE (μg) CpG (μg) Poly I:C (μg) In Out BW006 2395 ① gE 10 ② gE + CpG + Poly I:C 10 5 5 25 ③ gE + Alum 10 ④ gE + Freund's 10 ⑤ DOTAP-IN 10 5 5 25 ⑥ DOTAP-HALF 5 5 5 5 25 ⑦ DOTAP-OUT 10 5 5 25 ⑧ PLGA 10 5 5 25 ⑨ DOPC 10 5 5 25 ①-④ are mixtures, and ⑤-⑨ are nanoparticles all contain gE+CpG+Poly I:C. For DOTAP-IN (⑤), gE only exists in the inner water phase of the (water/oil/water) emulsions; for DOTAP-OUT (⑦), gE only exists in the outer water phase; for DOTAP-HALF (⑥), gE exists in both water phases.
Table 1. Composition of each dose of the designed vaccines.
The extracellular domain of gE expressed in CHO cells was supplied by AtaGenix Laboratory (Wuhan, China). Phosphodiesters CpG ODN BW006 (class B, 5'-tcg acg ttc gtc gtt cgt cgt tc-3') and 2395 (Class C, 5'-tcg tcg ttt tcg gcg c:gc gcc g-30) were synthesized and purified via high-performance liquid chromatography (HPLC) by Sangon Biotech Co., Ltd. (Shanghai, China). Low molecular weight (LMW) poly I:C was purchased from InvivoGen (tlrl-picw-250), alum adjuvant was purchased from Thermo Fisher (77161), and Freund's adjuvant was purchased from Merck (F5581 & F5506). For groups ① gE, ② gE + CpG + Poly I:C, ③ gE + Alum and ④ gE + Freund's, vaccines were formulated by mixing these agents in phosphate-buffered saline (PBS, pH 7.4) with a pipettor.
Nanoparticle vaccines based on PLGA were formulated with a double-emulsion (W/O/W) solvent evaporation method (Liu et al. 2016a). Briefly, 1 mL of dichloromethane (DCM, Merk, 270997) containing 30 mg of PLGA (Merk, 719897) without (for group ⑧ PLGA) or with 6.5 mg of the cationic lipid 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP, Merk, 890890P, for groups ⑤-⑦, i.e., DOTAPIN/HALF/OUT) or 6.5 mg of the neutral lipid 1, 2-dioleoylsn-glycero-3-phosphocholine (DOPC, Merk, 850375P for group ⑨ DOPC) were mixed with PBS solution containing gE, CpG ODNs and Poly I:C and sonicated over an ice bath with a microtip probe sonicator (Sonics & Material Inc., Connecticut, USA) at a power of 30 kW for 1 min. After the addition of a secondary aqueous phase, i.e., 5 mL of 2% w/v polyvinyl alcohol (Merk, P8136) in deionized water, the primary water-in-oil (W/O) emulsion was further sonicated for 5 min to obtain a secondary emulsion (W/O/W) and agitated overnight with a magnetic stir bar at room temperature to evaporate the DCM completely. The final product was collected by centrifugation at 21, 000 rpm for 30 min, washed with distilled water and lyophilized. For groups containing gE on the surface of nanoparticles (groups ⑥ DOTAP-HALF and ⑦ DOTAP-OUT), aqueous solutions containing gE were gently stirred with DOTAP-PLGA nanoparticles overnight at 4 ℃ for adsorption. The gE-ad-sorbed nanoparticles were washed three times with distilled water via centrifugation at 21, 000 rpm for 30 min and lyophilized.
With PLGA as a control, the prepared vaccines were dissolved in 0.1 mol/L NaOH and 0.1% sodium dodecyl sulfate (SDS) overnight at room temperature. Loaded gE was detected with a bicinchoninic acid (BCA) protein assay kit (Beyotime, P0012). Loaded nucleic acid adjuvants were detected with a Quant-iT OliGreen ssDNA Reagent Kit (Thermo Fisher, O11492). Before immunization, the nanoparticles were suspended in PBS and observed under a transmission electron microscope (TEM, Hitachi Ltd., Tokyo, Japan) (Liu et al. 2015).
Female specific pathogen-free (SPF) C57BL/6N mice at 5 weeks of age (16–20 g) were supplied by Vital River Laboratory Animal Technology Ltd. (Beijing, China) and maintained under SPF conditions at the Central Animal Services of Medical Biology of the Chinese Academy of Medical Sciences, Peking Union Medical College. The animals were randomly divided into 10 groups with 8 mice in each group (N = 8). The mice were immunized intramuscularly in the thigh muscle three times with 50 μL of immunogen at 2-week intervals. Blood samples (via heart puncture) and spleens were collected 2 weeks after the final immunization. After clotting at 4 ℃ overnight, serum was collected after centrifugation at 3000 rpm for 10 min.
gE (2 μg/mL) was coated on 96-well plates (Corning, USA) at 4 ℃ overnight. After blocking with 5% (w/v) skim milk at 37 ℃ for 1 h, the plates were incubated with serial dilutions of mouse sera at 37 ℃ for 1 h. Bound antibodies were detected with goat-anti-mouse IgG-HRP (horseradish peroxidase) conjugate (1:5000, Bio-Rad, 5178-2504) as a secondary antibody. Ten minutes after the addition of the substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB, BD, 555214), 1 mol/L phosphoric acid was added to terminate the reaction. Absorbance at 450 nm was detected with a spectrophotometer (BioTek, USA).
Spleens were dispersed with a 70 μm cell strainer (BD, USA), and splenocytes were separated with a Ficoll-Isopaque density gradient centrifugation kit (Multi Sciences, 70-LSM01). After calculation, splenocytes were suspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher, 61870127) with 10% v/v fetal bovine serum (FBS, Biological Industries, 04-001-1ACS) and penicillin-streptomycin (Thermo Fisher, 15140122) at a final concentration of 1 × 107 cell/mL. Then, 100 μL of cells was added into each well of a 96-well plate (Corning, USA) for further analysis.
Splenocytes from 8 mice in each group were further divided randomly into two subgroups, one for the detection of antigen-specific IL-2-producing cells and the other for the detection of IFN-γ-producing cells. With ELISPOT assay kits (551076 for IL-2 and 551083 for IFN-γ, both from BD) and according to the manufacturers' protocol, tests were carried out as follows: 1 × 106 cells were incubated with 10 μg/mL gE, 10 μg/mL pooled peptides or a mixture of 5 μg/mL gE and 5 μg/mL pooled peptides for 16-18 h at 37 ℃ with 5% CO2. Pooled peptides were those T cell epitopes of gE predicted with tools from the Immune Epitope Database at http://tools.iedb.org and matched with previous reports (Garcia-Valcarcel et al. 1997). Four of these peptides (corresponding to sequences: gE (11-30): VLMGFGIITGTLRITNPVRA; gE (71-90): SRKAYDHNSPYIWPRNDYDG; gE (91-110): FLENAHEHHGVYNQGRGIDS; gE (106-125): GRIDSGERLMQPTQMSAQED) were selected and synthesized by GL Biochem Co., Ltd (Shanghai, China) with purity ≥ 95% to stimulate gE-specific T cell responses.Spots were counted with an ELISPOT reader system (Autoimmun Diagnostika GmbH, Germany) after immunoimaging.
A total of 1 × 106 splenocytes were incubated with 5 μg/mL gE or 5 μg/mL pooled peptides together with 5 μL/well FastImmune (BD, 347690) at 37 ℃ with 5% CO2 for 2 h, and brefeldin A (Biolegend, 420601) was added and incubated overnight under the same conditions to block cytokine release. After washing with staining buffer (Biolegend, 420201), 5 μg/mL CD16/32 antibodies (Biolegend, 101326) were added and incubated at 4 ℃ for 10 min to block nonspecific binding of Fc. After incubation with FITC antimouse CD4 antibodies (Biolegend, 100405) and PerCP/ Cyanine5.5 anti-mouse CD8a antibodies (Biolegend, 100733) at 4 ℃ for 30 min, the cells were fixed with fixation buffer (Biolegend, 420801) in the dark at room temperature for 20 min. After washing with permeabilization wash buffer (Biolegend, 421002), APC anti-mouse IL-2 antibodies (Biolegend, 503809) and PE anti-mouse IFN-γ antibodies (Biolegend, 505807) were added and kept in the dark at room temperature for 30 min. After one wash with permeabiliza- tion wash buffer and one wash with PBS, the cells were analyzed with a BD Accuri C6 Flow Cytometer and FlowJo software (BD, USA).
Significant differences among experimental groups were analyzed using one-way analysis variance (ANOVA) followed by Dunnett's multiple comparisons test, compare the mean of each group with the mean of PLGA group as control (GraphPad Prism 7.0 software, GraphPad Software Inc., La Jolla, CA, USA). *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001. ns, no significant difference.
Vaccine Preparation and Characterization
Immunization of Mice
Enzyme-Linked Immunosorbent Assay (ELISA) for gE-Specific IgG
Preparation of Splenocytes
Enzyme-Linked Immunospot Assay (ELISPOT)
The compositions of the prepared nanoparticle vaccines are shown in Table 2, and the data were obtained as the average of three measurements. To test gE presented on the surface of group ⑥ DOTAP-HALF, a portion of the semifinished nanoparticles (after gE encapsulation but before gE absorption) were collected and deducted from the finished nanoparticles. The final contents of CpG ODN BW006, 2395 and Poly I:C were calculated by their designed proportion in Table 1 and the total nucleic acid contents tested.
Vaccine group gE (mg) CpG (mg) Poly I:C (mg) In Out BW006 2395 ⑤ DOTAP-IN 0.53 0.19 0.19 0.98 ⑥ DOTAP-HALF 0.32 0.30 0.30 0.30 1.53 ⑦ DOTAP-OUT 0.61 0.27 0.27 1.38 ⑧ PLGA 0.55 0.21 0.21 1.07 ⑨ DOPC 0.5 0.17 0.17 0.86
Table 2. Composition of 50 doses of the nanoparticle vaccines.
Two typical forms of nanoparticles are shown under TEM. One has a diameter of approximately 200 nm (shown by the arrows hit to the right in Fig. 1), which is the approximate diameter of VZV, i.e., 180–200 nm, and is mainly present among nanoparticles without cationic lipids (groups PLGA and DOPC). Nanoparticles with cationic lipids (groups DOTAP-IN, DOTAP-HALF and DOTAP-OUT) are mainly composed of particles with much smaller diameters (shown by the arrows hit to the left in Fig. 1).
Figure 1. Nanoparticles under a transmission electron microscope. Particles with diameters of approximately 200 nm are shown by the arrows pointing to the right, which are mainly present among nanoparticles without cationic lipids (groups PLGA and DOPC). And particles with diameters of approximately 50 nm are shown by the arrows pointing to the left, which are mainly present among nanoparticles with cationic lipids (DOTAP). Scale bar, 250 nm.
Two weeks after the last immunization, blood of tested C57BL/6N mice was collected (Fig. 2A). Serum samples from the mice in group PLGA showed the highest gEspecific IgG titers (592981) among the mice immunized with nanoparticles; these titers were only lower (P = 0.4339) than those of the mice in the Freund's adjuvanted group (816702) with pipettor-mixed vaccines. The titer was much higher than that of the DOTAP-HALF nanoparticle group (140972, P = 0.0077) or the group treated with the pipettor-mixed vaccine with the same antigens and nucleic acid immune stimulators (gE + CpG + Poly I:C group in Fig. 2B, 214633, P = 0.0370).
Similar numbers of IFN-γ-producing splenocytes were detected in each group of mice immunized after stimulation with 10 μg/mL gE (Fig. 3A) or with 10 μg/mL pooled peptides (Fig. 3B). This similarity was also observed for IL-2-producing splenocytes (Fig. 3D, 3E). A mixture of 5 μg/mL gE and 5 μg/mL pooled peptides as stimulants showed no prominent effects on the numbers of either IFN-γ-producing (Fig. 3C) or IL-2-producing (Fig. 3F) splenocytes.
Figure 3. Enzyme-linked immunospot assay (ELISPOT) of splenocytes. A–C IFN-γ-producing splenocytes after stimulation with gE, pooled peptides, or mixtures of gE and pooled peptides. D–F IL-2-producing splenocytes after stimulation with gE, pooled peptides, or mixtures of gE and pooled peptides. Spot numbers of each group were compared with that of the PLGA group.*P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001. ns, no significant difference.
The PLGA group showed both the most IFN-γ-producing splenocytes and the most IL-2-producing splenocytes among all of the immunized groups (including nanoparticle vaccines and pipettor-mixed vaccines). While loading of antigens and nucleic acid adjuvants with PLGA promoted a limited increase in IFN-γ-producing splenocytes (PLGA group versus gE + CpG + Poly I:C group in Fig. 3A–3C), nanoparticle formation was quite helpful for increasing in IL-2-producing splenocytes (PLGA group versus gE + CpG + Poly I:C group in Fig. 3D–3F). While encapsulation of PLGA-nucleic acid adjuvant systems in either neutral (group DOPC) or cationic lipids (groups with DOTAP) reduced not only IFN-γ-producing but also IL-2-producing splenocytes to different extents, presentation of gE in nanoparticles that contained cationic lipids could reduce this disadvantage to a certain extent. In contrast to the DOTAP-IN and DOTAP-OUT groups, the DOTAP-HALF group showed higher IFN-γ- and IL-2-producing splenocytes but still fewer than the PLGA group after stimulation with different combinations, but no significant differences were detected (Fig. 3).
After stimulation with 5 μg/mL gE (Fig. 4A, 4C) or 5 μg/mL pooled peptides (Fig. 4B, 4D), higher proportions of antigenspecific CD4+ (Fig. 4A–4B) and CD8+ (Fig. 4C–4D) T cells (including those producing IFN-γ alone, those producing L-2 alone and those producing both IFN-γ and IL-2) were detected in splenocytes of both the mice immunized with PLGA nanoparticles and the mice immunized with DOTAPHALF nanoparticles. Although there was no significant difference in the proportion of antigen-specific CD8+ T cells among splenocytes between the PLGA and DOTAP-HALF nanoparticle-immunized mice, much higher proportions of gE-specific (P = 0.0106, Fig. 4A) and peptide-specific (P = 0.0305, Fig. 4B) CD4+ T cells were detected in the splenocytes of the PLGA nanoparticle-immunized mice.
Figure 4. Flow cytometry assay for antigen-specific T cells. A–B Proportion of IFN-γ- and IL-2-producing CD4+ T cells among splenocytes after stimulation with gE or pooled peptides stimulation. C–D Proportion of IFN-γ- and IL-2-producing CD8+ T cells among splenocytes after stimulation with gE or pooled peptides. Percentage of cytokine-producing T cells of each group was compared with that of the PLGA group. *P < 0.05. ns, no significant difference.