Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice
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The newly emerged mosquito-borne Zika virus (ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles (VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane (prM) and envelope (E) proteins were co-expressed in insect cells and self-assembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.
KeywordsZIKV Baculovirus expression system Virus-like particles (VLPs) Neutralizing antibodies
Zika virus (ZIKV), first discovered in 1947 (Dick et al. 1952), is a mosquito-borne flavivirus and has posed a great threat to public health over the past three decades (Lessler et al. 2016). It has been reported that ZIKV infection is associated with microcephaly and serious neurological complications, such as Guillain–Barre syndrome (Mlakar et al. 2016; Miner et al. 2016). There are no specific vaccines or drugs available; thus, the rapid development of a safe and effective vaccine is a high priority.
Zika viruses are spherical virions that package a single-stranded, positive-sense RNA genome complexed with multiple copies of the capsid protein (C), surrounded by a host-derived lipid envelope that involves two viral structural proteins: the pre-membrane/membrane (prM/M) and envelope (E) proteins (Heinz and Stiasny 2012). High-resolution structures of mature/immature ZIKV and the ectodomain of the E protein have revealed that ZIKV displays a similar structure to other known flaviviruses (Sirohi et al. 2016; Prasad et al. 2017; Dai et al. 2016).
During infection of host cells, the viral genome is translated in the cytoplasm of host cells as a single open reading frame that is subsequently cleaved into three structural proteins (C, prM and E) and seven non-structural proteins by viral and host proteases (Heinz and Stiasny 2012). The new virions assemble in the endoplasmic reticulum (ER) and bud as non-infectious immature particles consisting of 60 trimeric spikes of E-prM heterodimers (Zhang et al. 2003). They are transported through the exocytic pathway of host cell until they reach the trans-Golgi network (TGN). In the low-pH environment of TGN, the E-prM heterodimers are reorganized into E homodimers (Yu et al. 2008) and the cleavage site of prM is exposed for digestion by a host furin-like protease. After prM is cleaved, the virions become infectious mature particles composed of 180 copies of the E and M proteins on the envelope. Subsequently, the newly synthesized virions are transported to the cell surface for exocytosis.
The E glycoprotein is involved in receptor binding, attachment and virus fusion during cell entry, and it represents a major target for neutralizing antibodies, which play a critical role in protection against flaviviruses (Heinz and Stiasny 2012). Thus, the E protein is the primary antigen for ZIKV vaccine development. Neutralization studies of ZIKV-confirmed convalescent human serum or plasma samples indicate that the different lineages of ZIKV represent a single serotype, with little antigenic variation and high sequence homology, suggesting that antigens produced from one lineage would provide protection against all contemporary circulating strains (Dowd et al. 2016a). Recently, the development of ZIKV vaccines has been accelerated by the exploration of various antigen-delivery approaches, including inactivated and live attenuated virus vaccines, DNA vaccines, RNA vaccines, protein subunits and other viral vectors-based vaccines. These candidates have been shown to provide protection against ZIKV challenge in mice and nonhuman primates (Pierson and Graham 2016; Durbin 2016; Abbink et al. 2016; Larocca et al. 2016; Pardi et al. 2017; Shan et al. 2017; Dowd et al. 2016b), and some have entered early clinical trials. However, safety concerns may limit the licensing of these ZIKV vaccine candidates.
Virus-like particles (VLPs) are empty, multi-protein structures resembling native virions, but they are non-infectious due to a lack of viral genetic material (Rodriguez-Limas et al. 2013). The antigens are present in their native conformation (without involving a replicating virus). VLPs can serve as excellent platforms for the development of efficient vaccines, since they have the ability to induce strong humoral and cellular responses, allow rapid testing of multiple candidate antigen designs and are associated with a safer manufacturing process (Liu et al. 2016). Studies of ZIKV (Boigard et al. 2017; Yang et al. 2017; Garg et al. 2017) and other flaviviruses, such as tick-borne encephalitis virus (TBEV) (Allison et al. 1995), dengue fever virus (DENV) (Shang et al. 2012) and Japanese encephalitis virus (JEV) (Du et al. 2015), have showed that expression of the structural proteins prM and E is sufficient for the assembly and release of VLPs that are morphologically and antigenically similar to the native virions. ZIKV VLPs have been studied in mammalian cells (Boigard et al. 2017; Garg et al. 2017) and plants (Yang et al. 2017), and these studies showed that co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, or displaying the ZIKV E protein domain III on VLPs based on the hepatitis B core antigen (HBcAg), stimulated immunized mice to generate high levels of virus neutralizing antibody (NAb) titers. In addition to mammalian cells and plants, baculovirus-insect cell systems are extensively utilized for VLP production due to a number of advantages, such as lower costs, large-scale cultivation capacity, post-translational modification of the recombinant proteins that is similar to mammalian cells and the rapid growth of insect cells in animal-product-free media, which prevents contamination by mammalian pathogens (Zeltins 2013).
In this study, we introduced a method to construct ZIKV VLPs based on the baculovirus expression system. Co-expression of the prM and E proteins in insect cells enabled the formation of VLPs that were similar to ZIKV virions. The VLPs represent a promising vaccine candidate due to their potential to induce immune responses in mice.
Materials and Methods
Cells and Viruses
Vero cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C in 5% CO2. Spodoptera frugiperda Sf9 cells were cultured in Grace’s insect medium (Gibco, Grand Island, NY, USA), pH 6.0, supplemented with 10% FBS at 27 °C. The ZIKV strain SZ-WIV01 (GenBank accession no.: KU963796), which was isolated from the serum of an imported ZIKV case in China (Deng et al. 2016), was obtained from Microorganisms & Viruses Culture Collection Centre (MVCCC) of Wuhan Institute of Virology. The virus was propagated in Vero cells and stored at − 80 °C. Virus titers were determined using Vero cells by the microtitration method, and they were expressed as the 50% tissue culture infective dose (TCID50) according to the Reed-Muench method.
Construction of Recombinant Baculovirus
Preparation of Polyclonal Antibodies
The nucleotide sequences encoding the full-length E protein (504 aa) and prM protein (164 aa) were cloned into a pET32a vector (Novagen, Carlsbad, CA, USA). The corresponding His-tag fusion proteins were expressed in Escherichia coli BL21(DE3) and were purified by affinity chromatography using nickel-charged resin (Roche Diagnostics, Indianapolis, IN, USA). The purified proteins were used as antigens to generate rabbit polyclonal antiserum (anti-E and anti-prM) in our lab according to a previously reported method (Deng et al. 2007). Polyclonal antibodies (pAb) against GP64 and VP39 were also used (Wang et al. 2008).
Western Blot Analysis of Protein Expression
The Sf9 cells were infected with the recombinant baculovirus vAc-prME or the control baculovirus vAc-hsp70-egfp at a multiplicity of infection (MOI) of 5, and the lysates were collected at 72 h post infection (h.p.i.) and prepared for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with Tris-buffered saline (TBS) containing 5% nonfat milk, the membranes were incubated with anti-E and anti-prM pAb as primary antibodies, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Sigma, St. Louis, MO, USA) as the secondary antibody. Protein band signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
Sf9 cells were infected with the recombinant baculovirus vAc-prME at an MOI of 5. At 72 h.p.i., the cells were fixed for 10 min with 4% paraformaldehyde-PBS. The fixed cells were incubated in 0.2% Triton X-100-PBS for permeabilization, and they were then blocked with 5% bovine serum albumin (BSA; Biosharp, Hefei, China). The cells were then treated with primary antibodies for 1 h at room temperature and stained with goat anti-rabbit IgG-fluorescein isothiocyanate (FITC; Abcam, Cambridge, UK) for 1 h at room temperature. For visualization of the nuclei, the cells were incubated with Hoechst 33,258 (Beyotime, Shanghai, China) for 3 min at room temperature.
Production and Purification of ZIKV Virions and ZIKV VLPs
Vero cells were infected with ZIKV at an MOI of 0.1. The medium was collected 4 days post infection (d.p.i.), inactivated with β-propiolactone (1:4000 v/v), cleared of cell debris and concentrated using tangential flow filtration. The sample was then loaded onto a discontinuous sucrose gradient (20%, 50%) and subjected to ultracentrifugation at 150,000 ×g (SW41 rotor; Beckman, Fullerton, CA, USA) for 3 h. The band between the surface of the 20% and 50% sucrose was then extracted and concentrated at 150,000 ×g (SW41 rotor; Beckman) for 3 h.
ZIKV VLPs were produced by Sf9 cells infected with the recombinant baculovirus vAc-prME at an MOI of 5. At 3 d.p.i., 100 mL cells (2 × 106 cells/mL) were harvested by centrifugation at 3000 ×g for 5 min and resuspended in 10 mL cell lysis buffer (NaCl-Tris-Ethylenediaminetetraacetic acid [NTE] buffer, comprising 120 mmol/L NaCl, 10 mmol/L Tris–HCl and 1 mmol/L ethylenediaminetetraacetic acid [EDTA], pH 7.5), followed by sonication for 1 min and centrifugation at 13,000 ×g for 30 min. The supernatant was passed through a 0.22-μm filter to remove the debris and then concentrated using a 20% sucrose cushion at 150,000 ×g (SW41 rotor; Beckman) for 3 h. The pellets were resuspended in NTE buffer, sonicated for 30 s and subjected to a continuous sucrose gradient (10%–60%). After ultracentrifugation at 150,000 ×g (SW41 rotor; Beckman) for 3 h, 12 fractions were taken (from top to bottom) for western blot analysis and the E and prM antigen-enriched fractions were pelleted again at 150,000 ×g (SW41 rotor; Beckman) for 3 h. The pellets were resuspended in 100 μL NTE buffer for subsequent transmission electron microscopy (TEM) assays.
TEM and Immune-Electron Microscopy (IEM)
To observe VLPs within cells, Sf9 cells were infected with vAc-prME at an MOI of 5. Infected cells were harvested at 72 h.p.i. and processed for electron microscopy as previously described (Vanlent et al. 1990) with slight modification. Sf9 cells infected with vAc-hsp70-egfp and healthy cells were used as controls.
After purification, the VLPs or ZIKV particles were adsorbed onto formvar-coated copper grids for 5 min, negatively stained using 2% phosphotungstic acid (PTA) for 1 min and then examined with a transmission electron microscope (H-7000 FA; Hitachi, Japan).
For IEM, purified particles were adhered onto carbon-coated nickel grids (200 mesh) and blocked with 5% BSA. The primary antibodies were anti-prM and anti-E pAb and rabbit pre-immune serum. The 12-nm Colloidal Gold-AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) was used as the secondary antibody. Subsequently, the grids were negatively stained and examined with TEM as described above.
Four groups of 6–8-week-old female BALB/c mice (n = 6 in each group) were vaccinated with 50 μg ZIKV VLPs, 5 μg purified inactivated ZIKV vaccine (designated PIV, for positive control), 50 μg vAc-hsp70-egfp-infected Sf9 cell lysates (designated NC, for negative control; these cells were treated under exactly the same conditions as the ZIKV VLPs) or PBS (blank control). The level of E and prM proteins in purified inactivated ZIKV and VLPs used in a dose were analyzed by Western blot analysis and quantified by ImageJ software. All vaccines were adjuvanted with aluminum hydroxide (Thermo Fisher, Waltham, MA, USA) using a 1:1 volume ratio, and they were administered via the intramuscular (i.m.) route at weeks 0, 2 and 4. Serum samples were taken before and 2 weeks after each vaccination to monitor the humoral immune response. All the animals were euthanized, and the splenocytes were harvested at week 6 for cellular immune response studies. The animal experiment procedures were approved by the ethics committees of Wuhan Institute of Virology, Chinese Academy of Sciences (approval number: WIVA33201601).
The neutralization test was carried out using a microneutralization assay in Vero cells. The sera were incubated at 56 °C for 30 min to inactivate the complement. Vero cells in 96-well plates were cultured overnight to 80% confluence. For each well, 50 μL of a serial two-fold dilution of the serum was mixed with 50 μL (equal volume) of 100 TCID50 of ZIKV and incubated at 37 °C for 1 h to neutralize the infectious viruses. The mixtures were then transferred to the Vero cell monolayers. After incubation for 5 days at 37 °C, the NAb titer, defined as the highest dilution of serum that resulted in a 50% reduction in the cytopathic effect, was recorded.
Serum Enzyme-Linked Immunosorbent Assay (ELISA)
ZIKV-specific antibodies (IgG, IgG1, IgG2a, IgG2b, IgG2C, IgG3 and IgM) in sera were determined by ELISA. Microplates (Xiamen Labware, Xiamen, China) were coated with 50 μL/well of purified and inactivated ZIKV (2 μg/mL) or VLPs (5 μg/mL) at 4 °C overnight. After extensive plate washing and blocking with 5% (w/v) BSA in PBS with Tween 20 (PBST) for 1 h at 37 °C, the serum was serially diluted and added to the wells in triplicate. Following 1 h incubation at 37 °C, the plates were washed and HRP-conjugated goat anti-mouse secondary antibodies (Jackson Immuno Research, West Grove, PA, USA) were added. After 1 h of incubation at 37 °C, the plates were washed and developed by adding 100 μL/well of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate in the dark at room temperature for 20 min and stopped with 50 μL of 2 mol/L H2SO4. The optical density at 450 nm (OD450) of the plates was read using an ELISA plate reader (BioTek, USA). The titer for each group was determined as the reciprocal of the highest serum dilution with OD value 2σ above the mean of the negative control.
Memory immune response was measured at 2 weeks after the last immunization using a Mouse IFN-γ Precoated ELISPOT kit (Dakewe Bioengineering, Beijing, China) according to the manufacturer’s instructions. Briefly, 3 × 105 splenocytes/well (in duplicate) were cultured with inactivated ZIKV (5 μg/mL) or ZIKV VLPs (10 μg/mL) as an antigenic stimulator. Phorbol 12-myristate 13-acetate (PMA; 50 ng/mL) with ionomycin (1 μg/mL) was used as the positive control. Spots were counted using a Bio-Reader (ByoSys, German).
To detect cytokine production, 2 × 106 splenocytes/well were cultured in 24-well plates with 0.5 mL Roswell Park Memorial Institute (RPMI) 1640 medium (containing 10% FBS) and stimulated with ZIKV (5 μg/mL) or ZIKV VLPs (10 μg/mL). After incubation at 37 °C for 48 h, the media were collected, softly centrifuged at 1000 rpm for 5 min and assayed for interferon (IFN)-γ, interleukin (IL)-2, IL-4 and IL-10 production using commercially available ELISA kits (4A Biotech, Beijing, China). All the assays were performed in triplicate.
For the statistical analysis of antibody titers, the titers were first transformed to log10.
Data are shown as the mean ± standard deviations (SD) of six mice per group. Statistical significance was determined by Student’s t test, with P value < 0.05 considered to be statistically significant.
Recombinant Baculovirus Expressing ZIKV Proteins
The DNA fragment encoding ZIKV prME was inserted into AcMNPV bacmid under the control of the polyhedrin promoter, which generated the recombinant bacmid Ac-prME (Fig. 1A). After transfection and infection, the recombinant baculovirus, vAc-prME, was generated and confirmed using western blot and immunofluorescence assays (IFA) (Fig. 1) using specific antibodies. As shown in Fig. 1B, separate bands corresponding to prM (18 kDa) and E (50 kDa) proteins were detected, indicating that digestion processing performed by host cell signalase had indeed occurred at the native cleavage site. In addition, a 70-kDa band corresponding to the uncleaved polyprotein prME was also detected. In the ZIKV infected Vero cells, which were used as positive control, the prME band was not detected. This could because the cleavage of prME in ZIKV-sensitive Vero cells is more efficient and complete.
ZIKV VLPs Were Generated by the Baculovirus Expression System
ZIKV VLPs Elicited Neutralizing Antibodies and Virus-Specific IgG
To demonstrate whether the antibodies induced by VLPs were able to neutralize live ZIKV, a microneutralization assay was carried out and the geometric mean titer (GMT) was calculated for each group (Fig. 4B). In VLP-immunized mice, neutralization titers after primary immunization were significantly increased compared with the pre-immune serum (week 0). Antibody titers at week 6 (2 weeks after the second booster) were similar to those at week 4 (2 weeks after the first booster), suggesting that the last immunization did not significantly further boost the neutralization response. The neutralization titers elicited by VLPs were lower than those elicited by inactivated ZIKV, but significantly different from those elicited in the other two control groups.
ZIKV VLPs Induced Virus-Specific T cell Responses
Recently, several types of ZIKV vaccine candidates, including inactivated and live attenuated viruses, DNA and RNA vaccines, have been shown to have protective efficacy (Durbin and Wilder-Smith 2017). While these vaccine candidates show great promise, difficulties remain to be overcome regarding their licensing, especially on account of safety. The use of live viruses increases the risk of viral dissemination, and these vaccines increase the risk of side effects due to infection with viral nucleic acids. As alternative ZIKV vaccine platform, ZIKV VLPs have been produced in mammalian cells and plants and have been demonstrated to be potentially safe and effective (Boigard et al. 2017; Yang et al. 2017). VLPs are highly ordered multiprotein structures and carry many characteristics of viruses that can be used in vaccine development. Their good safety profile makes ZIKV VLPs an appropriate choice for immunocompromised individuals and pregnant women, as the World Health Organization has identified women of child-bearing age (including pregnant women) as the primary target population for ZIKV vaccination. Furthermore, VLPs have high immunogenicity and efficacy due to their unique structures. Flavivirus infection usually elicits neutralizing antibodies that bind only to complex quaternary epitopes that are only displayed on intact particles, and not to recombinant monomeric E proteins (de Alwis et al. 2012). ZIKV VLPs display E proteins with proper folding and conformation, which means that they have advantages over subunit vaccines (such as the single E protein vaccine) regarding the induction of neutralizing antibodies.
In this study, we described a new strategy for generating ZIKV VLPs consisting of prM and E proteins in insect Sf9 cells using recombinant baculovirus, and these VLPs can potentially induce strong humoral and cellular immune responses in mice. The VLPs were efficiently isolated using sucrose gradient purification. TEM and IEM revealed that the VLPs appear as rough spherical particles and have similar morphology and antigenicity to native virions. However, the lack of an inner shell of capsid protein and the viral genome leads to a smaller size (30–50 nm) compared with native virions.
Immune responses elicited by the VLP vaccine and controls
We propose that ZIKV VLPs produced by insect cells using recombinant baculovirus should be further developed as a safe and effective vaccine candidate to protect humans against ZIKV outbreaks.
This work was supported by the Science and Technology Basic Work Program (2013FY113500) from the Ministry of Science and Technology of China, and the strategic priority research program of the Chinese Academy of Sciences (ZDRW-ZS-2016-4). We acknowledge the Core Facility and Technical Support of Wuhan Institute of Virology for technical assistance.
HW and FD designed the experiments. SD and YZ carried out the experiments. SD and TZ analyzed the data. SD, HW and FD wrote the paper. All the authors approved the final manuscript.
Compliance with Ethical Standards
Conflict of interest
The authors declared that they have no conflict of interests.
Animal and Human Rights Statement
This study was approved of the Wuhan Institute of Virology, Chinese Academy of Sciences. All institutional and national guidelines for the care and use of animals were followed.
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