Tetravalent recombinant dengue virus-like particles as potential vaccine candidates: immunological properties
© Liu et al.; licensee BioMed Central. 2014
Received: 2 April 2014
Accepted: 19 August 2014
Published: 18 December 2014
Currently, a licensed vaccine for Dengue Virus (DENV) is not yet available. Virus-like particles (VLP) have shown considerable promise for use as vaccines and have many advantages compared to many other types of viral vaccines. VLPs have been found to have high immunogenic potencies, providing protection against various pathogens.
In the current study, four DENV-VLP serotypes were successfully expressed in Pichia pastoris, based on co-expression of the prM and E proteins. The effects of a tetravalent VLP vaccine were also examined. Immunization with purified, recombinant, tetravalent DENV1-4 VLPs induced specific antibodies against all DENV1-4 antigens in mice. The antibody titers were higher after immunization with the tetravalent VLP vaccine compared to titers after immunization with any of the dengue serotype VLPs alone. Indirect immunofluorescence assay (IFA) results indicated that sera from VLP immunized mice recognized the native viral antigens. TNF-α and IL-10 were significantly higher in mice immunized with tetravalent DENV-VLP compared to those mice received PBS. The tetravalent VLP appeared to stimulate neutralizing antibodies against each viral serotype, as shown by PRNT50 analysis (1:32 against DENV1 and 2, and 1:16 against DENV3 and 4). The highest titers with the tetravalent VLP vaccine were still a little lower than the monovalent VLP against the corresponding serotype. The protection rates of tetravalent DENV-VLP immune sera against challenges with DENV1 to 4 serotypes in suckling mice were 77, 92, 100, and 100%, respectively, indicating greater protective efficacy compared with monovalent immune sera.
Our results provide an important basis for the development of the dengue VLP as a promising non-infectious candidate vaccine for dengue infection.
Dengue virus (DENV), a mosquito-borne RNA virus, belonged to the genus Flavivirus of the family Flaviviridae, is the cause of a range of well described clinical diseases, dengue fever (DF), dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF) ,. Globally, over 2.5 billion people are at risk for DENV infection in tropical and subtropical countries and regions, and approximately 50 to 100 million new cases of dengue infection and 500,000 cases of DHF and/or DSS occur annually . Prevention and control of widespread dengue infection has been a priority of the World Health Organization (WHO) for three decades; however, a licensed vaccine for DENV is not yet available .
There are four antigenically distinct serotypes of DENV, designated as DENV1-4. Dengue fever can be caused by any one of these four serotypes and life-long immunity against a distinct serotype can be established after infection. However, the severe forms of DENV infection, DHF and DSS, often occur when individuals are infected a second time by a different serotype -. The increased severity of subsequent infections is believed to result, at least in part, from antibody-dependent enhancement (ADE) of DENV infection, in which antibody-mediated neutralization does not occur. Instead, virus-antibody complexes facilitate viral entry into cells that express the Fcγ receptor, such as monocytes and macrophages -. For these reasons, an effective dengue vaccine should be able to induce long-lived protective immunity simultaneously against all four DENV serotypes.
Three structural proteins (capsid (C), premembrane/membrane (prM/M) and envelope (E)) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) are encoded in the RNA genome of the dengue virus. The C protein forms the main structural component of the nucleocapsid  and the formation of M, from prM, appears to be the crucial, terminal event in virion morphogenesis ,. The E protein is located on the surface of mature dengue virions and is the principal glycosylated structural protein (MW 51,000 to 60,000). The E protein mediates viral entry and induces a protective immune response -. The antibody response to DENV infection is mainly directed against the E and prM glycoproteins that are present on the virion surface -. Antibodies have been found to both neutralize and enhance DENV infectivity, in vivo and in vitro, and thus appear to play a dual role in controlling DENV infection ,-. Presumably, this adverse association might predict a potential risk for development of DHF or DSS in an individual who had previously been vaccinated with a specific DENV serotype and then is subsequently infected with a different serotype. Indeed, the majority of DENV vaccines under development are multivalent and aimed at producing immunity potent enough to protect against all four DENV serotypes .
Recent advances in genetic manipulation and vaccinology have lead to renewed hope for the development of live attenuated vaccines, subunit vaccines, DNA vaccines and viral-vector vaccines. Of particular interest is the effort to develop recombinant subunit vaccines, based on the viral proteins, capable of mimicking the overall structure of viral particles and inducing an optimal immune response. To this end, the virus-like particles (VLP) approach appears promising and advantageous over many other structural forms of vaccines. VLPs have been found to provide high immunogenic potency in protecting against various pathogens, such as human papillomavirus .
It has been demonstrated that co-expression of flavivirus prM and E, or alternatively, C, prM, and E led to production of VLPs that were composed of spherical membrane vesicles containing prM/M and E embedded in a lipid bilayer with or without a nucleocapsid, similar to the morphology of natural virions. These VLPs showed immunogenicity and were able to elicit neutralizing antibodies and virus-specific cytotoxic T lymphocytes -. Our previous studies demonstrated that co-expression of the prM and E proteins of DENV1, DENV2, and DENV3 produced antigenic DENV-VLP -. In the current study, DENV4-VLP was constructed and expressed, and the immunological properties and protection level of tetravalent DENV-VLP were evaluated in mice challenged with all four DENV serotypes. The data suggest that antibody responses and cellular immunity induced by monovalent and tetravalent DENV-VLP were comparable. Tetravalent DENV-VLP had better protective efficacy compared to monovalent VLP in suckling mice. The results indicate that tetravalent DENV-VLP could be a promising vaccine candidate.
Cells and viruses
C6/36 Aedes albopictus (ATCCNo.CRL-1660) was cultured at 28°C in MEM (Gibco, Guangzhou, China) supplemented with 0.11% of sodium bicarbonate and 10% newborn calf serum (Gibico, USA). BHK-21 Mesocricetus auratus was cultured in DMEM (Gibico, Guangzhou, China) supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C in 5% CO2. Each dengue virus serotype was passaged and propagated in C6/36 cells and titrated in BHK-21 cells. The DENV1 strain, GZ01/95 (GenBank accession No. EF032590), and DENV2 strain, ZS01/01 (GenBank accession no. EF051521), had been previously isolated, sequence-verified and preserved in our laboratory. DENV3 strain H87 and DENV4 strain H241 were supplied by the Institute for Viral Disease Control and Prevention, China CDC. The viral titer was determined after removal of cell debris via centrifugation. The virions used for mice immunization were inactivated with 1:2000 β-propionolactone and the viron concentration was subsequently detected using the BCA method (Biocolor, Shanghai, China).
Construction of DENV-VLP expression plasmids
The P. pastois host strain, X33 (Invitrogen, Guangzhou, China), and the expression vector, pGAPZαA (Invitrogen), have been previously described in detail ,. The cDNA of virions of each DENV serotype was obtained by RT-PCR and the genes coding for the prM and E proteins were amplified. The amplified prM-E genes were subsequently linearized and ligated into the pGAPZαA (Invitrogen) vectors in frame with the α-factor secretion signal (for DENV1/2-VLP expression) or the signal peptide of prM (for DENV3/4-VLP espression). The recombinant plasmids for expressing DENV1-4 VLP were named pGAPZα-prME-D1, pGAPZα-prME-D2, pGAPZ-sprM/E-D3, and pGAPZ-sprM/E-D4.
Expression and purification of DENV-VLP
Expression and purification of DENV-VLP was done as previously described -. Briefly, the four recombinant plasmids were electroporated into the Pichia pastoris host strain, X33. The yeast cells were then harvested and disrupted with glass beads in breaking buffer (50 mmol/L sodium phosphate, pH 7.4; 1 mmol/L ethylene diamine tetraacetic acid (EDTA); 1 mmol/L phenylmethyl sulfonylfluoride (PMSF); and 5% glycerol). The lysates were subjected to ultracetrifugation at 153 000 × g for 6 hours at 4°C (HATICHI, P80AT rotor, Ibaraki Prefecture, Japan) using a 5 ~ 50% sucrose density gradient before the sucrose fractions were collected and analyzed via Western blot, using the MAb 15 F3-1 (ATCC, USA), 3H5-1 (ATCC, USA), 5D4-11 (ATCC, USA), and IH10-6 (ATCC, USA) to detect E proteins expression for each DENV-VLP serotype. The formation of VLP was demonstrated using electron microscopy. Finally, protein concentration was assessed using the BCA method (Biocolor, Shanghai, China).
Virus titer assay
Virus quantitation was determined using a standard plaque assay as previously described ,. Briefly, 0.25 mL of DENV dilutions (10-fold) was added to duplicate wells of BHK-21 cells, which were cultured overnight in 24-well plates before the media was removed. The plates were incubated for 2 h after which the media was aspirated and replaced with 0.8 mL of 0.8% methyl-cellulose medium (with 4% newborn calf serum). The plates were then incubated for 7 days, the media was removed and the cells were fixed in 4% formaldehyde and stained with crystal violet. Plaques were counted visually and the concentrations of plaque-forming units per mL (PFU/mL) were calculated.
Experiments with mice were conducted in compliance with a protocol approved by the Institutional Animal Care and Use Committee of Sun Yat-sent University based on the Ethical Principles in Animal Experimentation. Specific pathogen-free female BALB/c mice, aged three to four weeks, were supplied by the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China) and were divided into ten groups according to immunogen. The mice were inoculated intraperitoneally (i.p.) with monovalent DENV-VLP (25 μg per dose and in 15 mice (n = 15)) of a specific serotype or a tetravalent combination (25 μg of each serotype per dose and in 30 mice (n = 30)). Freund’s complete adjuvant (Sigma) was used for priming and Freund’s incomplete adjuvant was used for boosters. Booters were twice at an interval of 2 weeks. Equal amounts of PBS (n = 30) or inactivated DENV virions (25 μg per dose and in 15 mice (n = 15)) were used as controls. Blood samples were collected on days 0, 14 and 28, via the tail vein, for measurement of serum IgG. Seven days after the last inoculation, 1/3 of the mice in each group were euthanized. The spleen of each mouse was removed and splenocytes were isolated in order to test cytokine profiles and serum was collected for further immunological analysis.
Enzyme-linked immunosorbent assay
Antigen specific serum IgG antibodies were titered using an amplified sandwich ELISA system. Briefly, 96 wells polystyrene plates (Costar, USA) were coated over night at 4°C with 100 μL/well of 5 ng/ml inactivated dengue virus antigen or VLP antigen and then blocked in coating buffer containing 5% fat-free milk powder for 1 h at 37°C. The plates were then incubated with 100 μl/well of sera from each group, along with a 2-fold serial dilution of PBS-T (starting from 1:100), at 37°C for 1 h. Bound IgG was detected using goat anti-mouse IgG-peroxidase conjugate (Santa Cruz, USA). A volume of 3,3′, 5,5′-tetramethly benzidine (TMB) substrate was then added and plates were incubated at 37°C for 15 min. The reaction was stopped with 50 μl of 2 M H2SO4. Absorbance was measured at 450 nm using an automated ELISA reader (EL×800 BioTek). An absorbance value two-fold above the mean pre-vaccine serum value plus two standard deviations was considered to be a positive result.
C6/36 cells were passaged in MEM medium containing 10% fatal bovine serum (FBS) and used to be infected with 100 PFU of each DENV serotype. Cells were harvested 2 ~ 4 days post-infection, resuspended in MEM containing 10% newborn calf serum, dropped onto slides and incubated at 37°C for 8 h. The slides were then fixed with acetone at −20°C for 15 min. Antisera diluted 1:80, was then added to the slides and the slides were incubated at 37°C for 1 h. Normal mouse sera, diluted 1:80, was used as the negative control. Lastly, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG was added and the slides were incubated at 37°C for 45 min. Positive cells were detected using a fluorescent microscope.
Cytokines ELISPOT assay
The cytokines, IFN-γ, TNF-α, and IL-10, from immunized mice were measured using ELISPOT kits (Ucytech, The Netherlands), according to the manufacturer’s instructions. Briefly, ELISPOT 96-well plates (Millipore, USA) were coated with 100 μl of anti-mouse IFN-γ, anti-mouse IL-10, or anti-mouse TNF-α (5 μg/ml in coating buffer). The plates were washed twice and blocked with blocking solution for 2 h. A volume of 100 μL freshly isolated splenocytes (2 × 105 cells) from the immunized mice were subsequently transferred to each well and stimulated with dengue virus at 37°C for 24 h. The cells were then washed away, and a secondary biotinylated anti-cytokine mAb was added to each well, followed by a streptavidin-HRP and AEC substrate solution system. Finally, the spots were counted using an ImmunoSpot® Analyzer (Cellular Technology Ltd. USA).
Antibody neutralization assay
The neutralizing ability of antibodies was measured using a 50% plaque reduction neutralization test (PRNT50). Briefly, BHK-21 cells were grown to 80% confluence in 24-well plates and infected with 200 μL DENV1 to 4 (150 ~ 200 PFU/mL). The plates were then pre-incubated with two-fold serial dilutions of mouse serum (1:4 ~ 1:64) at 37°C for 1 h. The virus-serum mixture was aspirated, added along with 0.8 mL overlay medium, and incubated at 37°C with 5% CO2 for 7 days. Dengue virus plaques were counted by naked eye or by scanning the cluster plate into Adobe Photoshop CS for further magnification. PRNT50 titers are defined as the maximal serum dilution that inhibits 50% of plaque formation compared to the number of plaques in infected cell wells with no sera.
Suckling mice passive protection assay
Newborn (1 day old) BALB/c mice were purchased from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China) and were divided into twelve groups. The 1:10 sera dilution was incubated with each DENV serotype (400PFU) at 37°C for 1 h. Suckling mice were injected intracerebrally with 20 μL of sera-virus mixture. Morbidity (paralysis, ruffling, slowing of activity, kyphoscoliosis and death) and/or mortality were recorded daily, for 3 weeks, post-challenge in all mice.
Data were analyzed for statistical significance using SPSS software (version 13.0). Statistical comparisons among groups were analyzed by one way ANOVA. Kaplan-Meier survival curves used for survival analysis and were analyzed by the log rank test.
Expression and purification of DENV-VLP
Immunogenicity of purified tetravalent DENV-VLP
Cytokine production measured by ELISPOT
Spleen cells were isolated on day 34 (7 days after the last immunization) to assess the cellular immune response to tetravalent DENV VLP. IFN-γ, TNF-α, and IL-10 cytokine secretion was analyzed, via ELISPOT, following their ex vivo stimulation of the cells with inactivated DENV1-4 virions.
Virus neutralizing antibodies induced by DENV-VLP in mice
Protection of suckling mice from viral challenge
VLPs have similar structural and physicochemical features to infectious virions, however they are non-infectious and have advantages in terms of safety and manufacturing. An important characteristic of VLPs is that they can elicit strong humoral and cellular immune responses against viruses -. Recombinant flaviviruses VLPs have been shown to be able to be efficiently produced by co-expression of prM and E proteins, either with or without C proteins ,. Our previous study demonstrated that DENV-VLP could be successfully expressed in Pichia pastoris, based on co-expression of prM and E proteins -. In that study, it was shown that vaccination with DENV-VLP efficiently elicited virus-specific humoral and cellular immune responses in BALB/c mice. In the current study, the immunogenicity and protective effect of a vaccine made up of four DENV-VLP serotypes was evaluated.
The analysis of humoral immune responses revealed that each of the four dengue VLP serotypes induced antigen specific IgG, similar to the corresponding inactivated dengue virions. The antibody levels were higher in DENV1 and 2-VLP vaccinated groups than in the DENV3 and 4-VLP groups and in the tetravalent VLP vaccinated group than in monovalent groups. Indirect immunofluorescence assay results showed that the antisera from the dengue VLP vaccinated groups could bind with natural dengue virus antigen. In addition, cross-reactions were found between the immune sera from the four DENV-VLP serotypes and the natural DENV virions (data not shown). The results suggest that joint immunization with polyvalent antigens may have a synergistic effect in stimulation of the antibody production.
VLPs not only induce antibody production, but they also have the ability to induce cellular immunity ,. In the present study, cellular immune responses were assessed by the ability of VLPs-stimulated spleen cells to release IFN-γ, TNF-α, and IL-10 cytokines. The number of TNF-α and IL-10 secreting splenocytes was significantly higher in mice that received tetravalent DENV-VLP compared to PBS controls. However, neither IFN-γ nor TNF-α secreting splenocytes were significantly higher in the monovalent DENV-VLP groups (data not shown). IFN-γ is a type of Th1 cytokine, that has an anti-viral effect and also enhances TNF-α production by DENV-infected cells. In addition, high systemic levels of TNF-α have been shown to cause capillary leakage . Serum levels of TNF-α and its receptors have been shown to be elevated in DENV infection and to correlate with DENV disease severity ,. In particular, it has been suggested that IL-10, a Th2 cytokine, to suppress type I IFN-mediated antiviral activity . In addition to the above three cytokines, many other cytokine/chemokines are considered to be correlated with dengue disease severity, including IL-1 β, IL-2, IL-6, IL-8, IL-10, IL-13, IL-18, IFN-γ, TNF-α, and MCP1 ,-. Results of the current study are not enough to evaluate Th1 or Th2 immune responses produced by tetravalent DENV-VLP or the corresponding dynamic variation. Further studies of T-cell responses are needed to understand the immune status of mice immunized with the tetravalent DENV-VLP.
The protective efficacy of tetravalent DENV-VLP was demonstrated with an antibody neutralization assay in vitro and in suckling mice in vivo. PRNT50 results showed that immunization with either monovalent or tetravalent DENV-VLP induced neutralization antibodies against dengue virus. The PRNT50 titers from immune sera of mice immunized with DENV1 and 2-VLP were higher than titers from immune sera of mice immunized with DENV3 and 4-VLP; however, these titers were all higher than immune sera titers reported after immunization with the tandem DENV1-4 domain III of the E protein  and higher than the DENV1-4 VLP level expressed in 293 T cell immune sera . The DENV-VLP in the present study showed good immunogenicity and neutralization efficacy, though there are still some differences in efficacy among the four DENV serotypes. The PRNT50 titers of immune sera from mice immunized with tetravalent DENV-VLP were a little lower than those from mice immunized with the corresponding monovalent DENV-VLP. Since the antibody levels detected by ELISA and IFA were also higher in the tetravalent VLP group than in the monovalent groups, we deduced that there was no significant relationship between antibody level and neutralization efficacy, particularly since more non-neutralizing antibody against a certain dengue serotype might exist in the tetravalent DENV-VLP immune sera.
The protective efficacy of the tetravalent DENV-VLP vaccine candidate was further tested in a suckling mouse model. The results showed that immunization with either monovalent or tetravalent DENV1-4 VLPs conferred at least some protection, in suckling mice, against DENV1-4 challenge. Furthermore, although the neutralizing antibody level detected was a little lower in the tetravalent formula of DENV1-4 VLP in vitro, the tetravalent formula provided more protective efficacy in vivo than did the monovalent vaccination. This suggests that the in vivo immune response is more complex, and that in vivo protective efficacy might be dependent on various immunologic mechanisms and not just the neutralizing antibody. Moreover, the tetravalent formula had better protective efficacy against DENV3 and 4 compared to DENV1 or 2, suggesting that further study of the compatibility of tetravalent DENV1-4 VLP is needed.
In conclusion, the construction of a tetravalent DENV1-4 VLP vaccine candidate, based on Pichia pastoris, is described. The tetravalent DENV-VLP induced humoral immune responses against all four dengue virus serotypes as measured by ELISA or IFA and also induced cellular immunity. The antibody levels were higher, though the neutralizing titers were somewhat lower with tetravalent than with monovalent immunization. Moreover, protective efficacy in the suckling mouse model was better with the tetravalent than with the monovalent vaccination. Further study is needed to adjust the compatibility of the tetravalent DENV1-4 VLP formula and to clarify the amount of cellular immunity that is induced.
This work was supported by grants from the National Science Foundation (No. U0632002, U1132002, 31270974, 81261160323), National High Technology Research Development Program of China (No. 2006AA02A223), and Guangdong Province Science and Technology Project (2011B031800360).
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