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E and NS1: Their immunity and protective efficacy in mice
Molecular Immunology 54 (2013) 109–114
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Preliminary evaluation of DNA vaccine candidates encoding dengue-2 prM/E and NS1: Their immunity and protective efficacy in mice Hui Lu a , Xiao-Feng Xu b , Na Gao a , Dong-Ying Fan a , Juan Wang a , Jing An a,∗ a b
Department of Microbiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, PR China Departments of Medical Genetics, Third Military Medical University, Chongqing 400038, PR China
a r t i c l e
i n f o
Article history: Received 10 July 2012 Received in revised form 15 November 2012 Accepted 18 November 2012 Available online 25 December 2012 Keywords: Dengue virus DNA vaccine GM-CSF
a b s t r a c t Public health is still seriously threatened by dengue virus (DENV) and no vaccine against DENV is yet available for clinical use till now. In this study, DNA vaccine candidates encoding DENV serotype 2 (DENV2) prM/E (premembrane and envelope proteins) and NS1 (non-structural 1 protein) with or without a gene adjuvant, granulocyte-macrophage colony-stimulating factor (GM-CSF), were evaluated in the aspects of immunity and protective efficacy in mice. We constructed three plasmids, pCAG-prM/E (which only expressed DENV2 prM/E), pCAG-prM/E/NS1 (which only expressed DENV2 prM/E/NS1) and pCAGDG (which co-expressed DENV2 prM/E/NS1 and GM-CSF). The expressions of the recombined plasmids were analyzed by immuno-staining in Vero cells. Antibody responses and neutralization activity of the sera from the mice were assayed by ELISA and plaque reduction neutralization test after immunization with the plasmids. Immunized BALB/c mice were intracerebrally challenged with DENV2 to evaluate protective efficacy of the plasmids. The recombinant plasmids could be efficiently expressed in Vero cells and induced different levels of specific anti-DENV2 immune responses. The immunized mice were partially protected. The highest survival rate was observed in the pCAG-DG group although the antiDENV2 titer and neutralization antibody titer were not the highest among the three groups. Our data suggested that pCAG-DG offered better protection against DENV2 infection. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Dengue viruses (DENV), belonging to the Flaviviridae family, are composed of four distinct serotypes (DENV1–4). The genome of the viruses is a single-stranded positive-sense RNA of approximately 11 kb that encodes three structural proteins, capsid (C), premembrane (prM) and envelope (E) proteins, and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). DENV are transmitted to humans by the mosquitoes, Aedes aegypti, and can cause the self-limiting dengue fever (DF), the more severe dengue hemorrhagic fever (DHF) or fatal dengue shock syndrome (DSS) throughout tropical and subtropical regions of the world. It is estimated that up to 100 million infections of dengue occur annually, resulting in about 500,000 cases of DHF and 24,000 deaths each year (Rigau-Pérez et al., 1998). Dengue infection is still a severe public health problem. Considerable research had been done toward the development of DENV vaccines (Chambers et al., 1997). The most exciting among these was the chimeric Yellow fever dengue tetravalent vaccine (CYD, ChimeriVax), which has already entered clinical trial
∗ Corresponding author. Tel.: +86 10 8391 1741; fax: +86 10 8391 1496. E-mail addresses: [email protected], [email protected] (J. An). 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.11.007
(Guy et al., 2008). Although CYD, ChimeriVax was very promising, potential complications remain, and studies have shown that the tetravalent vaccine formulations elicit unbalanced immune responses due to viral interference (Guirakhoo et al., 2002). Such interference is particularly an important concern for dengue vaccines because the unbalanced immune responses may lead to increased disease severity when the vaccinated host acquires an infection with one of the four serotypes for which the induction of immunity is insufficient (Imoto and Konishi, 2007). Therefore, none of the traditional vaccine against DENV is yet available for clinical use till now. DNA vaccines are able to induce long-lasting cellular and humoral immunity against some pathogens including flaviviruses (Donnelly et al., 1997). One of the potential advantages was the expression of viral proteins in situ after DNA immunization, leading to proper folding and posttranslational modifications, which originally occur during the course of natural viral infections (Schlesinger et al., 1987). In studies of DENV DNA vaccines, prM/M, E, NS1 and NS3 proteins were usually used as target antigens because they could elicit protective immune responses. The E protein, the viral major surface protein, contains important neutralizing epitopes. The prM protein is necessary for the proper processing and expression of the E protein. The NS1 protein associates with the E protein in the ER (endoplasmic reticulum) lumen and is involved in virion
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maturation. It can induce a strong antibody response and protect hosts through an Fc-dependent complement-mediated manner (Schlesinger et al., 1993). In fact, several investigations reported inoculation of plasmids containing flaviviruses prM, E, or NS1 gene to elicit specific immune responses in mice (Schlesinger et al., 1987; Ulmer et al., 1993; Donnelly et al., 1997; Colombage et al., 1998). But the efficiency of these DNA vaccines was poor. In our previous studies, we found that granulocyte-macrophage colony-stimulating factor (GM-CSF) was an effective gene adjuvant, which enhanced the immune and protective effects against Japanese encephalitis virus (JEV) and DENV1 when co-expressed with their antigens (Gao et al., 2010; Zheng et al., 2011). In this report, we constructed three recombinant plasmids containing either prM/E of DENV2 (pCAGprM/E), prM/E/NS1 of DENV2 (pCAG-prM/E/NS1) or prM/E/NS1 fused to GM-CSF (pCAG-DG) and analyzed their immunogenicity and protective role in mice. This will allow us to evaluate whether this strategy of DNA vaccine construction could be more widely applied in other DENV serotypes and viruses.
subcloned into the vectors. The expression of these plasmids was further confirmed by indirect immuno-fluorescence in Vero cells. For immunization, the plasmids were extracted and purified with an endotoxin-free plasmid extraction kit (Omega, Norcross, America) from transformed Escherichia coli JM109. Then, the purified plasmids were dissolved in sterile saline and adjusted to 1.0 mg/ml before use. 2.3. Mouse experiments All of the experiments with mice were conducted in compliance with Ethical Principles in Animal Experimentation. For DNA immunization, 6-week-old female BALB/c mice were used. To enhance the uptake of plasmid DNA, mice were prior injected with 50 l 0.25% lidocaine hydrochloride in each quadriceps muscle the day before immunization. Each mouse was inoculated intramuscularly with 100 g of the plasmids at 3-week intervals for three times. Mice injected with pCAG served as negative controls. The mice were finally euthanized. For the protection test, three weeks after the last immunization, the mice were challenged intracerebrally with a lethal dose (50× LD50) of DENV2. The mice were monitored daily for the morbidity and mortality for 3 weeks.
2. Materials and methods 2.1. Cells lines, virus, and mice Aedes albopictus mosquito cells (C6/36) were grown at 28 ◦ C in RPMI (Roswell Park Memorial Institute) 1640 containing 10% fetal bovine serum (FBS, Gibco, Auckland, New Zealand) and were used to propagate DENV-2 (strain TR1751) which was isolated from a patient with DF and was kindly provided by Dr. A. Oya (National Institute of Infectious Disease, Japan). Vero cells were cultivated at 37 ◦ C in Eagle’s minimal essential medium (MEM, Gibco, Buffalo, America) supplemented with 5% FBS. Female inbred BALB/c mice were purchased from the Academy of Military Medical Sciences (Beijing, China). All of the mice were maintained in specific pathogen-free environments.
2.4. Antibody assay Mice sera samples were collected by tail bleeding at three weeks after the last immunization and analyzed for the presence of DENV2 antibodies by an enzyme-linked immunosorbent assay (ELISA). Briefly, Two-fold serial dilutions of the serum samples were added to wells coated with concentrated virus. After incubation and washing with TPBS, each well was incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (KPL, Houston, America). Afterward, the plates were washed five times with TPBS, and the color was generated by using the HRP substrate, orthophenylene diamine (OPD), and the action was stopped by addition of H2 SO4 . After measurement of the absorbance at 490 nm, the end-point titers of anti-DENV2 antibodies were determined as the reciprocal of the highest dilution, giving an optical density (OD) twice that of the non-immune serum. To determine the IgG subclass, antibody isotype ELISAs were similarly performed using anti-mouse IgG1-HRP or anti-mouse IgG2a-HRP (SBA, Oregon, America) replacing the former anti-mouse IgG-HRP.
2.2. Construction of plasmids We used pCAG and pIRES (Qiagen) to construct the DNA vaccines. First, viral RNA was isolated from DENV2-infected C6/36 cells with the Biozol reagent (Bioflux, Tokyo, Japan), according to the manufacturer’s protocol, and reversely transcribed to cDNA using primer DENV (−): CTC CCG CTC ATC AAG AAT AA by reverse transcriptase M-MLV (Invitrogen, Grand Island, America). Different segments of DENV2 were then amplified by PCR using the primers shown in Table 1. Then, the amplified fragments were cloned into the pCAG vector using the multiple cloning sites. Furthermore, we constructed a bicistronic plasmid expressing simultaneously DENV2 prM-E-NS1 and GM-CSF under the control of a single promoter. Briefly, DENV-2 prM-E-NS1 and GM-CSF fragments were inserted into the multiple cloning sites A and B of the pIRES, respectively. Then, the total prM-E-NS1-IRES-GM-CSF fragment was digested with the XhoI and NotI restriction enzymes (MBI, Glen Burnie, America) and subcloned into pCAG. DNA sequencing was used to verify the cloned fragments and they were proper
2.5. Plaque reduction neutralization test (PRNT) To determine the presence of the neutralizing antibodies (NAbs) in the sera of inoculated mice, sera were diluted twofold in MEM containing 2% FBS. Sera were heated at 56 ◦ C for 30 min to inactivate complement. Serial dilutions were incubated with equal volumes of DENV2 virus at 37 ◦ C for 1 h. Then the mixtures containing 100 PFU/well of the virion were added in duplicate to Vero cell monolayers seeded in 24-well plates. After adsorption for 1 h, Vero cells were then washed and incubated in MEM containing 5% FBS and 1.3% methyl-cellulose. After 1 week of incubation at 37 ◦ C in 5%
Table 1 Constructions of plasmids including the components of DENV-2. Recombinant plasmids
Amplified regions
pCAG-prM/E
prM/E (367-2421)
pCAG-prM/E/NS1
prM/E/NS1 (367-3477)
pIRES-prM/E/NS1
prM/E/NS1 (367-3477)
Primers
Restriction sites
5 -ggcc ctcgag ATG CTG AAC ATC TTG AAC A-3 5 -cgta gcggccgc CTA GGC CTG CAC CAT AAC TCC-3 5‘-ggcc ctcgag ATG CTG AAC ATC TTG AAC A-3 5 -cgta gcggccgc CTA GGC TGT GAC CAA GGA GTT-3 5 -ggcc ctcgag ATG CTG AAC ATC TTG AAC A-3 5 -cggc acgcgt CTA GGC TGT GAC CAA GGA GTT-3
XhoI NotI XhoI NotI XhoI MluI
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CO2, the plaques were stained with crystal violet and counted. The NAb titer was calculated as the reciprocal of the maximum dilution of serum that yielded a 90% plaque reduction (PRNT90) and a 50% plaque reduction (PRNT50) when compared to the virus controls without serum. 2.6. Indirect Immuno-fluorescence Staining The DENV2-infected or plasmids transfected Vero cells were used for the experiments of indirect immuno-fluorescence staining. In brief, purified DENV2 was used as antigen and BALB/c were immunized to induce specific anti-DENV2 antibodies (Chen et al., 2009). Cells were fixed with 4% polyformaldehyde and permeabilized with 0.2% Triton X-100. After washing and blocking, cells were incubated with monoclonal anti-DENV2 E antibody (abcam, Tokyo, Japan), rat anti-GM-CSF antibody (BD, Franklin Lakes, America) or mouse anti-DENV2 antibodies at 4 ◦ C overnight. The following day, the cells were incubated with FITC-conjugated goat anti-mouse IgG1 (Sigma, Santa Clara, America). The specimens were analyzed by fluorescent microscope (Leica). Images were processed using Adobe Photoshop (Adobe Systems). 2.7. Statistical analysis All of the data were analyzed using SPSS software (version 12.0). Data were considered to be statistically significant when P < 0.05. 3. Results 3.1. Immunizations with DNA vaccine candidates could elicit mouse antibody responses To test the expression of the recombinant plasmids in eukaryotic cells, Vero cells were transfected with pCAG-prM/E,
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pCAG-prM/E/NS1, pCAG-DG and pCAG followed by indirect immuno-fluorescence analysis with a monoclonal anti-DENV2 E antibody (Fig. 1a) and rat anti-GM-CSF (Fig. 1b) antibody. Results show that specific reactive signals were observed in the cytoplasm after transfection with pCAG-prM/E, pCAG-prM/E/NS1 or pCAG-DG while no specific fluorescent signals were detectable after transfection with the control plasmid pCAG. This indicated that all plasmids vaccines candidates could be efficiently expressed and were suitable for the subsequent experiments. In antibodies analysis, the mice inoculated with pCAG-prM/E, pCAG-prM/E/NS1 and pCAG-DG showed higher specific antibody titers compared to the control group, pCAG (P < 0.05). Among the three DNA vaccines, the group of pCAG-prM/E induced the highest anti-DENV2 antibody titer (1:3, 200), whereas the antibody titer in the pCAG-DG group (1:1, 600) was twofold lower. The mice vaccinated with pCAG-prM/E/NS1 elicited only moderate anti-DENV2 titer (1:800) (Fig. 2). Furthermore, the sera obtained from the mouse experiments were subjected to indirect immunofluorescence staining. As shown in Fig. 3, sera of mice immunized with the three DNA vaccine candidates could specifically recognize the antigens in the DNEV2-infected Vero cells while the serum from the control only showed a faint fluorescent background signal. These results suggested that our three DNA vaccine candidates could efficiently and specifically elicit immune responses against DNEV2 in mice. In addition, DENV2-specific IgG1 and IgG2a in the sera were also examined by ELISA. In this test, the mice inoculated with pCAG-prM/E, pCAG-prM/E/NS1 and pCAG-DG showed higher specific IgG1 and IgG2a antibody titers compared to the control group, pCAG (P < 0.05). Among these groups, pCAG-prM/E and pCAG-DG induced similar levels of anti-DENV2 IgG1 and IgG2a responses, while pCAG-prM/E/NS1 showed a decreased IgG1/IgG2a ratio in the anti-DENV2 antibody (Fig. 2). Since the IgG1 isotype is generated by the Th2 immune response and the IgG2a isotype by Th1
Fig. 1. (a) The indirect immuno-fluorescence analysis with a monoclonal anti-DENV2 E antibody of the expression of recombinant plasmids in Vero cells. (b) The indirect immuno-fluorescence analysis with a monoclonal rat anti-GM-CSF of the expression of recombinant plasmids in Vero cells.
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3500
*
Nab titers
3000
PRNT90 PRNT50
2500 2000
*
1500
*
1000 500 0
* pCAG
pCAG-prM/E
* pCAGprM/E/NS1
* pCAG-DG
Fig. 4. Nab titers induced by DNA vaccine candidates by ELISA.
morbidity (Fig. 5). Interestingly, the group of pCAG-DG had the highest survival rate although its anti-DENV2 titer and NAb titer were not among the highest. Fig. 2. Anti-DENV2 antibody titers elicited by the DNA vaccine candidates.
in mice, our data suggested that the plasmid vaccinations could induce mixed Th1/Th2 immune responses and the NS1 of DENV2 might induce the more Th1 immune response. 3.2. Vaccination with DENV2 DNA vaccines elicits antibodies with neutralizing activity To assay NAb titers induced by DNA vaccine candidates, PRNTs were performed with sera samples from mice at three weeks after the final immunization. With the subtraction of the control’s background noise, the NAb titers of pCAG-DG were 80 in PRNT90 and 870 in PRNT50, 170 in PRNT90 and 1050 in PRNT50 for pCAG-prM/E and 100 in PRNT90 and 2770 in PRNT50 for pCAG-prM/E/NS1, respectively (Fig. 4). All of our DNA vaccine candidates obviously induced NAbs compared with the control group, pCAG, (P < 0.05), but the levels of NAbs were different among the three immune groups. 3.3. DNA vaccines candidates partially protect immunized mice from DENV2 challenge To evaluate the protection of the DNA vaccine candidates, vaccinated BALB/c mice were challenged three weeks after the final immunization with 50× LD50 of DENV2. None of the control mice immunized with pCAG survived after the DENV2 challenge in this group. In contrast, the mice immunized with pCAG-prM/E, pCAG-prM/E/NS1 and pCAG-DG were partially protected with respectively 30%, 40% and 60% survival rate and a 2-day delay in
4. Discussion Public health is still seriously threatened by DENV though great effort has been carried out to develop DENV vaccines (Rigau-Pérez et al., 1998). Since there are no safe and efficient vaccines against DENV available, we and other researchers attempt to develop a new vaccine devoid of the shortcomings of the traditional vaccine candidates. In this research, we had constructed three DNA vaccine candidates expressing DENV2 proteins in eukaryotic cells including one, pCAG-DG with co-expression a gene adjuvant, GM-CSF. As the data of intracerebrally challenge experiment show, the three DNA vaccine candidates could partially protected BALB/c mice from the lethal infection of DENV2. The strategy of co-expression a gene adjuvant, GM-CSF, with the DENV2 prM/E/NS1 gene provided promising results since a survival rate of 60% was obtained. Although it did not induce the highest antibody response and NAb titer, pCAG-DG might induce a more efficient and balanced humoral and cellular response. In fact, pCAG-prM/E could induce a higher anti-DENV2 antibody titer while pCAG-prM/E/NS1 elicited a stronger Th1 immune response. However, the protection with pCAG-prM/E in mice was not as good as with pCAG-prM/E/NS1 after intracerebrally challenge. Although M and E proteins of DNVE are the major targets of neutralizing antibodies since they play important roles in receptor binding and membrane fusion, they also possess specific epitopes present in different DENV serotypes (Costa et al., 2006; Kaufman et al., 1989). This determines that the vaccines used M and E proteins both as immunogens increase the humoral immune response and the risks of ADE. On the other hand, immunization with the NS1 protein can confer protection without this risk (Huisman et al., 2009; Krishna et al., 2009), so NS1 was
Fig. 3. DNEV2-infected Vero cells stained with sera of mice immunized with DNA vaccine candidates.
H. Lu et al. / Molecular Immunology 54 (2013) 109–114
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The survival rates (%)
100 90 80 70 60 50 40 30 20 10 0
1
2
3
4
5
pCAG
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21
pCAG-prM/E
pCAG-prM/E/NS1
pCAG-DG
Fig. 5. The survival rates of immunized mice with plasmids.
also considered for development of DNA vaccines even though its ability of elicitation of NAbs was much weaker. The ability of DNA immunization to elicit both antibody and CTL immunity makes it an ideal vaccination approach to stimulate the host defense against viral infection. Despite the potential advantages of a DNA vaccine, it is sometimes difficult to induce a sufficient effective immunity against viral antigens. Numbers of strategies have been explored to improve the immune response of DNA vaccine. Cytokines, as a novel molecule adjuvant, has shown great promise in promoting immune responses. Given its central role in antigen presentation and recruitment of antigen presenting cells, GM-CSF represented an attractive adjuvant candidate and was a good choice in many studies (Whitehead et al., 2007; Sabchareon et al., 2004; Sun et al., 2006). Previous studies have shown that co-administration of plasmid GM-CSF as an adjuvant, using different protocols, could increase the immune response and improve the protective efficacy against challenge, including DNA vaccines of DENV (Raviprakash et al., 2003). Considering this information, plasmids expressing prM-E-NS1 with or without the GM-CSF adjuvant were constructed, and their immunogenicity and protective effects were investigated in this study. Compared with our previous work (Gao et al., 2010; Zheng et al., 2011), pCAG-DG did not induce high anti-DENV2 antibody and NAb titer and this result was different from those of the constructions with DENV1 and JEV. This suggests that the components of DENV2 might have mechanism to induce the immune responses that are different from the mechanisms involved in DNA vaccines from the other flaviviruses. Further work should be carried out to unravel this interesting problem. 5. Conclusion We had successfully constructed three DNA vaccine candidates expressing components of DENV2 viruses. The DNA vaccine candidates could express DENV2 proteins, elicit specific immune responses and partially protect BALB/c mice from a DENV2 intracerebrally challenge. pCAG-DG, co-expressing prM/E/NS1 of DENV2 and GM-CSF, had the highest survival rate though its antiDENV2 antibody titer and NAb were not the highest compared to those obtained with pCAG-prM/E and pCAG-prM/E/NS1. Our data showed that the strategy of developing a DNA vaccine with GM-CSF as a gene adjuvant shows promising prospects in the battle against DENV2. Acknowledgments This work was supported by grants 2011CB504703 from the National Key Programs on Basic Research of China and 31100131 from the National Natural Science Foundation of China.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Preliminary evaluation of DNA vaccine candidates encoding dengue-2 prM/E and NS1: Their immunity and protective efficacy in mice Hui Lu a , Xiao-Feng Xu b , Na Gao a , Dong-Ying Fan a , Juan Wang a , Jing An a,∗ a b
Department of Microbiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, PR China Departments of Medical Genetics, Third Military Medical University, Chongqing 400038, PR China
a r t i c l e
i n f o
Article history: Received 10 July 2012 Received in revised form 15 November 2012 Accepted 18 November 2012 Available online 25 December 2012 Keywords: Dengue virus DNA vaccine GM-CSF
a b s t r a c t Public health is still seriously threatened by dengue virus (DENV) and no vaccine against DENV is yet available for clinical use till now. In this study, DNA vaccine candidates encoding DENV serotype 2 (DENV2) prM/E (premembrane and envelope proteins) and NS1 (non-structural 1 protein) with or without a gene adjuvant, granulocyte-macrophage colony-stimulating factor (GM-CSF), were evaluated in the aspects of immunity and protective efficacy in mice. We constructed three plasmids, pCAG-prM/E (which only expressed DENV2 prM/E), pCAG-prM/E/NS1 (which only expressed DENV2 prM/E/NS1) and pCAGDG (which co-expressed DENV2 prM/E/NS1 and GM-CSF). The expressions of the recombined plasmids were analyzed by immuno-staining in Vero cells. Antibody responses and neutralization activity of the sera from the mice were assayed by ELISA and plaque reduction neutralization test after immunization with the plasmids. Immunized BALB/c mice were intracerebrally challenged with DENV2 to evaluate protective efficacy of the plasmids. The recombinant plasmids could be efficiently expressed in Vero cells and induced different levels of specific anti-DENV2 immune responses. The immunized mice were partially protected. The highest survival rate was observed in the pCAG-DG group although the antiDENV2 titer and neutralization antibody titer were not the highest among the three groups. Our data suggested that pCAG-DG offered better protection against DENV2 infection. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Dengue viruses (DENV), belonging to the Flaviviridae family, are composed of four distinct serotypes (DENV1–4). The genome of the viruses is a single-stranded positive-sense RNA of approximately 11 kb that encodes three structural proteins, capsid (C), premembrane (prM) and envelope (E) proteins, and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). DENV are transmitted to humans by the mosquitoes, Aedes aegypti, and can cause the self-limiting dengue fever (DF), the more severe dengue hemorrhagic fever (DHF) or fatal dengue shock syndrome (DSS) throughout tropical and subtropical regions of the world. It is estimated that up to 100 million infections of dengue occur annually, resulting in about 500,000 cases of DHF and 24,000 deaths each year (Rigau-Pérez et al., 1998). Dengue infection is still a severe public health problem. Considerable research had been done toward the development of DENV vaccines (Chambers et al., 1997). The most exciting among these was the chimeric Yellow fever dengue tetravalent vaccine (CYD, ChimeriVax), which has already entered clinical trial
∗ Corresponding author. Tel.: +86 10 8391 1741; fax: +86 10 8391 1496. E-mail addresses: [email protected], [email protected] (J. An). 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.11.007
(Guy et al., 2008). Although CYD, ChimeriVax was very promising, potential complications remain, and studies have shown that the tetravalent vaccine formulations elicit unbalanced immune responses due to viral interference (Guirakhoo et al., 2002). Such interference is particularly an important concern for dengue vaccines because the unbalanced immune responses may lead to increased disease severity when the vaccinated host acquires an infection with one of the four serotypes for which the induction of immunity is insufficient (Imoto and Konishi, 2007). Therefore, none of the traditional vaccine against DENV is yet available for clinical use till now. DNA vaccines are able to induce long-lasting cellular and humoral immunity against some pathogens including flaviviruses (Donnelly et al., 1997). One of the potential advantages was the expression of viral proteins in situ after DNA immunization, leading to proper folding and posttranslational modifications, which originally occur during the course of natural viral infections (Schlesinger et al., 1987). In studies of DENV DNA vaccines, prM/M, E, NS1 and NS3 proteins were usually used as target antigens because they could elicit protective immune responses. The E protein, the viral major surface protein, contains important neutralizing epitopes. The prM protein is necessary for the proper processing and expression of the E protein. The NS1 protein associates with the E protein in the ER (endoplasmic reticulum) lumen and is involved in virion
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maturation. It can induce a strong antibody response and protect hosts through an Fc-dependent complement-mediated manner (Schlesinger et al., 1993). In fact, several investigations reported inoculation of plasmids containing flaviviruses prM, E, or NS1 gene to elicit specific immune responses in mice (Schlesinger et al., 1987; Ulmer et al., 1993; Donnelly et al., 1997; Colombage et al., 1998). But the efficiency of these DNA vaccines was poor. In our previous studies, we found that granulocyte-macrophage colony-stimulating factor (GM-CSF) was an effective gene adjuvant, which enhanced the immune and protective effects against Japanese encephalitis virus (JEV) and DENV1 when co-expressed with their antigens (Gao et al., 2010; Zheng et al., 2011). In this report, we constructed three recombinant plasmids containing either prM/E of DENV2 (pCAGprM/E), prM/E/NS1 of DENV2 (pCAG-prM/E/NS1) or prM/E/NS1 fused to GM-CSF (pCAG-DG) and analyzed their immunogenicity and protective role in mice. This will allow us to evaluate whether this strategy of DNA vaccine construction could be more widely applied in other DENV serotypes and viruses.
subcloned into the vectors. The expression of these plasmids was further confirmed by indirect immuno-fluorescence in Vero cells. For immunization, the plasmids were extracted and purified with an endotoxin-free plasmid extraction kit (Omega, Norcross, America) from transformed Escherichia coli JM109. Then, the purified plasmids were dissolved in sterile saline and adjusted to 1.0 mg/ml before use. 2.3. Mouse experiments All of the experiments with mice were conducted in compliance with Ethical Principles in Animal Experimentation. For DNA immunization, 6-week-old female BALB/c mice were used. To enhance the uptake of plasmid DNA, mice were prior injected with 50 l 0.25% lidocaine hydrochloride in each quadriceps muscle the day before immunization. Each mouse was inoculated intramuscularly with 100 g of the plasmids at 3-week intervals for three times. Mice injected with pCAG served as negative controls. The mice were finally euthanized. For the protection test, three weeks after the last immunization, the mice were challenged intracerebrally with a lethal dose (50× LD50) of DENV2. The mice were monitored daily for the morbidity and mortality for 3 weeks.
2. Materials and methods 2.1. Cells lines, virus, and mice Aedes albopictus mosquito cells (C6/36) were grown at 28 ◦ C in RPMI (Roswell Park Memorial Institute) 1640 containing 10% fetal bovine serum (FBS, Gibco, Auckland, New Zealand) and were used to propagate DENV-2 (strain TR1751) which was isolated from a patient with DF and was kindly provided by Dr. A. Oya (National Institute of Infectious Disease, Japan). Vero cells were cultivated at 37 ◦ C in Eagle’s minimal essential medium (MEM, Gibco, Buffalo, America) supplemented with 5% FBS. Female inbred BALB/c mice were purchased from the Academy of Military Medical Sciences (Beijing, China). All of the mice were maintained in specific pathogen-free environments.
2.4. Antibody assay Mice sera samples were collected by tail bleeding at three weeks after the last immunization and analyzed for the presence of DENV2 antibodies by an enzyme-linked immunosorbent assay (ELISA). Briefly, Two-fold serial dilutions of the serum samples were added to wells coated with concentrated virus. After incubation and washing with TPBS, each well was incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (KPL, Houston, America). Afterward, the plates were washed five times with TPBS, and the color was generated by using the HRP substrate, orthophenylene diamine (OPD), and the action was stopped by addition of H2 SO4 . After measurement of the absorbance at 490 nm, the end-point titers of anti-DENV2 antibodies were determined as the reciprocal of the highest dilution, giving an optical density (OD) twice that of the non-immune serum. To determine the IgG subclass, antibody isotype ELISAs were similarly performed using anti-mouse IgG1-HRP or anti-mouse IgG2a-HRP (SBA, Oregon, America) replacing the former anti-mouse IgG-HRP.
2.2. Construction of plasmids We used pCAG and pIRES (Qiagen) to construct the DNA vaccines. First, viral RNA was isolated from DENV2-infected C6/36 cells with the Biozol reagent (Bioflux, Tokyo, Japan), according to the manufacturer’s protocol, and reversely transcribed to cDNA using primer DENV (−): CTC CCG CTC ATC AAG AAT AA by reverse transcriptase M-MLV (Invitrogen, Grand Island, America). Different segments of DENV2 were then amplified by PCR using the primers shown in Table 1. Then, the amplified fragments were cloned into the pCAG vector using the multiple cloning sites. Furthermore, we constructed a bicistronic plasmid expressing simultaneously DENV2 prM-E-NS1 and GM-CSF under the control of a single promoter. Briefly, DENV-2 prM-E-NS1 and GM-CSF fragments were inserted into the multiple cloning sites A and B of the pIRES, respectively. Then, the total prM-E-NS1-IRES-GM-CSF fragment was digested with the XhoI and NotI restriction enzymes (MBI, Glen Burnie, America) and subcloned into pCAG. DNA sequencing was used to verify the cloned fragments and they were proper
2.5. Plaque reduction neutralization test (PRNT) To determine the presence of the neutralizing antibodies (NAbs) in the sera of inoculated mice, sera were diluted twofold in MEM containing 2% FBS. Sera were heated at 56 ◦ C for 30 min to inactivate complement. Serial dilutions were incubated with equal volumes of DENV2 virus at 37 ◦ C for 1 h. Then the mixtures containing 100 PFU/well of the virion were added in duplicate to Vero cell monolayers seeded in 24-well plates. After adsorption for 1 h, Vero cells were then washed and incubated in MEM containing 5% FBS and 1.3% methyl-cellulose. After 1 week of incubation at 37 ◦ C in 5%
Table 1 Constructions of plasmids including the components of DENV-2. Recombinant plasmids
Amplified regions
pCAG-prM/E
prM/E (367-2421)
pCAG-prM/E/NS1
prM/E/NS1 (367-3477)
pIRES-prM/E/NS1
prM/E/NS1 (367-3477)
Primers
Restriction sites
5 -ggcc ctcgag ATG CTG AAC ATC TTG AAC A-3 5 -cgta gcggccgc CTA GGC CTG CAC CAT AAC TCC-3 5‘-ggcc ctcgag ATG CTG AAC ATC TTG AAC A-3 5 -cgta gcggccgc CTA GGC TGT GAC CAA GGA GTT-3 5 -ggcc ctcgag ATG CTG AAC ATC TTG AAC A-3 5 -cggc acgcgt CTA GGC TGT GAC CAA GGA GTT-3
XhoI NotI XhoI NotI XhoI MluI
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CO2, the plaques were stained with crystal violet and counted. The NAb titer was calculated as the reciprocal of the maximum dilution of serum that yielded a 90% plaque reduction (PRNT90) and a 50% plaque reduction (PRNT50) when compared to the virus controls without serum. 2.6. Indirect Immuno-fluorescence Staining The DENV2-infected or plasmids transfected Vero cells were used for the experiments of indirect immuno-fluorescence staining. In brief, purified DENV2 was used as antigen and BALB/c were immunized to induce specific anti-DENV2 antibodies (Chen et al., 2009). Cells were fixed with 4% polyformaldehyde and permeabilized with 0.2% Triton X-100. After washing and blocking, cells were incubated with monoclonal anti-DENV2 E antibody (abcam, Tokyo, Japan), rat anti-GM-CSF antibody (BD, Franklin Lakes, America) or mouse anti-DENV2 antibodies at 4 ◦ C overnight. The following day, the cells were incubated with FITC-conjugated goat anti-mouse IgG1 (Sigma, Santa Clara, America). The specimens were analyzed by fluorescent microscope (Leica). Images were processed using Adobe Photoshop (Adobe Systems). 2.7. Statistical analysis All of the data were analyzed using SPSS software (version 12.0). Data were considered to be statistically significant when P < 0.05. 3. Results 3.1. Immunizations with DNA vaccine candidates could elicit mouse antibody responses To test the expression of the recombinant plasmids in eukaryotic cells, Vero cells were transfected with pCAG-prM/E,
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pCAG-prM/E/NS1, pCAG-DG and pCAG followed by indirect immuno-fluorescence analysis with a monoclonal anti-DENV2 E antibody (Fig. 1a) and rat anti-GM-CSF (Fig. 1b) antibody. Results show that specific reactive signals were observed in the cytoplasm after transfection with pCAG-prM/E, pCAG-prM/E/NS1 or pCAG-DG while no specific fluorescent signals were detectable after transfection with the control plasmid pCAG. This indicated that all plasmids vaccines candidates could be efficiently expressed and were suitable for the subsequent experiments. In antibodies analysis, the mice inoculated with pCAG-prM/E, pCAG-prM/E/NS1 and pCAG-DG showed higher specific antibody titers compared to the control group, pCAG (P < 0.05). Among the three DNA vaccines, the group of pCAG-prM/E induced the highest anti-DENV2 antibody titer (1:3, 200), whereas the antibody titer in the pCAG-DG group (1:1, 600) was twofold lower. The mice vaccinated with pCAG-prM/E/NS1 elicited only moderate anti-DENV2 titer (1:800) (Fig. 2). Furthermore, the sera obtained from the mouse experiments were subjected to indirect immunofluorescence staining. As shown in Fig. 3, sera of mice immunized with the three DNA vaccine candidates could specifically recognize the antigens in the DNEV2-infected Vero cells while the serum from the control only showed a faint fluorescent background signal. These results suggested that our three DNA vaccine candidates could efficiently and specifically elicit immune responses against DNEV2 in mice. In addition, DENV2-specific IgG1 and IgG2a in the sera were also examined by ELISA. In this test, the mice inoculated with pCAG-prM/E, pCAG-prM/E/NS1 and pCAG-DG showed higher specific IgG1 and IgG2a antibody titers compared to the control group, pCAG (P < 0.05). Among these groups, pCAG-prM/E and pCAG-DG induced similar levels of anti-DENV2 IgG1 and IgG2a responses, while pCAG-prM/E/NS1 showed a decreased IgG1/IgG2a ratio in the anti-DENV2 antibody (Fig. 2). Since the IgG1 isotype is generated by the Th2 immune response and the IgG2a isotype by Th1
Fig. 1. (a) The indirect immuno-fluorescence analysis with a monoclonal anti-DENV2 E antibody of the expression of recombinant plasmids in Vero cells. (b) The indirect immuno-fluorescence analysis with a monoclonal rat anti-GM-CSF of the expression of recombinant plasmids in Vero cells.
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3500
*
Nab titers
3000
PRNT90 PRNT50
2500 2000
*
1500
*
1000 500 0
* pCAG
pCAG-prM/E
* pCAGprM/E/NS1
* pCAG-DG
Fig. 4. Nab titers induced by DNA vaccine candidates by ELISA.
morbidity (Fig. 5). Interestingly, the group of pCAG-DG had the highest survival rate although its anti-DENV2 titer and NAb titer were not among the highest. Fig. 2. Anti-DENV2 antibody titers elicited by the DNA vaccine candidates.
in mice, our data suggested that the plasmid vaccinations could induce mixed Th1/Th2 immune responses and the NS1 of DENV2 might induce the more Th1 immune response. 3.2. Vaccination with DENV2 DNA vaccines elicits antibodies with neutralizing activity To assay NAb titers induced by DNA vaccine candidates, PRNTs were performed with sera samples from mice at three weeks after the final immunization. With the subtraction of the control’s background noise, the NAb titers of pCAG-DG were 80 in PRNT90 and 870 in PRNT50, 170 in PRNT90 and 1050 in PRNT50 for pCAG-prM/E and 100 in PRNT90 and 2770 in PRNT50 for pCAG-prM/E/NS1, respectively (Fig. 4). All of our DNA vaccine candidates obviously induced NAbs compared with the control group, pCAG, (P < 0.05), but the levels of NAbs were different among the three immune groups. 3.3. DNA vaccines candidates partially protect immunized mice from DENV2 challenge To evaluate the protection of the DNA vaccine candidates, vaccinated BALB/c mice were challenged three weeks after the final immunization with 50× LD50 of DENV2. None of the control mice immunized with pCAG survived after the DENV2 challenge in this group. In contrast, the mice immunized with pCAG-prM/E, pCAG-prM/E/NS1 and pCAG-DG were partially protected with respectively 30%, 40% and 60% survival rate and a 2-day delay in
4. Discussion Public health is still seriously threatened by DENV though great effort has been carried out to develop DENV vaccines (Rigau-Pérez et al., 1998). Since there are no safe and efficient vaccines against DENV available, we and other researchers attempt to develop a new vaccine devoid of the shortcomings of the traditional vaccine candidates. In this research, we had constructed three DNA vaccine candidates expressing DENV2 proteins in eukaryotic cells including one, pCAG-DG with co-expression a gene adjuvant, GM-CSF. As the data of intracerebrally challenge experiment show, the three DNA vaccine candidates could partially protected BALB/c mice from the lethal infection of DENV2. The strategy of co-expression a gene adjuvant, GM-CSF, with the DENV2 prM/E/NS1 gene provided promising results since a survival rate of 60% was obtained. Although it did not induce the highest antibody response and NAb titer, pCAG-DG might induce a more efficient and balanced humoral and cellular response. In fact, pCAG-prM/E could induce a higher anti-DENV2 antibody titer while pCAG-prM/E/NS1 elicited a stronger Th1 immune response. However, the protection with pCAG-prM/E in mice was not as good as with pCAG-prM/E/NS1 after intracerebrally challenge. Although M and E proteins of DNVE are the major targets of neutralizing antibodies since they play important roles in receptor binding and membrane fusion, they also possess specific epitopes present in different DENV serotypes (Costa et al., 2006; Kaufman et al., 1989). This determines that the vaccines used M and E proteins both as immunogens increase the humoral immune response and the risks of ADE. On the other hand, immunization with the NS1 protein can confer protection without this risk (Huisman et al., 2009; Krishna et al., 2009), so NS1 was
Fig. 3. DNEV2-infected Vero cells stained with sera of mice immunized with DNA vaccine candidates.
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The survival rates (%)
100 90 80 70 60 50 40 30 20 10 0
1
2
3
4
5
pCAG
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21
pCAG-prM/E
pCAG-prM/E/NS1
pCAG-DG
Fig. 5. The survival rates of immunized mice with plasmids.
also considered for development of DNA vaccines even though its ability of elicitation of NAbs was much weaker. The ability of DNA immunization to elicit both antibody and CTL immunity makes it an ideal vaccination approach to stimulate the host defense against viral infection. Despite the potential advantages of a DNA vaccine, it is sometimes difficult to induce a sufficient effective immunity against viral antigens. Numbers of strategies have been explored to improve the immune response of DNA vaccine. Cytokines, as a novel molecule adjuvant, has shown great promise in promoting immune responses. Given its central role in antigen presentation and recruitment of antigen presenting cells, GM-CSF represented an attractive adjuvant candidate and was a good choice in many studies (Whitehead et al., 2007; Sabchareon et al., 2004; Sun et al., 2006). Previous studies have shown that co-administration of plasmid GM-CSF as an adjuvant, using different protocols, could increase the immune response and improve the protective efficacy against challenge, including DNA vaccines of DENV (Raviprakash et al., 2003). Considering this information, plasmids expressing prM-E-NS1 with or without the GM-CSF adjuvant were constructed, and their immunogenicity and protective effects were investigated in this study. Compared with our previous work (Gao et al., 2010; Zheng et al., 2011), pCAG-DG did not induce high anti-DENV2 antibody and NAb titer and this result was different from those of the constructions with DENV1 and JEV. This suggests that the components of DENV2 might have mechanism to induce the immune responses that are different from the mechanisms involved in DNA vaccines from the other flaviviruses. Further work should be carried out to unravel this interesting problem. 5. Conclusion We had successfully constructed three DNA vaccine candidates expressing components of DENV2 viruses. The DNA vaccine candidates could express DENV2 proteins, elicit specific immune responses and partially protect BALB/c mice from a DENV2 intracerebrally challenge. pCAG-DG, co-expressing prM/E/NS1 of DENV2 and GM-CSF, had the highest survival rate though its antiDENV2 antibody titer and NAb were not the highest compared to those obtained with pCAG-prM/E and pCAG-prM/E/NS1. Our data showed that the strategy of developing a DNA vaccine with GM-CSF as a gene adjuvant shows promising prospects in the battle against DENV2. Acknowledgments This work was supported by grants 2011CB504703 from the National Key Programs on Basic Research of China and 31100131 from the National Natural Science Foundation of China.
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