- Email: [email protected]
Development of an effective Japanese encephalitis virus-specific DNA vaccine
Microbes and Infection 8 (2006) 2578e2586 www.elsevier.com/locate/micinf
Original article
Development of an effective Japanese encephalitis virus-specific DNA vaccine Chang Jer Wu a,b,*, Tsung Lin Li a,b, Hui Wen Huang a, Mi Hua Tao c, Yi Lin Chan d b
a Department of Food Science, National Taiwan Ocean University, 2, Pei Ning Road, Keelung 202, Taiwan Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University, 2, Pei Ning Road, Keelung 202, Taiwan c Division of Cancer Research, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan d Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
Received 3 February 2006; accepted 5 June 2006 Available online 7 August 2006
Abstract Intramuscular immunization with DNA vaccines has been shown to induce a broad range of immune responses and protective immunity in many animal models, but it is less effective in primates. One reason for this may be the low expression of vector-encoded antigen in cells. Here we report that the use of vaccine vector (pCJ-3) containing two regulatory elements, a chimeric intron and a bovine growth hormone (BGH) polyadenylation signal, markedly increased antigen expression both in vitro and in vivo. A positive correlation was seen between the level of expression of Japanese encephalitis virus (JEV) envelope proteins and the levels of antibodies in C3H/HeN mice. Immunization of mice with pCJ-3/ME (pCJ-3 containing the entire membrane and envelope protein genes) with or without cardiotoxin pretreatment resulted in higher antibody titers than immunization with vector containing only envelope protein and conferred full protection against infection with JEV. Electron microscopy showed that pCJ-3/ME expression resulted in the production of virus-like particles of JEV in vitro. The particles enhanced the production of higher titers of neutralizing antibodies and thus provided immunity against JEV. Consequently, the efficacy of the newly developed DNA vaccines was validated. This should pave the way to clinical trials in man. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Japanese encephalitis virus; DNA vaccine; Intramuscular injection; Virus-like particle
1. Introduction Japanese encephalitis virus (JEV) is a mosquito-transmitted, zoonotic flavivirus that affects a large part of Asia occupied by 40% of the world’s population. Encephalitis caused by JEV has a high mortality rate [1]. The use of a mouse brain-derived, formalin-fixed killed vaccine has reduced the number of encephalitis cases to 10e30 per year compared to
Abbreviations: JEV, Japanese encephalitis virus; PBS, phosphate-buffered saline. * Corresponding author. Department of Food Science, National Taiwan Ocean University, 2, Pei Ning Road, Keelung 202, Taiwan. Tel.: þ886 2 2462 2192x5137; fax: þ886 2 2463 4203. E-mail address: [email protected] (C.J. Wu). 1286-4579/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2006.06.010
a thousand cases per year previously in vaccine-preventable countries, including Taiwan [2]. However, due largely to the increased awareness of animal rights, the in vivo production of this vaccine using large numbers of animals is becoming less acceptable. In addition, adverse effects, such as allergy and neurotoxicity, can occur [1,3]. The cost of production is relatively high, and long-term immunity is not achieved [4]; furthermore, three doses of JEV vaccine are required to achieve adequate seroconversion and antibody titers [5]. A new vaccine of equal efficacy, but with a longer lasting effect and free of adverse side effects, would be preferable. A recently described vaccine technology, DNA vaccine, uses naked DNA, rather than protein, in the vaccine formulation [6,7]. In fact, in animals ranging from mice to non-human primates, immunization with antigen-encoding plasmid DNA has been
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
demonstrated to induce a broad range of immune responses, including humoral immune responses and cell-mediated immunity against challenge with the antigen-bearing pathogen, e.g., influenza virus [7], rabies virus [8], malaria parasites [9], and Mycobacterium tuberculosis [10]. Although high responsiveness is generally seen in mice, in other species, the efficacy of DNA vaccines in terms of providing protective immunity is reported to range from 28 to 100% [11]. To improve the efficacy of DNA vaccines, factors such as transcriptional elements, DNA dose, immunization schedule, injection route, and method of DNA delivery must all be taken into account. One group of factors that has not received much attention is regulatory elements. For instance, promoters, a major type of regulatory element, can be somewhat tissuespecific, and their use can result in variable amounts of mRNA production in different tissues [12]. A better DNA vaccine may therefore be developed by the systematic evaluation of regulatory elements. The most commonly used promoter in DNA vaccines in mammalian systems is derived from cytomegalovirus (CMV), and vaccines in which this is used show a good performance when injected into the muscle or skin in test animals. The effect of introns on expression has been ascribed primarily to an enhanced efficiency of RNA polyadenylation and RNA splicing, but may also reflect the presence of transcriptional enhancers within the intron [12,13]. Furthermore, transcriptional terminators are not widely recognized as gene regulatory elements, but their status may have changed in that they are now known to affect the processing efficiency of a primary RNA transcript [14]. For an ideal plasmid suitable for clinical use, the presence of an antibiotic resistance gene and some backbone elements of a vector must also be considered. For example, expression of the Ampr gene, a common selection marker, or other b-lactam resistance genes can provoke allergic reactions in certain groups of subjects. The FDA’s ‘‘Points to consider on plasmid DNA for preventive infectious disease indications’’ (1996) recommend the Kanr gene for such application, as kanamycin and other aminoglycoside antibiotics are less often used in the treatment of clinical infections. Enveloped JEV particles contain a single positive-stranded RNA genome 11 kb in length. This long single parent sequence encodes three structural proteins, the capsid (C), membrane (M), and envelope (E) proteins, together with seven non-structural proteins [15]. The non-glycosylated M protein is derived from a glycosylated precursor, premembrane protein (prM), and the E protein, which is involved in membrane association and assembly, is produced via a co-synthetic process with prM, which helps the E protein fold correctly [16]. Compared to immunization with vector coding for both the entire M and E proteins, immunization with pE, which contains a full-length E protein gene and a fragment encoding the C-terminal 15 amino acids of the M protein as the required signal sequence, resulted in a lower plaque reduction neutralization test (PRNT) titer [17]; this lower efficacy may be due to the E protein’s not being able to fold properly in the absence of the intact M protein. Here, we report the development of a new high-level expression JEV DNA vaccine carrying full-length M and E
2579
genes. A higher performance was achieved by modifying two regulatory elements, the intron and the polyadenylation signal. Moreover, this vaccine was equally effective with or without cardiotoxin pretreatment in a murine model. 2. Materials and methods 2.1. Viruses and animals The Beijing-1 JEV strain, maintained in suckling mouse brain, was used to prepare a virus stock which was used for the cloning of the JEV genes and for setting up a JEV challenge model. Groups of five 6- to 8-week-old female C3H/ HeN mice were used for evaluating induction of antibody and protective immunity. Female C3H/HeN mice, purchased from the National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, were housed at the Laboratory Animal Facility of the College of Life Sciences, National Taiwan Ocean University, Keelung, Taiwan. The LD50 of Beijing-1 JEV in 12- to 14-week-old C3H/HeN mice was found to be 3.0 105 PFU. For a lethal challenge experiment, C3H/HeN mice were injected intraperitoneally with 50 times the LD50 of JEV Beijing-1 and intracerebrally with PBS, then were observed for symptoms of viral encephalitis and death every day for 30 days. 2.2. Construction of expression vectors The eukaryotic expression vector, pcDNA3 (Invitrogen, San Diego, CA, USA), commonly used in our laboratory, contains the CMV early promoter and enhancer sequence, the bovine growth hormone polyadenylation signal, the ampicillin resistance gene, and the neomycin resistance selection gene. Vector p3224 (kindly provided by Dr. N.S. Yang, Academia Sinica, Taiwan), which has been used in clinical trials of gene therapy, contains the CMV early promoter and enhancer sequence, CMV intron A, the bovine growth hormone (BGH) polyadenylation signal, and the kanamycin resistance gene. We ligated the BglII/BamHI fragment of pRL-CMV (Promega, Madison, WI, USA) and the XbaI/SalI fragment of p3224 to construct the chimeric vector, pCJ-1. The Renilla luciferase gene of the vector pCJ-1 was removed and the resultant plasmid designated pCJ-2. Since pCJ-2 contains only one NotI cloning site, a multiple cloning site was inserted, generating pCJ-2 0 . This vector contains the cytomegalovirus early promoter and enhancer sequence, the chimeric intron, the SV40 polyadenylation signal, and the kanamycin resistance gene. The SV40 polyadenylation signal of pCJ-2 0 was replaced by the BGH polyadenylation signal to generate plasmid pCJ-3. The level of expression of the different constructed vectors was checked using the luciferase reporter system. We inserted the firefly luciferase gene (from pGL3-basic; Promega, Madison, WI, USA) into pcDNA3, p3224, pCJ-2 0 , and pCJ-3 to construct, respectively, plasmids pcDNA3/Luc, p3224/Luc, pCJ-2 0 /Luc, and pCJ-3/Luc. The cDNA for the JEV envelope (E) gene was obtained by reverse transcription and PCR
2580
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
amplification of Beijing-1 JEV genomic RNA. The PCR reaction for the E gene was performed using the primer set 5 0 -GAT GAAGCTTGCCATGGTGGTATTCACCATCCTC-3 0 (positive sense) and 5 0 -TCCGAATTCAAGCATGCACATTGG TAG-3 0 (negative sense) to obtain a fragment encoding the entire E protein and a 15-amino-acid signal peptide derived from the C-terminus of the M protein. The PCR reaction for the M and E protein (ME) genes was performed using the primer set 5 0 -ATGAAGCTTCCACCATGTGGCTCGCG-3 0 (positive sense) and 5 0 -CTGCAGAATTCAAGCATGCACA TTGGT-3 0 (negative sense) to obtain a fragment encoding the entire M and E proteins and a 15-amino-acid signal peptide derived from the C-terminus of the core protein. The blunt-end fragment of the E or ME gene was cloned into the Klenow fragment-treated plasmids of pcDNA3, p3224, pCJ-2 0 , or pCJ-3 to give pE, pME, p3224/E, p3224/ME, pCJ-2 0 /E, pCJ-2 0 /ME, pCJ-3/E, or pCJ-3/ME respectively. Plasmid DNA was purified from transformed Escherichia coli DH5a using Qiagen Plasmid Giga Kits (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions and stored at 70 C as pellets. For use, the DNA was dissolved at a concentration of 1 mg/ml in sterile saline.
2.3. Cell transfection and luciferase assay About 2 105 BHK cells (ATCC CCL-10), seeded into each well of a 6-well plate and maintained in Dulbecco’s modified Eagle’s medium and 10% bovine calf serum (BCS) (Invitrogen, San Diego, CA, USA), were incubated overnight, then the medium was replaced with fresh medium four hours before transfection using the calcium phosphate precipitation method. DNA (5 mg) dissolved in 220 ml of 0.1 TE buffer (1 mM TriseHCl, 0.1 mM EDTA, pH 8.0) was mixed with 250 ml of 2 HBS buffer (280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4, 50 mM HEPES, 12 mM dextrose, pH 7.05), then 31 ml of 2 M CaCl2 was added and the mixture vortexed immediately, then left to stand for 25 min at room temperature. The transfection solution was added dropwise to the cell cultures, which were then incubated at 37 C for 5 h. The transfection medium was then removed and 15% glycerol in 1 HBS added for glycerol shock for 2 min at room temperature, then the glycerol was removed and the cells washed twice with PBS. Fresh medium was then added and the cells incubated at 37 C. Forty-eight hours after transfection, the cells were washed twice with PBS and incubated for 15 min at room temperature with 200 ml of 1 reporter lysis buffer (Promega, Madison, WI, USA). The cell lysates were briefly centrifuged to pellet large debris, and the protein concentration of the supernatant was measured using BCA protein assay reagent (Pierce, USA). Samples of cell lysate (20 ml, 25 mg of protein) were mixed with 100 ml of luciferase assay reagent (Promega, USA) at room temperature in a luminometer (TD-20/20, Promega, USA) and plasmid expression quantified automatically as relative luciferase activity units (RLU) per 25 mg of protein.
2.4. Immunization and antibody assay For intramuscular DNA immunization, all mice were immunized at 6 to 8 weeks of age. In brief, unless otherwise stated, groups of five mice were anesthetized and injected three times at 3-week intervals with 100 mg of DNA [50 mg in each quadricep muscle injected 1 week earlier with 100 ml of 10 mM cardiotoxin (Sigma, St. Louis, MO, USA)]. In some cases, no cardiotoxin pretreatment was used, and a single injection of vaccine was also used in some mice. Serum samples were collected by tail bleeding at various time points and analyzed for the presence of JEV E-specific antibodies by ELISA as described previously [17,18]. Endpoint titers were defined as the highest serum dilution that gave an absorbance value equal to, or greater than, twice the value of 0.05 given by non-immune serum. Samples below the limit of detection were assigned a value of 10, since the most concentrated sample of serum tested was a 10-fold dilution. 2.5. Dot blot and Western blot analysis Monoclonal antibody E3 against E protein of JEV was produced as ascites in BALB/c mice by injection of the producing hybridoma [19] and purified by protein A chromatography. For the dot blot assay, the transiently transfected cells were washed twice with cold PBS and lysed by addition of 300 ml of NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris, pH 8.0, protease inhibitor cocktail [Boehringer Mannheim, Mannheim, Germany]). After centrifugation for 10 min at 4 C at 12,000 g, the protein concentration of the supernatant was measured using BCA Protein Assay Reagent (Pierce, USA). Different amounts of protein (1, 5, or 20 mg) were applied to a nitrocellulose membrane by vacuum; then the membrane was incubated for 2 h at room temperature with blocking buffer (5% skimmed milk, 150 mM NaCl, 50 mM Tris, pH 8.0), then for 1 h at room temperature with monoclonal antibody E3 (1 mg/ml) diluted in PBS containing 1% BSA (PBSB). After 4 5 min washes in washing buffer (0.05% Tween 20, 1% skimmed milk, 150 mM Tris, pH 8.0), the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG Fc (1:1,000; Cappel, Organon Teknika, Veedijk, Belgium) in PBSB; then, after 6 5 min washes, the blots were developed using an enhanced chemiluminescence Western blot detection system (Amersham, Little Chalfont, UK) and exposed to X-ray film. For Western blots, JEV-infected cells were lysed with lysis buffer and the lysates separated by SDS-PAGE and transferred to a nitrocellulose membrane in transfer buffer. The membrane was then incubated with blocking buffer for 2 h at room temperature and clamped in a multiscreen apparatus (Bio-Rad, Hercules, CA, USA). Serum samples collected by tail bleeding at different time points or JEV anti-E MAb E3 (1 mg/ml) were added to each channel for 1 h at room temperature, then, after 4 5 min washes in washing buffer, the membranes were processed as above.
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
2581
2.6. Plaque reduction neutralization test (PRNT)
2.8. Statistical analysis
The neutralization test was carried out using BHK-21 cells and the 50% plaque reduction technique. Two-fold dilutions of sera were prepared in PBS containing 5% bovine calf serum (BCS) (Invitrogen, San Diego, CA, USA), incubated at 56 C for 30 min to inactivate complement, and mixed with an equal volume of infectious JEV in minimum essential medium (MEM; Invitrogen, San Diego, CA, USA) containing 5% BCS to yield a mixture containing approximately 1000 PFU of virus/ml. The virus-antibody mixtures were incubated at 4 C for 18e21 h, then were added to triplicate wells of 6-well plates containing confluent monolayers of BHK-21 cells, which were then incubated at 37 C for 1 h with gentle shaking every 15 min. The wells were then overlaid with 4 ml of 1% agarose (SeaPlaque, FMC, USA) in MEM containing 2% BCS, and the plates incubated for 3 days at 37 C in 5% CO2, when the cells were fixed with formalin and stained with 1% crystal violet, and plaque numbers counted. The neutralizing antibody titer was calculated as the reciprocal of the highest dilution resulting in a 50% reduction in the number of plaques compared to a control of virus mixed with normal sera from na€ıve C3H/HeN mice of the same age.
The graphs and statistical analyses were performed using SigmaPlotÒ and SigmaStatÒ. One-way ANOVA and Tukey HSD test were used for the statistical analyses between groups of tested animals. The survival of tested animals was depicted using KaplaneMeier curves, and the corresponding analyses were performed by log rank test. Differences were considered significant if the P value was <0.05.
2.7. Electron microscopy JEV-infected or plasmid-transfected BHK cells were collected as cell pellets at 48 h postinfection, fixed for 60 min in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, then washed overnight in the same buffer. The cell pellets were then stained en bloc with uranyl acetate, postfixed with osmium, dehydrated with graded ethanol, and embedded in Eponate-12 resin. Thin sections were double stained with uranyl acetate and lead citrate and examined under a Zeiss 900 electron microscope (Carl Zeiss, Germany).
3. Results 3.1. Effect of various introns and poly(A) on luciferase expression Previous studies reported that the inclusion of a heterologous intron in the expression plasmid can enhance antigen expression in cultured cells or in transgenic mice [12,13,20]. In this study, we used three constructs, pcDNA3/Luc, p3224/Luc, and pCJ-3/Luc, containing the BGH poly(A) signal sequence (Fig. 1A) to examine the effect of two introns on antigen expression, CMV intron A (plasmid p3224/Luc) and a chimeric intron (plasmid pCJ-3/Luc), composed of a 5 0 -splice donor site from the first intron of the human b-globin gene and a 3 0 -splice acceptor site from the immunoglobulin heavy chain variable region gene. The expression of the constructed vectors was measured using a luciferase reporter system. Fig. 1A shows the averaged data for six independent transfection experiments in BHK cells, each in duplicate. pCJ-3/Luc gave the highest expression (1.6 107 RLU), p3224/Luc gave 3.4 106 RLU, while pcDNA3/Luc gave only 3.0 105 RLU. Compared to pcDNA3/Luc, about 53-fold or 11-fold higher expression was seen with pCJ-3/Luc or p3224/Luc. These results show that the vector containing the chimeric intron was more effective than that containing CMV intron A. Fig. 1B shows the expression levels of the same vectors measured using dot-blot assay, but using
Fig. 1. Efficacy of the different expression vectors in vitro. (A) BHK cells were transfected with 5 mg of each luciferase-encoding vector, and 25 mg of total cell lysate was analyzed by luciferase assay as described in Section 2. The data are the mean SD for 6 independent experiments. The asterisks indicate significant differences (P < 0.05 (*) and P < 0.01 (**)) compared to expression of pcDNA3/Luc. Abbreviations: CMV, human cytomegalovirus immediate early gene promoter/enhancer; CMV intron A, human cytomegalovirus intron A; chimeric intron, the 5 0 donor splice site of the human b-globin gene and the 3 0 acceptor splice site of an immunoglobulin gene heavy chain variable region; BGH pA, bovine growth hormone poly(A); SV40 pA, SV40 poly(A). (B) BHK cells were transfected with the different JEV E vectors, and after 48 h, cell lysates were tested by dot blot assay for JEV E protein using monoclonal E3 (anti-JEV E). ‘‘Virus’’ indicates an extract of JEV-infected BHK cells. The results shown are representative of those for 3 experiments.
2582
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
the E protein gene instead of the Luc gene. Expression using pCJ-3/E was 3-fold higher than that using p3224/E, which, in turn, was about 10-fold more efficient than pE. The created chimeric intron was again superior to CMV intron A in terms of E protein yield. The intron therefore plays a pivotal role during protein expression. In addition, to determine whether the poly(A) signal sequence affected protein expression, a vector containing the poly(A) signal from the SV40 late gene was compared to that containing the signal from the BGH gene; both of these poly(A) signals are often used in such applications. Each was inserted into a test vector consisting of the CMV promoter, the luciferase reporter gene, and the newly developed chimeric intron. As shown in Fig. 1A, the vector containing the BGH poly(A) signal (pCJ-3) gave 1.6 107 RLU, approximately 5-fold higher than that containing the late SV40 poly(A) signal (pCJ-2 0 ) (3.6 106 RLU). Measurement of JEV E protein expression using the dot blot assay showed 3fold higher expression of JEV E protein using the pCJ-3 vector compared to pCJ-2 0 (Fig. 1B).
3.2. Effect of introns and poly(A) on the antibody response and protection To determine which plasmid provided better protection against JEV infection, female C3H/HeN mice were immunized intramuscularly with plasmids encoding JEV Eprotein, then were injected with a lethal dose of JEV. C3H/HeN mice were chosen, as they are more sensitive to JEV than other inbred mouse species [21]. For the intramuscular DNA immunization, 3 doses of 100 mg of DNA were given intramuscularly at 3-week intervals as described in Section 2. Fig. 2A shows the antibody titers obtained using three injections of the different constructs. At week 3, there was no significant difference between the vectors in the production of JEV E-specific antibody. However, at week 6, the titer in the group immunized with pCJ-3/E was 3-, 2-, or 3-fold higher, respectively, than those in the groups immunized with pCJ-2 0 /E, p3224/E, or pE, while, at the peak of antibody production at week 8, the titer using pCJ-3/E was 1.6-, 1-, or 2-fold higher than using pCJ-2 0 /E, p3224/E, and pE, respectively. In terms of survival rates of immunized mice challenged with JEV virus at week 16, 100% protection (10 of 10, P < 0.01, versus pcDNA3 group) was seen with pCJ-3/ E-immunized mice, while mice immunized with p3224/E or pCJ-2 0 /E showed lower protection (80% survival, 8 of 10, P < 0.01), and those immunized with pE, only a 60% survival rate (6 of 10, P < 0.05). As expected, none of the mice in the control pcDNA3 groups survived the JEV challenge (0 of 10) (Fig. 2B). Taken together, these data clearly indicate that the simultaneous use of the chimeric intron and the BGH polyadenylation signal increased the efficiency of muscle-targeted gene expression in vitro and the generation of immunity in vivo.
Fig. 2. Protective effects of the different JEV E expression vectors. (A) C3H/ HeN mice were given three intramuscular injections of the different plasmids at 3-week intervals, and anti- JEV E antibodies were measured at the indicated time points by ELISA. The concentration of anti-E antibodies was calculated from a standard curve generated from a serially diluted calibrated pool of antiE antisera and expressed as arbitrary units per milliliter (1 EU ¼ 50% maximal optical density; 1 EU/ml is roughly equal to 22 ng/ml of anti-E antibody). The asterisks indicate significant differences (P < 0.01 (**)) compared to the antibody of pE at week 8. (B) The immunized mice were challenged with 50 LD50 of JEV Beijing-1 at 16 weeks after the first immunization, then survival was measured over 30 days.
3.3. Factors that affect the antibody response and immune protection In our previous study [22], a JEV DNA vaccine given intramuscularly was less effective in the absence of cardiotoxin pretreatment. Cardiotoxin or bupivacaine pretreatment, which can induce both muscle necrosis and regeneration, is believed to increase the efficiency of DNA plasmid uptake and expression in muscles, but is not acceptable for clinical use. Since co-production of M protein seems to help generate the correct structure for E protein [16], we examined whether the generation of the proper conformation could enhance the effectiveness of the JEV DNA vaccines in the absence of cardiotoxin pretreatment by injecting 100 mg of pE or pME or pCJ-3/ME
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
DNA in the quadricep muscle of non-pretreated () or cardiotoxin-pretreated (þ) C3H/HeN mice. As shown in Fig. 3, pE()-treated mice did not produce antibody production, at either week 6 or 8, while pE(þ)-treated mice produced low levels of antibody. pCJ-3/ME(þ)-treated mice produced nearly 3 times more antibody than pCJ-3/ME()-treated mice at week 6, but both produced similar amounts of antibody at week 8, showing that this vector could produce high titers in the absence of cardiotoxin pretreatment. pCJ-3/ ME()-treated mice or pME()-treated mice produced 197or 164-fold more antibody than pE()-treated mice at week
2583
6 and 72.4- or 55.7-fold more antibody at week 8, respectively, (Fig. 3A), showing that the adoption of the correct conformation by E protein was an important factor. pCJ-3/ME()treated or pME()-treated mice showed full protection (10 of 10, P < 0.01, versus pCJ-3(þ) group) when challenged with virus at week 8, whereas pE(þ)-treated mice had only a 60% survival rate (6 of 10, P < 0.01, versus pE() group), and pE()-treated mice were not protected (0 of 10) (Fig. 3B). Thus, JEV DNA vaccines carrying both the M and E genes induce higher antibody titers and thereby confer full protection against JEV challenge in the presence or absence of cardiotoxin pretreatment. To confirm that antibody in the sera of mice immunized without cardiotoxin pretreatment recognized E protein, C3H/ HeN mice were injected intramuscularly with pE or pCJ-3/ ME three times at 3-week intervals, and the sera collected at weeks 3, 6, and 8 were tested on Western blots of lysates of JEV-infected cells. The characteristic 52-kD band of E protein was clearly labeled with the serum of pCJ-3/ME-immunized mice at weeks 3, 6, and 8, but not with serum from pE-immunized mice. In addition, the titer of anti-JEV E antibody was time-dependent (data not shown). We also examined the morphology of pCJ-3/ME-transfected BHK-21 cells by transmission electron microscopy. Beijing-1 strain JEV-infected and pCJ-3/ME-transfected BHK-21 cells showed a normal gross appearance, similar to that of parental cells (not shown). At the suborganelle level, many small particulate structures were seen in the cisternae of the endoplasmic reticulum (ER) and the Golgi apparatus in the virus-infected cells (Fig. 4A), but not in non-infected or non-transfected cells (data not shown). The viral particles were spherical, 40 to 60 nm in diameter, and consisted of an electron-dense core (filled with viral genome) surrounded by a lipid bilayer (Fig. 4A). In the pCJ-3/ME-transfected BHK21 cells, the expression of M and E proteins triggered the formation of virus-like particles (VLPs) (Fig. 4B), which were bigger than wild type virus at 80 to 100 nm in diameter. 3.4. Effect of DNA vaccine on the antibody titer and the provoked immunity
Fig. 3. Effect of the correct structural conformation of E protein on JEV DNA vaccine-induced protective immunity with or without cardiotoxin pretreatment. (A) Cardiotoxin-pretreated (þ) or control () mice were given three intramuscular injections of plasmid DNA at 3-week intervals and serum samples were collected at different time-points and assayed for the presence of antiJEV E antibodies. The concentration of anti-E antibodies was calculated from a standard curve generated from serially diluted control antibodies and expressed as units per milliliter. The asterisks indicated significant differences (P < 0.01 (**)). (B) Two weeks after the last immunization, animals were challenged with 50 LD50 of Beijing-1 strain JEV, then observed for 30 days and the percentage of survivors calculated. In A and B, cardiotoxin-pretreated mice immunized with pCJ-3 by three i.m. injections at 3-week intervals were used as negative controls.
To evaluate the effectiveness of a single immunization with the created DNA vaccines, non-cardiotoxin-pretreated mice were given a single injection of pME or pCJ-3/ME. Cardiotoxin-pretreated mice injected with pE served as positive controls and untreated mice injected with a single dose of pCJ-3 served as negative controls. All mice were challenged with 50 LD50 (1.5 107 PFU) of JEV Beijing-1 at 8 weeks after immunization. As shown in Fig. 5A, no significant difference in anti-JEV E antibody titer was seen between the pME()and pCJ-3/ME()-treated groups at either week 3 or 6, although the pCJ-3/ME()-treated group had a 1.3-fold higher antibody titer than the pME()-treated group at week 8. In contrast, the pCJ-3/ME()-treated group had a 44-, 10.6-, and 9.4-fold higher antibody titer than the pE(þ)-treated group at week 3, 6, and 8, respectively. Unsurprisingly, none of the pCJ-3 mice survived JEV challenge (0 of 10), while the
2584
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
Fig. 4. Electron microscopy of JEV-infected or pCJ-3/ME-transfected BHK cells. BHK-21 cells were infected with JEV Beijing-1 strain at an MOI of 1 (A) or transfected with pCJ-3/ME by the calcium phosphate precipitation method (B), then incubated for 48 h. (A) JEV viral particles are seen as spherical electron-dense structures within the cisternae of the ER. (B) The spherical virus-like particles (arrows) consist of an electron-dense membrane surrounded by envelope protein within the Golgi apparatus. Magnification, 50,000; bar, 200 nm.
survival rate in the pE(þ)-treated group was 60% (6 of 10, P < 0.01, versus pCJ-3(þ) group) and those in the pME()and pCJ-3/ME()-treated groups were 100% (10 of 10, P < 0.01, versus pE(þ) group)(>30 days after virus challenge) (Fig. 5B). The ability of the serum from the different immunized groups to neutralize JEV infection in vitro was examined using PRNT. Groups of 6- to 8-week-old C3H/HeN mice were immunized intramuscularly either once or three times at 3week intervals with pCJ-3, pE, or pCJ-3/ME with or without cardiotoxin pretreatment, then, at week 8 after the first immunization, the sera were collected and checked for JEV-neutralizing antibodies. As shown in Table 1, no antibody was detectable (<1:10) in mice immunized with pE with or
Fig. 5. Effect of one dose of JEV DNA vaccine on protective immunity. (A) C3H/HeN mice with (þ) or without () cardiotoxin pretreatment were injected i.m. with a single dose of plasmid DNA, then serum samples were collected at weeks 3, 6, and 8, and the anti-JEV E antibody titers analyzed by ELISA assay. The asterisks indicate significant differences (P < 0.05 (*)). (B) Dose-effect of DNA vaccine-induced protective immunity. Animals were challenged at week 8 with 50 LD50 Beijing-1 JEV. Mice immunized with pCJ-3 three times at 3-week intervals were used as negative controls. Following challenge, the mice were observed for 30 days and the percentage of survivors was calculated.
Table 1 Induction of neutralizing antibodies in mice immunized with JEV DNA vaccines No. of injectionsa
Neutralizing antibodyb pCJ-3(þ)
pE()
pE(þ)
PCJ-3/ME()
PCJ-3/ME(þ)
1 3
<1/10 <1/10
<1/10 <1/10
1/10 1/10
1/160 >1/320
1/160 >1/320
a Groups of 6-to 8-week-old C3H/HeN mice with (þ) or without () cardiotoxin pretreatment were immunized intramuscularly either once or three times at 3 week intervals with pCJ-3, pE, or pCJ-3/ME. b JEV neutralizing antibody titers in serum collected 8 weeks after the first immunization expressed as the reciprocal of the serum dilution yielding a 50% reduction in plaque number.
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
without cardiotoxin pretreatment, while titers of 160 and >320 were seen with single and multiple injections, respectively, in pCJ-3/ME-immunized mice with or without cardiotoxin pretreatment. These results show that, in terms of neutralizing antibody, immunization with either one or three doses of pCJ-3/ME without cardiotoxin pretreatment was far more effective than identical treatment with pE with cardiotoxin pretreatment. 4. Discussion In small animal models, DNA vaccination has shown promise against an array of infectious diseases [7e10]. However, in primates, a relatively low level of induced antigen expression has been found, and the induced immune responses have been much lower than anticipated. In fact, the intrinsic efficacy of DNA vaccination may largely depend on the expression level of antigen (reviewed in ref. [23]). Many efforts have being made since the onset of DNA vaccination to increase the level of expression of a given gene. Gene regulatory elements, such as promoter-enhancer complexes, introns, and polyadenylation signals, may be important factors in gene expression. In addition, the prokaryotic antibiotic resistance gene and backbone elements have been altered and undesired viral sequences removed to produce a plasmid more acceptable for future clinical use (FDA, 1996). In the present study, we examined the effects of two regulatory elements, the intron and the polyadenylation signal, with the hope of increasing antigen expression. The level of expression of the engineered DNA plasmids in our cell-based assay was determined using a typical firefly luciferase reporter system or dot blots. The results showed that better expression of the introduced gene could be achieved by selecting an appropriate promoter/enhancer element, the choice being made based on cell type targeted and how the vector is constructed [24]. Almost all mammalian DNA vaccines developed to date have used CMV-derived promoters, with which high efficiency is observed across mammalian species, especially in tissues such as muscle and skin. Introduction or replacement of introns is reported to increase gene expression [12,13,20]. This may result from enhancement of RNA polyadenylation and nuclear transportassociated RNA splicing [13] or the presence of other transcriptional enhancers within the intron [12]. The largest intron of the CMV immediate early gene, intron A, followed by the CMV immediate early promoter/enhancer, has been shown to increase expression of heterologous genes in vitro [12]. Based on these observations, we created a vector, pCJ-3, containing a modified chimeric intron situated immediately downstream of the CMV enhancer/promoter region. The chimeric intron consisted of a 5 0 -splice donor site derived from the first intron of the human b-globin gene and a 3 0 -splice acceptor site derived from the immunoglobulin heavy chain variable region gene. The sequences of the donor and acceptor splice sites, along with the branchpoint site were aligned, and consensus sequences selected to act as the basis of the chimeric intron. Our results showed that cDNA flanked by the chimeric intron gave higher protein expression (Fig. 1). The polyadenylation
2585
signal tells RNA polymerase II to terminate transcription and to add 200e250 adenosine bases to the 3 0 -end of the RNA transcript [14]. The replacement of the SV40 late gene poly(A) signal with the BGH poly(A) signal resulted in a 5fold increase in luciferase expression (pCJ-2 0 vs. pCJ-3; Fig. 1A). This newly developed vector, pCJ-3, may soon be deployed in the front line against diseases. DNA vaccines have been demonstrated in many animal models to induce a broad range of immune responses, including antibodies, CD8þ CTLs, CD4þ helper T (Th) lymphocytes, and protective immunity against challenge with the pathogen. By using gene knockout mice, adoptive transfer experiments and T cell depletion experiments, Pan et al. [18] demonstrate that the anti-E antibody is the most critical protective component in this JEV challenge model, and production of anti-E antibody by pE DNA vaccine is dependent on the presence of CD4þT cells but independent of CD8þT cells. There is a positive correlation between the level of antigen expression and the levels of the corresponding antibody produced [23]. To estimate the intrinsic efficacy of these newly developed DNA vaccines, they were tested in C3H/HeN mice. The C3H/HeN mice injected intramuscularly with 100 mg of each of the plasmids coding for JEV E generated an anti-E-specific antibody, and the response was boosted by subsequent injections of 100 mg of vaccine. The results (Fig. 2A) demonstrated that there was no significant difference between the vectors in the production of JEV E-specific antibody at week 3, while, at week 6, pCJ-3/E generated 3-, 2-, and 3-fold more antibody than pCJ-2 0 /E, p3224/E, and pE, respectively, and at week 8, 1.6-, 1-, and 2-fold more than pCJ2 0 /E, p3224/E, and pE. The survival rates after virus challenge 16 weeks after primary immunization showed that pCJ-3/E provided full protection against challenge, p3224/E and pCJ2 0 /E both gave an 80% survival rate, and pE a 60% survival rate (Fig. 2B). Taken together, the data (Figs. 1 and 2) clearly indicate that pCJ-3, containing a chimeric intron and the BGH polyadenylation signal, increased the efficiency of muscletargeted gene expression in vitro and immunity in vivo. E protein is a major surface protein that plays a pivotal role in receptor binding and membrane fusion and is known to contain many protective epitopes [25]. M protein is found in infected cells as a glycosylated precursor, the premembrane protein (prM). During virion maturation in vertebrate cells, provirion particles are formed when portions of endoplasmic reticulum membrane containing prM and E envelop nucleocapsids consisting of the C protein and genomic RNA [26]. These poorly infectious provirions accumulate in the lumen of the exocytic pathway, and during virion maturation, prM is cleaved to M by a cellular protease, furin, located in the trans-Golgi network [27]. Maturation cleavage is accompanied by changes in the oligomerization of prM and M. E, which is essential for the development of matured virions, contains both hemagglutination and fusion activities [27]. Further support for the requirement for coordinated synthesis of prM and E proteins comes from the observation that prM and E form heterodimers in cell-associated forms of West Nile virus [28]. This feature explains the excellent vaccine potential of
2586
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
the JEV vector, which results in the production of virus-like particles which enhance antigenic stability and provide highdensity presentation to antigen-presenting cells such as macrophages, dendritic cells, and Langerhans’ cells [29]. When DNA is given by the intramuscular route, the majority of vaccine antigens are expressed by cells which do not present antigen to the immune system. The efficacy of a DNA vaccine is therefore dependent on transfection of antigen-presenting cells or the reprocessing of antigen derived from other cells [30]. In this study, muscle cells transfected by the JEV DNA vaccines synthesized and secreted virus-like particles, resulting in full protection and the generation of cellular and humoral immunity without cardiotoxin pretreatment (Fig. 3). A high expression capacity and the selection of a suitable antigen gene are the key factors for a competent DNA vaccine. The DNA vaccines developed in the present study clearly fulfill these criteria. This paves the way to their use in man, although an accurate and efficient delivery system has yet to be developed. Acknowledgements This work was supported financially by the National Science Council, Taiwan and the ADImmune Corporation, Tantz, Taichung, Taiwan. We also thank Dr Tom Barkas for critical evaluation of the English. References [1] D.S. Burke, T.P. Monath, in: D.M. Knipe, P.M. Howley (Eds.), Fields Virology, fourth ed. Lippincott Williams & Wilkins Publishers, Philadelphia, PA, 2001, pp. 1043e1125. [2] T.F. Tsai, G.J. Chang, Y.X. Yu, in: A.P. Stanley, W.A. Orenstein (Eds.), Vaccines, third ed. W.B. Saunders, Philadelphia, PA, 1999, pp. 672e710. [3] M. Sakaguchi, M. Yoshida, W. Kuroda, O. Harayama, Y. Matsunaga, S. Inouye, Systemic immediate-type reactions to gelatin included in Japanese encephalitis vaccines, Vaccine 15 (1997) 121e122. [4] J.D. Poland, C.B. Cropp, R.B. Craven, T.P. Monath, Evaluation of the potency and safety of inactivated Japanese encephalitis vaccine in US inhabitants, J. Infect. Dis. 161 (1990) 878e882. [5] R.F. Juang, Y. Okuno, T. Fukunaga, M. Tadano, K. Fukai, K. Baba, N. Tsuda, A. Yamada, H. Yabuuchi, Neutralizing antibody responses to Japanese encephalitis vaccine in children, Biken J. 26 (1983) 25e34. [6] D.C. Tang, M. DeVit, S.A. Johnston, Genetic immunization is a simple method for eliciting an immune response, Nature 356 (1992) 152e154. [7] J.B. Ulmer, J.J. Donnelly, S.E. Parker, G.H. Rhodes, P.L. Felgner, V.J. Dwarki, S.H. Gromkowski, R.R. Deck, C.M. DeWitt, A. Friedman, L.A. Hawe, K.R. Leander, D. Martinez, H.C. Perry, J.W. Shiver, D.L. Montgomery, M.A. Liu, Heterologous protection against influenza by injection of DNA encoding a viral protein, Science 259 (1993) 1745e1749. [8] Z.Q. Xiang, S. Spitalnik, M. Tran, W.H. Wunner, J. Cheng, H.C.J. Ertl, Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus, Virology 199 (1994) 132e140. [9] M. Sedegah, R. Hedstrom, P. Hobart, S.L. Hoffman, Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 9866e9870.
[10] R.E. Tascon, M.J. Colston, S. Ragno, E. Stavropoulos, D. Gregory, D.B. Lowrie, Vaccination against tuberculosis by DNA injection, Nat. Med. 2 (1996) 888e892. [11] E.F. Fynan, R.G. Webster, D.H. Fuller, J.R. Haynes, J.C. Santoro, H.L. Robinson, DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 11478e11482. [12] B.S. Chapman, R.M. Thayer, K.A. Vincent, N.L. Haigwood, Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells, Nucleic Acids Res. 19 (1991) 3979e3986. [13] M.T.F. Huang, C.M. Gorman, Intervening sequences increase efficiency of RNA 3 0 processing and accumulation of cytoplasmic RNA, Nucleic Acids Res. 18 (1990) 937e946. [14] N.J. Proudfoot, Poly(A) signals, Cell 64 (1991) 671. [15] B.D. Lindenbach, C.M. Rice, in: D.M. Knipe, P.M. Howley (Eds.), Fields Virology, fourth ed. Lippincott Williams & Wilkins Publishers, Philadelphia, PA, 2001, pp. 991e1041. [16] E. Konishi, P.W. Mason, Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein, J. Virol. 67 (1993) 1672e1675. [17] H.W. Chen, C.H. Pan, M.Y. Liau, R. Jou, C.J. Tsai, H.J. Wu, Y.L. Lin, M.H. Tao, Screening of protective antigens of Japanese encephalitis virus by DNA immunization: a comparative study with conventional viral vaccines, J. Virol. 73 (1999) 10137e10145. [18] C.H. Pan, H.W. Chen, H.W. Huang, M.H. Tao, Protective mechanisms induced by a Japanese encephalitis virus DNA vaccine: requirement for antibody but not CD8(þ) cytotoxic T-cell responses, J. Virol. 75 (2001) 11457e11463. [19] S.C. Wu, W.C. Lian, L.C. Hsu, M.Y. Liau, Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence, Virus Res. 51 (1997) 173e181. [20] T. Choi, M. Huang, C. Gorman, R. Jaenisch, A generic intron increases gene expression in transgenic mice, Mol. Cell Biol. 11 (1991) 3070e 3074. [21] K. Miura, T. Onodera, A. Nishida, N. Goto, Y. Fujisaki, A single gene controls resistance to Japanese encephalitis virus in mice, Arch. Virol. 112 (1990) 261e270. [22] C.J. Wu, S.C. Lee, H.W. Huang, M.H. Tao, In vivo electroporation of skeletal muscle increases the efficacy of Japanese encephalitis virus DNA vaccine, Vaccine 22 (2004) 1457e1464. [23] M.A. Barry, S.A. Johnston, Biological features of genetic immunization, Vaccine 15 (1997) 788e791. [24] J. Harms, G. Splitter, Interferon-g inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter, Hum. Gene Ther. 6 (1995) 1291e1297. [25] F.X. Heinz, Epitope mapping of flavivirus glycoproteins, Adv. Virus Res. 31 (1986) 103e168. [26] T.J. Chambers, C.S. Hahn, R. Galler, C.M. Rice, Flavivirus genome organization, expression, and replication, Annu. Rev. Microbiol. 44 (1990) 649e688. [27] K. Stadler, S.L. Allison, J. Schalich, F.X. Heinz, Proteolytic activation of tick-borne encephalitis virus by furin, J. Virol. 71 (1997) 8475e8481. [28] G. Wengler, G. Wengler, Cell-associated West Nile flavivirus is covered with E þ pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release, J. Virol. 63 (1989) 2521e2526. [29] C. Condon, S.C. Watkins, C.M. Celluzzi, K. Thompson, L.D. Falo Jr., DNA-based immunization by in vivo transfection of dendritic cells, Nat. Med. 2 (1996) 1122e1128. [30] E. Konishi, M. Yamaoka, I. Khin-Sane-WinKurane, P.W. Mason, Induction of protective immunity against Japanese encephalitis in mice by immunization with a plasmid encoding Japanese encephalitis virus premembrane and envelope genes, J. Virol. 72 (1998) 4925e4930.
Original article
Development of an effective Japanese encephalitis virus-specific DNA vaccine Chang Jer Wu a,b,*, Tsung Lin Li a,b, Hui Wen Huang a, Mi Hua Tao c, Yi Lin Chan d b
a Department of Food Science, National Taiwan Ocean University, 2, Pei Ning Road, Keelung 202, Taiwan Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University, 2, Pei Ning Road, Keelung 202, Taiwan c Division of Cancer Research, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan d Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
Received 3 February 2006; accepted 5 June 2006 Available online 7 August 2006
Abstract Intramuscular immunization with DNA vaccines has been shown to induce a broad range of immune responses and protective immunity in many animal models, but it is less effective in primates. One reason for this may be the low expression of vector-encoded antigen in cells. Here we report that the use of vaccine vector (pCJ-3) containing two regulatory elements, a chimeric intron and a bovine growth hormone (BGH) polyadenylation signal, markedly increased antigen expression both in vitro and in vivo. A positive correlation was seen between the level of expression of Japanese encephalitis virus (JEV) envelope proteins and the levels of antibodies in C3H/HeN mice. Immunization of mice with pCJ-3/ME (pCJ-3 containing the entire membrane and envelope protein genes) with or without cardiotoxin pretreatment resulted in higher antibody titers than immunization with vector containing only envelope protein and conferred full protection against infection with JEV. Electron microscopy showed that pCJ-3/ME expression resulted in the production of virus-like particles of JEV in vitro. The particles enhanced the production of higher titers of neutralizing antibodies and thus provided immunity against JEV. Consequently, the efficacy of the newly developed DNA vaccines was validated. This should pave the way to clinical trials in man. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Japanese encephalitis virus; DNA vaccine; Intramuscular injection; Virus-like particle
1. Introduction Japanese encephalitis virus (JEV) is a mosquito-transmitted, zoonotic flavivirus that affects a large part of Asia occupied by 40% of the world’s population. Encephalitis caused by JEV has a high mortality rate [1]. The use of a mouse brain-derived, formalin-fixed killed vaccine has reduced the number of encephalitis cases to 10e30 per year compared to
Abbreviations: JEV, Japanese encephalitis virus; PBS, phosphate-buffered saline. * Corresponding author. Department of Food Science, National Taiwan Ocean University, 2, Pei Ning Road, Keelung 202, Taiwan. Tel.: þ886 2 2462 2192x5137; fax: þ886 2 2463 4203. E-mail address: [email protected] (C.J. Wu). 1286-4579/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2006.06.010
a thousand cases per year previously in vaccine-preventable countries, including Taiwan [2]. However, due largely to the increased awareness of animal rights, the in vivo production of this vaccine using large numbers of animals is becoming less acceptable. In addition, adverse effects, such as allergy and neurotoxicity, can occur [1,3]. The cost of production is relatively high, and long-term immunity is not achieved [4]; furthermore, three doses of JEV vaccine are required to achieve adequate seroconversion and antibody titers [5]. A new vaccine of equal efficacy, but with a longer lasting effect and free of adverse side effects, would be preferable. A recently described vaccine technology, DNA vaccine, uses naked DNA, rather than protein, in the vaccine formulation [6,7]. In fact, in animals ranging from mice to non-human primates, immunization with antigen-encoding plasmid DNA has been
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
demonstrated to induce a broad range of immune responses, including humoral immune responses and cell-mediated immunity against challenge with the antigen-bearing pathogen, e.g., influenza virus [7], rabies virus [8], malaria parasites [9], and Mycobacterium tuberculosis [10]. Although high responsiveness is generally seen in mice, in other species, the efficacy of DNA vaccines in terms of providing protective immunity is reported to range from 28 to 100% [11]. To improve the efficacy of DNA vaccines, factors such as transcriptional elements, DNA dose, immunization schedule, injection route, and method of DNA delivery must all be taken into account. One group of factors that has not received much attention is regulatory elements. For instance, promoters, a major type of regulatory element, can be somewhat tissuespecific, and their use can result in variable amounts of mRNA production in different tissues [12]. A better DNA vaccine may therefore be developed by the systematic evaluation of regulatory elements. The most commonly used promoter in DNA vaccines in mammalian systems is derived from cytomegalovirus (CMV), and vaccines in which this is used show a good performance when injected into the muscle or skin in test animals. The effect of introns on expression has been ascribed primarily to an enhanced efficiency of RNA polyadenylation and RNA splicing, but may also reflect the presence of transcriptional enhancers within the intron [12,13]. Furthermore, transcriptional terminators are not widely recognized as gene regulatory elements, but their status may have changed in that they are now known to affect the processing efficiency of a primary RNA transcript [14]. For an ideal plasmid suitable for clinical use, the presence of an antibiotic resistance gene and some backbone elements of a vector must also be considered. For example, expression of the Ampr gene, a common selection marker, or other b-lactam resistance genes can provoke allergic reactions in certain groups of subjects. The FDA’s ‘‘Points to consider on plasmid DNA for preventive infectious disease indications’’ (1996) recommend the Kanr gene for such application, as kanamycin and other aminoglycoside antibiotics are less often used in the treatment of clinical infections. Enveloped JEV particles contain a single positive-stranded RNA genome 11 kb in length. This long single parent sequence encodes three structural proteins, the capsid (C), membrane (M), and envelope (E) proteins, together with seven non-structural proteins [15]. The non-glycosylated M protein is derived from a glycosylated precursor, premembrane protein (prM), and the E protein, which is involved in membrane association and assembly, is produced via a co-synthetic process with prM, which helps the E protein fold correctly [16]. Compared to immunization with vector coding for both the entire M and E proteins, immunization with pE, which contains a full-length E protein gene and a fragment encoding the C-terminal 15 amino acids of the M protein as the required signal sequence, resulted in a lower plaque reduction neutralization test (PRNT) titer [17]; this lower efficacy may be due to the E protein’s not being able to fold properly in the absence of the intact M protein. Here, we report the development of a new high-level expression JEV DNA vaccine carrying full-length M and E
2579
genes. A higher performance was achieved by modifying two regulatory elements, the intron and the polyadenylation signal. Moreover, this vaccine was equally effective with or without cardiotoxin pretreatment in a murine model. 2. Materials and methods 2.1. Viruses and animals The Beijing-1 JEV strain, maintained in suckling mouse brain, was used to prepare a virus stock which was used for the cloning of the JEV genes and for setting up a JEV challenge model. Groups of five 6- to 8-week-old female C3H/ HeN mice were used for evaluating induction of antibody and protective immunity. Female C3H/HeN mice, purchased from the National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, were housed at the Laboratory Animal Facility of the College of Life Sciences, National Taiwan Ocean University, Keelung, Taiwan. The LD50 of Beijing-1 JEV in 12- to 14-week-old C3H/HeN mice was found to be 3.0 105 PFU. For a lethal challenge experiment, C3H/HeN mice were injected intraperitoneally with 50 times the LD50 of JEV Beijing-1 and intracerebrally with PBS, then were observed for symptoms of viral encephalitis and death every day for 30 days. 2.2. Construction of expression vectors The eukaryotic expression vector, pcDNA3 (Invitrogen, San Diego, CA, USA), commonly used in our laboratory, contains the CMV early promoter and enhancer sequence, the bovine growth hormone polyadenylation signal, the ampicillin resistance gene, and the neomycin resistance selection gene. Vector p3224 (kindly provided by Dr. N.S. Yang, Academia Sinica, Taiwan), which has been used in clinical trials of gene therapy, contains the CMV early promoter and enhancer sequence, CMV intron A, the bovine growth hormone (BGH) polyadenylation signal, and the kanamycin resistance gene. We ligated the BglII/BamHI fragment of pRL-CMV (Promega, Madison, WI, USA) and the XbaI/SalI fragment of p3224 to construct the chimeric vector, pCJ-1. The Renilla luciferase gene of the vector pCJ-1 was removed and the resultant plasmid designated pCJ-2. Since pCJ-2 contains only one NotI cloning site, a multiple cloning site was inserted, generating pCJ-2 0 . This vector contains the cytomegalovirus early promoter and enhancer sequence, the chimeric intron, the SV40 polyadenylation signal, and the kanamycin resistance gene. The SV40 polyadenylation signal of pCJ-2 0 was replaced by the BGH polyadenylation signal to generate plasmid pCJ-3. The level of expression of the different constructed vectors was checked using the luciferase reporter system. We inserted the firefly luciferase gene (from pGL3-basic; Promega, Madison, WI, USA) into pcDNA3, p3224, pCJ-2 0 , and pCJ-3 to construct, respectively, plasmids pcDNA3/Luc, p3224/Luc, pCJ-2 0 /Luc, and pCJ-3/Luc. The cDNA for the JEV envelope (E) gene was obtained by reverse transcription and PCR
2580
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
amplification of Beijing-1 JEV genomic RNA. The PCR reaction for the E gene was performed using the primer set 5 0 -GAT GAAGCTTGCCATGGTGGTATTCACCATCCTC-3 0 (positive sense) and 5 0 -TCCGAATTCAAGCATGCACATTGG TAG-3 0 (negative sense) to obtain a fragment encoding the entire E protein and a 15-amino-acid signal peptide derived from the C-terminus of the M protein. The PCR reaction for the M and E protein (ME) genes was performed using the primer set 5 0 -ATGAAGCTTCCACCATGTGGCTCGCG-3 0 (positive sense) and 5 0 -CTGCAGAATTCAAGCATGCACA TTGGT-3 0 (negative sense) to obtain a fragment encoding the entire M and E proteins and a 15-amino-acid signal peptide derived from the C-terminus of the core protein. The blunt-end fragment of the E or ME gene was cloned into the Klenow fragment-treated plasmids of pcDNA3, p3224, pCJ-2 0 , or pCJ-3 to give pE, pME, p3224/E, p3224/ME, pCJ-2 0 /E, pCJ-2 0 /ME, pCJ-3/E, or pCJ-3/ME respectively. Plasmid DNA was purified from transformed Escherichia coli DH5a using Qiagen Plasmid Giga Kits (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions and stored at 70 C as pellets. For use, the DNA was dissolved at a concentration of 1 mg/ml in sterile saline.
2.3. Cell transfection and luciferase assay About 2 105 BHK cells (ATCC CCL-10), seeded into each well of a 6-well plate and maintained in Dulbecco’s modified Eagle’s medium and 10% bovine calf serum (BCS) (Invitrogen, San Diego, CA, USA), were incubated overnight, then the medium was replaced with fresh medium four hours before transfection using the calcium phosphate precipitation method. DNA (5 mg) dissolved in 220 ml of 0.1 TE buffer (1 mM TriseHCl, 0.1 mM EDTA, pH 8.0) was mixed with 250 ml of 2 HBS buffer (280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4, 50 mM HEPES, 12 mM dextrose, pH 7.05), then 31 ml of 2 M CaCl2 was added and the mixture vortexed immediately, then left to stand for 25 min at room temperature. The transfection solution was added dropwise to the cell cultures, which were then incubated at 37 C for 5 h. The transfection medium was then removed and 15% glycerol in 1 HBS added for glycerol shock for 2 min at room temperature, then the glycerol was removed and the cells washed twice with PBS. Fresh medium was then added and the cells incubated at 37 C. Forty-eight hours after transfection, the cells were washed twice with PBS and incubated for 15 min at room temperature with 200 ml of 1 reporter lysis buffer (Promega, Madison, WI, USA). The cell lysates were briefly centrifuged to pellet large debris, and the protein concentration of the supernatant was measured using BCA protein assay reagent (Pierce, USA). Samples of cell lysate (20 ml, 25 mg of protein) were mixed with 100 ml of luciferase assay reagent (Promega, USA) at room temperature in a luminometer (TD-20/20, Promega, USA) and plasmid expression quantified automatically as relative luciferase activity units (RLU) per 25 mg of protein.
2.4. Immunization and antibody assay For intramuscular DNA immunization, all mice were immunized at 6 to 8 weeks of age. In brief, unless otherwise stated, groups of five mice were anesthetized and injected three times at 3-week intervals with 100 mg of DNA [50 mg in each quadricep muscle injected 1 week earlier with 100 ml of 10 mM cardiotoxin (Sigma, St. Louis, MO, USA)]. In some cases, no cardiotoxin pretreatment was used, and a single injection of vaccine was also used in some mice. Serum samples were collected by tail bleeding at various time points and analyzed for the presence of JEV E-specific antibodies by ELISA as described previously [17,18]. Endpoint titers were defined as the highest serum dilution that gave an absorbance value equal to, or greater than, twice the value of 0.05 given by non-immune serum. Samples below the limit of detection were assigned a value of 10, since the most concentrated sample of serum tested was a 10-fold dilution. 2.5. Dot blot and Western blot analysis Monoclonal antibody E3 against E protein of JEV was produced as ascites in BALB/c mice by injection of the producing hybridoma [19] and purified by protein A chromatography. For the dot blot assay, the transiently transfected cells were washed twice with cold PBS and lysed by addition of 300 ml of NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris, pH 8.0, protease inhibitor cocktail [Boehringer Mannheim, Mannheim, Germany]). After centrifugation for 10 min at 4 C at 12,000 g, the protein concentration of the supernatant was measured using BCA Protein Assay Reagent (Pierce, USA). Different amounts of protein (1, 5, or 20 mg) were applied to a nitrocellulose membrane by vacuum; then the membrane was incubated for 2 h at room temperature with blocking buffer (5% skimmed milk, 150 mM NaCl, 50 mM Tris, pH 8.0), then for 1 h at room temperature with monoclonal antibody E3 (1 mg/ml) diluted in PBS containing 1% BSA (PBSB). After 4 5 min washes in washing buffer (0.05% Tween 20, 1% skimmed milk, 150 mM Tris, pH 8.0), the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG Fc (1:1,000; Cappel, Organon Teknika, Veedijk, Belgium) in PBSB; then, after 6 5 min washes, the blots were developed using an enhanced chemiluminescence Western blot detection system (Amersham, Little Chalfont, UK) and exposed to X-ray film. For Western blots, JEV-infected cells were lysed with lysis buffer and the lysates separated by SDS-PAGE and transferred to a nitrocellulose membrane in transfer buffer. The membrane was then incubated with blocking buffer for 2 h at room temperature and clamped in a multiscreen apparatus (Bio-Rad, Hercules, CA, USA). Serum samples collected by tail bleeding at different time points or JEV anti-E MAb E3 (1 mg/ml) were added to each channel for 1 h at room temperature, then, after 4 5 min washes in washing buffer, the membranes were processed as above.
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
2581
2.6. Plaque reduction neutralization test (PRNT)
2.8. Statistical analysis
The neutralization test was carried out using BHK-21 cells and the 50% plaque reduction technique. Two-fold dilutions of sera were prepared in PBS containing 5% bovine calf serum (BCS) (Invitrogen, San Diego, CA, USA), incubated at 56 C for 30 min to inactivate complement, and mixed with an equal volume of infectious JEV in minimum essential medium (MEM; Invitrogen, San Diego, CA, USA) containing 5% BCS to yield a mixture containing approximately 1000 PFU of virus/ml. The virus-antibody mixtures were incubated at 4 C for 18e21 h, then were added to triplicate wells of 6-well plates containing confluent monolayers of BHK-21 cells, which were then incubated at 37 C for 1 h with gentle shaking every 15 min. The wells were then overlaid with 4 ml of 1% agarose (SeaPlaque, FMC, USA) in MEM containing 2% BCS, and the plates incubated for 3 days at 37 C in 5% CO2, when the cells were fixed with formalin and stained with 1% crystal violet, and plaque numbers counted. The neutralizing antibody titer was calculated as the reciprocal of the highest dilution resulting in a 50% reduction in the number of plaques compared to a control of virus mixed with normal sera from na€ıve C3H/HeN mice of the same age.
The graphs and statistical analyses were performed using SigmaPlotÒ and SigmaStatÒ. One-way ANOVA and Tukey HSD test were used for the statistical analyses between groups of tested animals. The survival of tested animals was depicted using KaplaneMeier curves, and the corresponding analyses were performed by log rank test. Differences were considered significant if the P value was <0.05.
2.7. Electron microscopy JEV-infected or plasmid-transfected BHK cells were collected as cell pellets at 48 h postinfection, fixed for 60 min in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, then washed overnight in the same buffer. The cell pellets were then stained en bloc with uranyl acetate, postfixed with osmium, dehydrated with graded ethanol, and embedded in Eponate-12 resin. Thin sections were double stained with uranyl acetate and lead citrate and examined under a Zeiss 900 electron microscope (Carl Zeiss, Germany).
3. Results 3.1. Effect of various introns and poly(A) on luciferase expression Previous studies reported that the inclusion of a heterologous intron in the expression plasmid can enhance antigen expression in cultured cells or in transgenic mice [12,13,20]. In this study, we used three constructs, pcDNA3/Luc, p3224/Luc, and pCJ-3/Luc, containing the BGH poly(A) signal sequence (Fig. 1A) to examine the effect of two introns on antigen expression, CMV intron A (plasmid p3224/Luc) and a chimeric intron (plasmid pCJ-3/Luc), composed of a 5 0 -splice donor site from the first intron of the human b-globin gene and a 3 0 -splice acceptor site from the immunoglobulin heavy chain variable region gene. The expression of the constructed vectors was measured using a luciferase reporter system. Fig. 1A shows the averaged data for six independent transfection experiments in BHK cells, each in duplicate. pCJ-3/Luc gave the highest expression (1.6 107 RLU), p3224/Luc gave 3.4 106 RLU, while pcDNA3/Luc gave only 3.0 105 RLU. Compared to pcDNA3/Luc, about 53-fold or 11-fold higher expression was seen with pCJ-3/Luc or p3224/Luc. These results show that the vector containing the chimeric intron was more effective than that containing CMV intron A. Fig. 1B shows the expression levels of the same vectors measured using dot-blot assay, but using
Fig. 1. Efficacy of the different expression vectors in vitro. (A) BHK cells were transfected with 5 mg of each luciferase-encoding vector, and 25 mg of total cell lysate was analyzed by luciferase assay as described in Section 2. The data are the mean SD for 6 independent experiments. The asterisks indicate significant differences (P < 0.05 (*) and P < 0.01 (**)) compared to expression of pcDNA3/Luc. Abbreviations: CMV, human cytomegalovirus immediate early gene promoter/enhancer; CMV intron A, human cytomegalovirus intron A; chimeric intron, the 5 0 donor splice site of the human b-globin gene and the 3 0 acceptor splice site of an immunoglobulin gene heavy chain variable region; BGH pA, bovine growth hormone poly(A); SV40 pA, SV40 poly(A). (B) BHK cells were transfected with the different JEV E vectors, and after 48 h, cell lysates were tested by dot blot assay for JEV E protein using monoclonal E3 (anti-JEV E). ‘‘Virus’’ indicates an extract of JEV-infected BHK cells. The results shown are representative of those for 3 experiments.
2582
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
the E protein gene instead of the Luc gene. Expression using pCJ-3/E was 3-fold higher than that using p3224/E, which, in turn, was about 10-fold more efficient than pE. The created chimeric intron was again superior to CMV intron A in terms of E protein yield. The intron therefore plays a pivotal role during protein expression. In addition, to determine whether the poly(A) signal sequence affected protein expression, a vector containing the poly(A) signal from the SV40 late gene was compared to that containing the signal from the BGH gene; both of these poly(A) signals are often used in such applications. Each was inserted into a test vector consisting of the CMV promoter, the luciferase reporter gene, and the newly developed chimeric intron. As shown in Fig. 1A, the vector containing the BGH poly(A) signal (pCJ-3) gave 1.6 107 RLU, approximately 5-fold higher than that containing the late SV40 poly(A) signal (pCJ-2 0 ) (3.6 106 RLU). Measurement of JEV E protein expression using the dot blot assay showed 3fold higher expression of JEV E protein using the pCJ-3 vector compared to pCJ-2 0 (Fig. 1B).
3.2. Effect of introns and poly(A) on the antibody response and protection To determine which plasmid provided better protection against JEV infection, female C3H/HeN mice were immunized intramuscularly with plasmids encoding JEV Eprotein, then were injected with a lethal dose of JEV. C3H/HeN mice were chosen, as they are more sensitive to JEV than other inbred mouse species [21]. For the intramuscular DNA immunization, 3 doses of 100 mg of DNA were given intramuscularly at 3-week intervals as described in Section 2. Fig. 2A shows the antibody titers obtained using three injections of the different constructs. At week 3, there was no significant difference between the vectors in the production of JEV E-specific antibody. However, at week 6, the titer in the group immunized with pCJ-3/E was 3-, 2-, or 3-fold higher, respectively, than those in the groups immunized with pCJ-2 0 /E, p3224/E, or pE, while, at the peak of antibody production at week 8, the titer using pCJ-3/E was 1.6-, 1-, or 2-fold higher than using pCJ-2 0 /E, p3224/E, and pE, respectively. In terms of survival rates of immunized mice challenged with JEV virus at week 16, 100% protection (10 of 10, P < 0.01, versus pcDNA3 group) was seen with pCJ-3/ E-immunized mice, while mice immunized with p3224/E or pCJ-2 0 /E showed lower protection (80% survival, 8 of 10, P < 0.01), and those immunized with pE, only a 60% survival rate (6 of 10, P < 0.05). As expected, none of the mice in the control pcDNA3 groups survived the JEV challenge (0 of 10) (Fig. 2B). Taken together, these data clearly indicate that the simultaneous use of the chimeric intron and the BGH polyadenylation signal increased the efficiency of muscle-targeted gene expression in vitro and the generation of immunity in vivo.
Fig. 2. Protective effects of the different JEV E expression vectors. (A) C3H/ HeN mice were given three intramuscular injections of the different plasmids at 3-week intervals, and anti- JEV E antibodies were measured at the indicated time points by ELISA. The concentration of anti-E antibodies was calculated from a standard curve generated from a serially diluted calibrated pool of antiE antisera and expressed as arbitrary units per milliliter (1 EU ¼ 50% maximal optical density; 1 EU/ml is roughly equal to 22 ng/ml of anti-E antibody). The asterisks indicate significant differences (P < 0.01 (**)) compared to the antibody of pE at week 8. (B) The immunized mice were challenged with 50 LD50 of JEV Beijing-1 at 16 weeks after the first immunization, then survival was measured over 30 days.
3.3. Factors that affect the antibody response and immune protection In our previous study [22], a JEV DNA vaccine given intramuscularly was less effective in the absence of cardiotoxin pretreatment. Cardiotoxin or bupivacaine pretreatment, which can induce both muscle necrosis and regeneration, is believed to increase the efficiency of DNA plasmid uptake and expression in muscles, but is not acceptable for clinical use. Since co-production of M protein seems to help generate the correct structure for E protein [16], we examined whether the generation of the proper conformation could enhance the effectiveness of the JEV DNA vaccines in the absence of cardiotoxin pretreatment by injecting 100 mg of pE or pME or pCJ-3/ME
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
DNA in the quadricep muscle of non-pretreated () or cardiotoxin-pretreated (þ) C3H/HeN mice. As shown in Fig. 3, pE()-treated mice did not produce antibody production, at either week 6 or 8, while pE(þ)-treated mice produced low levels of antibody. pCJ-3/ME(þ)-treated mice produced nearly 3 times more antibody than pCJ-3/ME()-treated mice at week 6, but both produced similar amounts of antibody at week 8, showing that this vector could produce high titers in the absence of cardiotoxin pretreatment. pCJ-3/ ME()-treated mice or pME()-treated mice produced 197or 164-fold more antibody than pE()-treated mice at week
2583
6 and 72.4- or 55.7-fold more antibody at week 8, respectively, (Fig. 3A), showing that the adoption of the correct conformation by E protein was an important factor. pCJ-3/ME()treated or pME()-treated mice showed full protection (10 of 10, P < 0.01, versus pCJ-3(þ) group) when challenged with virus at week 8, whereas pE(þ)-treated mice had only a 60% survival rate (6 of 10, P < 0.01, versus pE() group), and pE()-treated mice were not protected (0 of 10) (Fig. 3B). Thus, JEV DNA vaccines carrying both the M and E genes induce higher antibody titers and thereby confer full protection against JEV challenge in the presence or absence of cardiotoxin pretreatment. To confirm that antibody in the sera of mice immunized without cardiotoxin pretreatment recognized E protein, C3H/ HeN mice were injected intramuscularly with pE or pCJ-3/ ME three times at 3-week intervals, and the sera collected at weeks 3, 6, and 8 were tested on Western blots of lysates of JEV-infected cells. The characteristic 52-kD band of E protein was clearly labeled with the serum of pCJ-3/ME-immunized mice at weeks 3, 6, and 8, but not with serum from pE-immunized mice. In addition, the titer of anti-JEV E antibody was time-dependent (data not shown). We also examined the morphology of pCJ-3/ME-transfected BHK-21 cells by transmission electron microscopy. Beijing-1 strain JEV-infected and pCJ-3/ME-transfected BHK-21 cells showed a normal gross appearance, similar to that of parental cells (not shown). At the suborganelle level, many small particulate structures were seen in the cisternae of the endoplasmic reticulum (ER) and the Golgi apparatus in the virus-infected cells (Fig. 4A), but not in non-infected or non-transfected cells (data not shown). The viral particles were spherical, 40 to 60 nm in diameter, and consisted of an electron-dense core (filled with viral genome) surrounded by a lipid bilayer (Fig. 4A). In the pCJ-3/ME-transfected BHK21 cells, the expression of M and E proteins triggered the formation of virus-like particles (VLPs) (Fig. 4B), which were bigger than wild type virus at 80 to 100 nm in diameter. 3.4. Effect of DNA vaccine on the antibody titer and the provoked immunity
Fig. 3. Effect of the correct structural conformation of E protein on JEV DNA vaccine-induced protective immunity with or without cardiotoxin pretreatment. (A) Cardiotoxin-pretreated (þ) or control () mice were given three intramuscular injections of plasmid DNA at 3-week intervals and serum samples were collected at different time-points and assayed for the presence of antiJEV E antibodies. The concentration of anti-E antibodies was calculated from a standard curve generated from serially diluted control antibodies and expressed as units per milliliter. The asterisks indicated significant differences (P < 0.01 (**)). (B) Two weeks after the last immunization, animals were challenged with 50 LD50 of Beijing-1 strain JEV, then observed for 30 days and the percentage of survivors calculated. In A and B, cardiotoxin-pretreated mice immunized with pCJ-3 by three i.m. injections at 3-week intervals were used as negative controls.
To evaluate the effectiveness of a single immunization with the created DNA vaccines, non-cardiotoxin-pretreated mice were given a single injection of pME or pCJ-3/ME. Cardiotoxin-pretreated mice injected with pE served as positive controls and untreated mice injected with a single dose of pCJ-3 served as negative controls. All mice were challenged with 50 LD50 (1.5 107 PFU) of JEV Beijing-1 at 8 weeks after immunization. As shown in Fig. 5A, no significant difference in anti-JEV E antibody titer was seen between the pME()and pCJ-3/ME()-treated groups at either week 3 or 6, although the pCJ-3/ME()-treated group had a 1.3-fold higher antibody titer than the pME()-treated group at week 8. In contrast, the pCJ-3/ME()-treated group had a 44-, 10.6-, and 9.4-fold higher antibody titer than the pE(þ)-treated group at week 3, 6, and 8, respectively. Unsurprisingly, none of the pCJ-3 mice survived JEV challenge (0 of 10), while the
2584
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
Fig. 4. Electron microscopy of JEV-infected or pCJ-3/ME-transfected BHK cells. BHK-21 cells were infected with JEV Beijing-1 strain at an MOI of 1 (A) or transfected with pCJ-3/ME by the calcium phosphate precipitation method (B), then incubated for 48 h. (A) JEV viral particles are seen as spherical electron-dense structures within the cisternae of the ER. (B) The spherical virus-like particles (arrows) consist of an electron-dense membrane surrounded by envelope protein within the Golgi apparatus. Magnification, 50,000; bar, 200 nm.
survival rate in the pE(þ)-treated group was 60% (6 of 10, P < 0.01, versus pCJ-3(þ) group) and those in the pME()and pCJ-3/ME()-treated groups were 100% (10 of 10, P < 0.01, versus pE(þ) group)(>30 days after virus challenge) (Fig. 5B). The ability of the serum from the different immunized groups to neutralize JEV infection in vitro was examined using PRNT. Groups of 6- to 8-week-old C3H/HeN mice were immunized intramuscularly either once or three times at 3week intervals with pCJ-3, pE, or pCJ-3/ME with or without cardiotoxin pretreatment, then, at week 8 after the first immunization, the sera were collected and checked for JEV-neutralizing antibodies. As shown in Table 1, no antibody was detectable (<1:10) in mice immunized with pE with or
Fig. 5. Effect of one dose of JEV DNA vaccine on protective immunity. (A) C3H/HeN mice with (þ) or without () cardiotoxin pretreatment were injected i.m. with a single dose of plasmid DNA, then serum samples were collected at weeks 3, 6, and 8, and the anti-JEV E antibody titers analyzed by ELISA assay. The asterisks indicate significant differences (P < 0.05 (*)). (B) Dose-effect of DNA vaccine-induced protective immunity. Animals were challenged at week 8 with 50 LD50 Beijing-1 JEV. Mice immunized with pCJ-3 three times at 3-week intervals were used as negative controls. Following challenge, the mice were observed for 30 days and the percentage of survivors was calculated.
Table 1 Induction of neutralizing antibodies in mice immunized with JEV DNA vaccines No. of injectionsa
Neutralizing antibodyb pCJ-3(þ)
pE()
pE(þ)
PCJ-3/ME()
PCJ-3/ME(þ)
1 3
<1/10 <1/10
<1/10 <1/10
1/10 1/10
1/160 >1/320
1/160 >1/320
a Groups of 6-to 8-week-old C3H/HeN mice with (þ) or without () cardiotoxin pretreatment were immunized intramuscularly either once or three times at 3 week intervals with pCJ-3, pE, or pCJ-3/ME. b JEV neutralizing antibody titers in serum collected 8 weeks after the first immunization expressed as the reciprocal of the serum dilution yielding a 50% reduction in plaque number.
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
without cardiotoxin pretreatment, while titers of 160 and >320 were seen with single and multiple injections, respectively, in pCJ-3/ME-immunized mice with or without cardiotoxin pretreatment. These results show that, in terms of neutralizing antibody, immunization with either one or three doses of pCJ-3/ME without cardiotoxin pretreatment was far more effective than identical treatment with pE with cardiotoxin pretreatment. 4. Discussion In small animal models, DNA vaccination has shown promise against an array of infectious diseases [7e10]. However, in primates, a relatively low level of induced antigen expression has been found, and the induced immune responses have been much lower than anticipated. In fact, the intrinsic efficacy of DNA vaccination may largely depend on the expression level of antigen (reviewed in ref. [23]). Many efforts have being made since the onset of DNA vaccination to increase the level of expression of a given gene. Gene regulatory elements, such as promoter-enhancer complexes, introns, and polyadenylation signals, may be important factors in gene expression. In addition, the prokaryotic antibiotic resistance gene and backbone elements have been altered and undesired viral sequences removed to produce a plasmid more acceptable for future clinical use (FDA, 1996). In the present study, we examined the effects of two regulatory elements, the intron and the polyadenylation signal, with the hope of increasing antigen expression. The level of expression of the engineered DNA plasmids in our cell-based assay was determined using a typical firefly luciferase reporter system or dot blots. The results showed that better expression of the introduced gene could be achieved by selecting an appropriate promoter/enhancer element, the choice being made based on cell type targeted and how the vector is constructed [24]. Almost all mammalian DNA vaccines developed to date have used CMV-derived promoters, with which high efficiency is observed across mammalian species, especially in tissues such as muscle and skin. Introduction or replacement of introns is reported to increase gene expression [12,13,20]. This may result from enhancement of RNA polyadenylation and nuclear transportassociated RNA splicing [13] or the presence of other transcriptional enhancers within the intron [12]. The largest intron of the CMV immediate early gene, intron A, followed by the CMV immediate early promoter/enhancer, has been shown to increase expression of heterologous genes in vitro [12]. Based on these observations, we created a vector, pCJ-3, containing a modified chimeric intron situated immediately downstream of the CMV enhancer/promoter region. The chimeric intron consisted of a 5 0 -splice donor site derived from the first intron of the human b-globin gene and a 3 0 -splice acceptor site derived from the immunoglobulin heavy chain variable region gene. The sequences of the donor and acceptor splice sites, along with the branchpoint site were aligned, and consensus sequences selected to act as the basis of the chimeric intron. Our results showed that cDNA flanked by the chimeric intron gave higher protein expression (Fig. 1). The polyadenylation
2585
signal tells RNA polymerase II to terminate transcription and to add 200e250 adenosine bases to the 3 0 -end of the RNA transcript [14]. The replacement of the SV40 late gene poly(A) signal with the BGH poly(A) signal resulted in a 5fold increase in luciferase expression (pCJ-2 0 vs. pCJ-3; Fig. 1A). This newly developed vector, pCJ-3, may soon be deployed in the front line against diseases. DNA vaccines have been demonstrated in many animal models to induce a broad range of immune responses, including antibodies, CD8þ CTLs, CD4þ helper T (Th) lymphocytes, and protective immunity against challenge with the pathogen. By using gene knockout mice, adoptive transfer experiments and T cell depletion experiments, Pan et al. [18] demonstrate that the anti-E antibody is the most critical protective component in this JEV challenge model, and production of anti-E antibody by pE DNA vaccine is dependent on the presence of CD4þT cells but independent of CD8þT cells. There is a positive correlation between the level of antigen expression and the levels of the corresponding antibody produced [23]. To estimate the intrinsic efficacy of these newly developed DNA vaccines, they were tested in C3H/HeN mice. The C3H/HeN mice injected intramuscularly with 100 mg of each of the plasmids coding for JEV E generated an anti-E-specific antibody, and the response was boosted by subsequent injections of 100 mg of vaccine. The results (Fig. 2A) demonstrated that there was no significant difference between the vectors in the production of JEV E-specific antibody at week 3, while, at week 6, pCJ-3/E generated 3-, 2-, and 3-fold more antibody than pCJ-2 0 /E, p3224/E, and pE, respectively, and at week 8, 1.6-, 1-, and 2-fold more than pCJ2 0 /E, p3224/E, and pE. The survival rates after virus challenge 16 weeks after primary immunization showed that pCJ-3/E provided full protection against challenge, p3224/E and pCJ2 0 /E both gave an 80% survival rate, and pE a 60% survival rate (Fig. 2B). Taken together, the data (Figs. 1 and 2) clearly indicate that pCJ-3, containing a chimeric intron and the BGH polyadenylation signal, increased the efficiency of muscletargeted gene expression in vitro and immunity in vivo. E protein is a major surface protein that plays a pivotal role in receptor binding and membrane fusion and is known to contain many protective epitopes [25]. M protein is found in infected cells as a glycosylated precursor, the premembrane protein (prM). During virion maturation in vertebrate cells, provirion particles are formed when portions of endoplasmic reticulum membrane containing prM and E envelop nucleocapsids consisting of the C protein and genomic RNA [26]. These poorly infectious provirions accumulate in the lumen of the exocytic pathway, and during virion maturation, prM is cleaved to M by a cellular protease, furin, located in the trans-Golgi network [27]. Maturation cleavage is accompanied by changes in the oligomerization of prM and M. E, which is essential for the development of matured virions, contains both hemagglutination and fusion activities [27]. Further support for the requirement for coordinated synthesis of prM and E proteins comes from the observation that prM and E form heterodimers in cell-associated forms of West Nile virus [28]. This feature explains the excellent vaccine potential of
2586
C.J. Wu et al. / Microbes and Infection 8 (2006) 2578e2586
the JEV vector, which results in the production of virus-like particles which enhance antigenic stability and provide highdensity presentation to antigen-presenting cells such as macrophages, dendritic cells, and Langerhans’ cells [29]. When DNA is given by the intramuscular route, the majority of vaccine antigens are expressed by cells which do not present antigen to the immune system. The efficacy of a DNA vaccine is therefore dependent on transfection of antigen-presenting cells or the reprocessing of antigen derived from other cells [30]. In this study, muscle cells transfected by the JEV DNA vaccines synthesized and secreted virus-like particles, resulting in full protection and the generation of cellular and humoral immunity without cardiotoxin pretreatment (Fig. 3). A high expression capacity and the selection of a suitable antigen gene are the key factors for a competent DNA vaccine. The DNA vaccines developed in the present study clearly fulfill these criteria. This paves the way to their use in man, although an accurate and efficient delivery system has yet to be developed. Acknowledgements This work was supported financially by the National Science Council, Taiwan and the ADImmune Corporation, Tantz, Taichung, Taiwan. We also thank Dr Tom Barkas for critical evaluation of the English. References [1] D.S. Burke, T.P. Monath, in: D.M. Knipe, P.M. Howley (Eds.), Fields Virology, fourth ed. Lippincott Williams & Wilkins Publishers, Philadelphia, PA, 2001, pp. 1043e1125. [2] T.F. Tsai, G.J. Chang, Y.X. Yu, in: A.P. Stanley, W.A. Orenstein (Eds.), Vaccines, third ed. W.B. Saunders, Philadelphia, PA, 1999, pp. 672e710. [3] M. Sakaguchi, M. Yoshida, W. Kuroda, O. Harayama, Y. Matsunaga, S. Inouye, Systemic immediate-type reactions to gelatin included in Japanese encephalitis vaccines, Vaccine 15 (1997) 121e122. [4] J.D. Poland, C.B. Cropp, R.B. Craven, T.P. Monath, Evaluation of the potency and safety of inactivated Japanese encephalitis vaccine in US inhabitants, J. Infect. Dis. 161 (1990) 878e882. [5] R.F. Juang, Y. Okuno, T. Fukunaga, M. Tadano, K. Fukai, K. Baba, N. Tsuda, A. Yamada, H. Yabuuchi, Neutralizing antibody responses to Japanese encephalitis vaccine in children, Biken J. 26 (1983) 25e34. [6] D.C. Tang, M. DeVit, S.A. Johnston, Genetic immunization is a simple method for eliciting an immune response, Nature 356 (1992) 152e154. [7] J.B. Ulmer, J.J. Donnelly, S.E. Parker, G.H. Rhodes, P.L. Felgner, V.J. Dwarki, S.H. Gromkowski, R.R. Deck, C.M. DeWitt, A. Friedman, L.A. Hawe, K.R. Leander, D. Martinez, H.C. Perry, J.W. Shiver, D.L. Montgomery, M.A. Liu, Heterologous protection against influenza by injection of DNA encoding a viral protein, Science 259 (1993) 1745e1749. [8] Z.Q. Xiang, S. Spitalnik, M. Tran, W.H. Wunner, J. Cheng, H.C.J. Ertl, Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus, Virology 199 (1994) 132e140. [9] M. Sedegah, R. Hedstrom, P. Hobart, S.L. Hoffman, Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 9866e9870.
[10] R.E. Tascon, M.J. Colston, S. Ragno, E. Stavropoulos, D. Gregory, D.B. Lowrie, Vaccination against tuberculosis by DNA injection, Nat. Med. 2 (1996) 888e892. [11] E.F. Fynan, R.G. Webster, D.H. Fuller, J.R. Haynes, J.C. Santoro, H.L. Robinson, DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 11478e11482. [12] B.S. Chapman, R.M. Thayer, K.A. Vincent, N.L. Haigwood, Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells, Nucleic Acids Res. 19 (1991) 3979e3986. [13] M.T.F. Huang, C.M. Gorman, Intervening sequences increase efficiency of RNA 3 0 processing and accumulation of cytoplasmic RNA, Nucleic Acids Res. 18 (1990) 937e946. [14] N.J. Proudfoot, Poly(A) signals, Cell 64 (1991) 671. [15] B.D. Lindenbach, C.M. Rice, in: D.M. Knipe, P.M. Howley (Eds.), Fields Virology, fourth ed. Lippincott Williams & Wilkins Publishers, Philadelphia, PA, 2001, pp. 991e1041. [16] E. Konishi, P.W. Mason, Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein, J. Virol. 67 (1993) 1672e1675. [17] H.W. Chen, C.H. Pan, M.Y. Liau, R. Jou, C.J. Tsai, H.J. Wu, Y.L. Lin, M.H. Tao, Screening of protective antigens of Japanese encephalitis virus by DNA immunization: a comparative study with conventional viral vaccines, J. Virol. 73 (1999) 10137e10145. [18] C.H. Pan, H.W. Chen, H.W. Huang, M.H. Tao, Protective mechanisms induced by a Japanese encephalitis virus DNA vaccine: requirement for antibody but not CD8(þ) cytotoxic T-cell responses, J. Virol. 75 (2001) 11457e11463. [19] S.C. Wu, W.C. Lian, L.C. Hsu, M.Y. Liau, Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence, Virus Res. 51 (1997) 173e181. [20] T. Choi, M. Huang, C. Gorman, R. Jaenisch, A generic intron increases gene expression in transgenic mice, Mol. Cell Biol. 11 (1991) 3070e 3074. [21] K. Miura, T. Onodera, A. Nishida, N. Goto, Y. Fujisaki, A single gene controls resistance to Japanese encephalitis virus in mice, Arch. Virol. 112 (1990) 261e270. [22] C.J. Wu, S.C. Lee, H.W. Huang, M.H. Tao, In vivo electroporation of skeletal muscle increases the efficacy of Japanese encephalitis virus DNA vaccine, Vaccine 22 (2004) 1457e1464. [23] M.A. Barry, S.A. Johnston, Biological features of genetic immunization, Vaccine 15 (1997) 788e791. [24] J. Harms, G. Splitter, Interferon-g inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter, Hum. Gene Ther. 6 (1995) 1291e1297. [25] F.X. Heinz, Epitope mapping of flavivirus glycoproteins, Adv. Virus Res. 31 (1986) 103e168. [26] T.J. Chambers, C.S. Hahn, R. Galler, C.M. Rice, Flavivirus genome organization, expression, and replication, Annu. Rev. Microbiol. 44 (1990) 649e688. [27] K. Stadler, S.L. Allison, J. Schalich, F.X. Heinz, Proteolytic activation of tick-borne encephalitis virus by furin, J. Virol. 71 (1997) 8475e8481. [28] G. Wengler, G. Wengler, Cell-associated West Nile flavivirus is covered with E þ pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release, J. Virol. 63 (1989) 2521e2526. [29] C. Condon, S.C. Watkins, C.M. Celluzzi, K. Thompson, L.D. Falo Jr., DNA-based immunization by in vivo transfection of dendritic cells, Nat. Med. 2 (1996) 1122e1128. [30] E. Konishi, M. Yamaoka, I. Khin-Sane-WinKurane, P.W. Mason, Induction of protective immunity against Japanese encephalitis in mice by immunization with a plasmid encoding Japanese encephalitis virus premembrane and envelope genes, J. Virol. 72 (1998) 4925e4930.