Mutation of putative N-Linked Glycosylation Sites in Japanese encephalitis Virus Premembrane and Envelope proteins enhances humoral immunity in BALB/C mice after DNA vaccination
© Zhang et al; licensee BioMed Central Ltd. 2011
Received: 4 December 2010
Accepted: 25 March 2011
Published: 25 March 2011
Swine are an important host of Japanese encephalitis virus (JEV). The two membrane glycoproteins of JEV, prM and E, each contain a potential N-linked glycosylation site, at positions N15 and N154, respectively. We constructed plasmids that contain the genes encoding wild-type prME (contain the signal of the prM, the prM, and the E coding regions) and three mutant prME proteins, in which the putative N-linked glycosylation sites are mutated individually or in combination, by site-directed mutagenesis. The recombinant plasmids were used as DNA vaccines in mice. Our results indicate that immunizing mice with DNA vaccines that contain the N154A mutation results in elevated levels of interleukin-4 secretion, induces the IgG1 antibody isotype, generates greater titers of anti-JEV antibodies, and shows complete protection against JEV challenge. We conclude that mutation of the putative N-glycosylation site N154 in the E protein of JEV significantly enhances the induced humoral immune response and suggest that this mutant should be further investigated as a potential DNA vaccine against JEV.
Japanese encephalitis virus
Tick-borne encephalitis virus
West Nile virus
complementary Deoxyribonucleic Acid
Polymerase Chain Reaction
- PNGase F:
Peptide N-Glycosidase F
Japanese encephalitis virus (JEV) belongs to the genus Flavivirus, and the genus Flavivirus include many clinically important pathogens, such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus, Murray Valley encephalitis virus, St.Louis encephalitis virus, and tick-borne encephalitis virus (TBEV). Japanese encephalitis virus (JEV) mostly causes infection of the central nervous system in humans and equines and stillbirths in swine [1, 2]. The virus is zoonotic, cycling between birds and mosquitoes, and is transmitted to humans by infected mosquitoes. Since swine serve as a reservoir and amplifier of the virus , the development of a swine vaccine against JEV is a high priority, as it could help prevent epidemics in humans.
JEV contains a single-stranded, plus-sense RNA genome of ~11 kb. It consists of a single open reading frame that codes for a large polyprotein of 3432 amino acids that is co- and post-translationally cleaved into three structural proteins (capsid, C; premembrane, prM; and envelope, E) and seven nonstructural proteins [4, 5]. Envelope is the major structural protein, and makes up the surface of the avivirus particle. E protein has numerous neutralization epitopes, which mediate attachment to host cells, and a putative receptor-binding domain that induces the host immune response [6, 7]. Though prM is able to fold independently of the E protein, correct folding of the E protein requires co-synthesis with prM . PrM interacts with E to form prM-E heterodimers, which are important for the formation of immature virions[9, 10], and the signal of the prM determine translocation and orientation of inserted protein, hence the topology of prM and E . Therefore, the signal of the prM and the prM protein play an important role in maintaining its native conformation of E protein.
N-linked glycans of viral proteins play important roles in modulating the immune response. Glycans can be important for maintaining the appropriate antigenic conformations, shielding potential neutralization epitopes, and may potentially alter the proteolytic susceptibility of proteins [12, 13]. In the JE viruses, the prM protein contains one putative N-linked glycosylation site, at N15. E protein also has one putative N-linked glycosylation site, at N154. Studies with JEV, TBEV and WNV have found that deletion of the N-linked glycosylation site in prM or E led to a decrease in virus release [14–16]. However, the effects of these putative N-linked glycosylation sites on the immune response to JEV remained elusive.
Our primary aim in this work was to investigate the role of the putative prME N-linked glycosylation sites in inducing an immune response. It is known that immunizing mice with plasmids encoding the prM and E glycoproteins of JEV provide varying degrees of protection against the virus . In this study, we constructed plasmids containing both the wild-type prME and mutant prME genes, in which the N-linked glycosylation sites are mutated individually or in combination. The immunogenicity of the three prME glycosylation mutants was evaluated in mice. We determined that mutating N154 of prME significantly enhanced the immune response in mice and propose that this mutant should be explored as a swine vaccine against JEV.
2. Materials and methods
2.1 Cells and virus
The NJ2008 strain (GQ918133) of JEV was isolated from brain tissues of aborted fetuses of sows, which were obtained from a piggery in the Jiangsu province in 2008. The NJ2008 strain of JEV was propagated in baby hamster kidney (BHK-21) cells (ATCC CCL-10) for the plaque reduction neutralization test (PRNT) and challenge test. The supernatants of the infected cells were clarified and stored at -80°C for animal challenge. The viral titers of the supernatants were approximately 7.3 × 108 PFU as determined by the PRNT. Monkey kidney cells (Vero) (ATCC) used for recombinant plasmid transfection were grown and maintained in Dubach's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/ml of streptomycin, and 100 μg/ml of penicillin.
2.2 Construction of site-directed prME mutants
pVAX-prME mutant vectors and predicted glycosylation patterns.
Asn Asn Thr
Asn Tyr Ser
Ala Ɵ Asn Thr
Asn Tyr Ser
Asn Asn Thr
Ala Ɵ Tyr Ser
Ala Ɵ Asn Thr
Ala Ɵ Tyr Ser
2.3 Transfection and Western blotting
BHK-21 cells were seeded at a concentration of 2.5 × 104 cells/well into 6-well tissue culture plate until the cells reached approximately 70-80% confluence. Transfection was performed with LipofectAMINE 2000 reagent (Invitrogen) as specified by the manufacturer. The transfected cells were collected at 48 h post-transfection and lysed in buffer L (50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl sulfate, pH 7.5). The lysates were centrifuged at 10,000 × g for 10 min to clear cellular debris and inactivated at 56°C for 1 h, and aliquots of each proteins were digested with 500 U of peptide N-glycosidase F (PNGase F, New England Biolabs) for 1 h at 37°C or mock digested as a negative control. Samples were then analyzed under denaturing conditions by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting was performed using JEV-positive serum (kept in our laboratory). Detection was performed using chemilumines.
2.4 Mouse immunization
Four-week old female BALB/c mice were purchased from the Animal Center of Nanjing Army Hospital, Nanjing, China. All mice were maintained in sterile cages in specific-pathogen-free environments. Five groups of mice (fourteen mice per group) were inoculated with one of the following plasmids: pVAX-prME-WT, pVAX-prME-M1, pVAXI-prME-M2, pVAX-prME-M3, and pVAX (control group). The mice received 50 μg of recombinant plasmids intramuscularly (IM) into each thigh (total dose 100 μg). All groups were inoculated three times at 2-week intervals. Serum samples were collected from the central tail vein before immunization on days 7, 21 and 35 after the prime immunization, and sera were stored at -20°C.
One week after the second boost immunization, four mice from each group were sacrificed and their spleens removed aseptically for in vitro splenocyte culture. The remaining mice were challenged by i.p. injection of a lethal dose of the JEV NJ2008 strain (5 × 106 PFU). Survival of the mice was monitored daily up to 15 days post-challenge. All animal experiments were conducted according to the guidelines approved by the Animal Ethical and Experimental Committee of the Nanjing Agricultural University.
2.5 ELISA assays to profile antibodies and measure cytokine production
Antibody levels were measured in sera were collected on days 7, 21 and 35 after the prime immunization. Antibody subtypes were analyzed by ELISA  with some modifications. The 96-well Maxi-sorpTM plates (Nunc) were coated overnight with purified JEV particles (10 ng/ml) in 0.1 M sodium carbonate (pH~9.5). The presence of IgG, IgG1 and IgG2a was measured using HRP-conjugated antibodies that recognized each of the subtypes. Production of IL-4 and IFN-γ was measured in serum samples collected from all five experiment groups 35 days after the prime immunization, using the commercially available mice cytokine ELISA kits (RD, USA).
2.6 Plaque reduction neutralization assay (PRNT)
Neutralization antibodies elicited in immunized mice were evaluated by PRNT as described previously . Two fold serial dilutions of murine sera starting at 1:5 were tested. The percentage neutralization was calculated from the number of plaques obtained in the presence or absence of serum. The reciprocal of the highest serum dilution giving at least 50% neutralization was taken as the JEV neutralization titer.
2.7 Statistical analysis
All data analyses were conducted using SPSS biostatistics software (version 16.0, SPSS Inc., Chicago, IL, USA).
3.1 Expressions of JEV prME-WT and three mutant proteins by Western blot analysis
3.2 Antibody isotype profiles
3.3 Cytokine response to prME mutants
3.4 Production of neutralizing antibodies
The neutralization titers of serum and protective immunity in vaccinated mice
Survival rate (%)
13 ± 4
15 ± 5
35 ± 9a
38 ± 8a
3.5 Protection against JEV challenge in immunized mice
To investigate the degree to which the immunized mice were protected from JEV, all immunized mice were exposed to a lethal dose of the JEV strain NJ2008 and evaluated for their ability to survive the challenge. Mice immunized with prME-M2 or prME-M3 showed complete protection against JEV challenge. Nine out of ten mice immunized with prME-WT and prME-M1 survived JEV challenge, whereas only one mouse in the control group survived (Table 2).
Japanese encephalitis (JE) is a serious disease prevalent throughout Asia  and is transmitted to humans by mosquito bite . Pigs are an important amplifier host for the virus  Vaccination of swine, therefore, can help prevent disease in humans .
DNA vaccines against JEV have shown great potential as preventative agents for their ability to elicit potent humoral and cytotoxic cellular immune responses against the plasmid-encoded protein in a broad range of hosts . A previous study demonstrated that plasmids carrying the JEV prM and E genes can induce high NEUT antibodies and protective immunity in mice. Most importantly, the signal of prM is included in DNA vaccine, thus it is likely that viral antigens can be secreted from transfected cells and these DNA vaccines can induce high levels of immune responses . In this study, we assessed the immunogenicity of several prME mutants to evaluate the potential of these mutants as DNA vaccines against JEV in swine.
The prM and E proteins of JEV are both exposed structural proteins. E is a major immunogenic antigen, and the prM is JEV induce protective immune additional components . It has been reported that the prM proteins of flaviviruses can form natively folded structures independent of the E protein, form hetero-dimers with the E protein, and appear to act as folding chaperones for E protein . In addition, the prME expression plasmids contain the signal of prM coding sequences for translocation into endoplasmic reticulum (ER), and the signal of prM can make the respective proteins are glycosylated or transported by the secretory pathway as supposed . To investigate the effect of mutating putative N-glycosylation sites on the immunogenicity of prM and E, these proteins must maintain their native conformation.
Previous studies have demonstrated that N-linked glycans on the glycoproteins of many viruses play important roles in modulating the immune response. Removal of N-glycosylation sites in the simian immunodeficiency virus envelope protein and influenza virus hemagglutinin protein have been observed to limit the neutralizing antibody response , while mutation of N-linked glycans in human immunodeficiency virus type 1 (HIV-1) envelope protein appears to enhance the production of CTL , and deletion of glycans in hepatitis C virus E1 can enhance cellular or humoral immune responses . The effects of the putative N-linked glycosylation sites in prM and E on the immune response to JEV are not known, however.
The JEV PrM protein contains one putative N-linked glycosylation site at N15, and the E protein also contains one putative N-linked glycosylation site, at N154. We demonstrate that immunization with the mutants prME-M2 and prME-M3, both of which contain the N154A mutation, induced a significantly enhanced antibody response, elevated IL-4 secretion levels, and full protection to lethal challenge of JEV compared to immunization with native prME, indicating that these mutations could elicit a stronger humoral immune response than the wild-type prME. We also demonstrate that mutating the N15 site (prME-M1) induces a humoral immune response comparable to that observed upon immunization with wild-type prME. This strongly suggests that mutating N15 in the prM glycoprotein does not strongly perturb the immune response to prME, but mutating N154 of the E glycoprotein does affect the immune response to prME.
Though DNA vaccines generally induce a stronger Th1 immune response, producing elevated levels of IFN-γ and IgG2a, the immune responses induced by DNA vaccines need to be improved. Our results show that a single mutation, N154A, significantly enhances the humoral immune response. We propose, therefore, that this highly immunogenic mutant could serve as a swine vaccine against JEV and should be further optimized for this purpose.
This work was supported by the National Special Research Programs for Non-Profit Trades, Ministry of Agriculture (No. 200803015)
- Shope RE: Medical significance of togaviruses: an overview of diseases caused by togaviruses in man and in domestic and wild vertebrate animals. In The togaviruses. Edited by: Schlesinger RW. New York: Academic Press; 1980:47–82.
- Monath TP: Pathobiology of the flaviviruses. In The Togaviridae and Flaviviridae. Edited by: Schlesinger S, Schlesinger MJ. New York: Plenum Press; 1986:375–440.
- Scherer WF, Moyer J, Izumi T, Gresser I, McCown J: Ecologic studies of Japanese encephalitis virus in Japan: VI, Swine infection. Am J Trop Med Hyg 1959, 8:698–706.PubMed
- Vrati S, Giri RK, Razdan A, Malik P: Complete nucleotide sequence of and Indian strain of Japanese encephalitis virus: sequence comparison with other strains and phylogenetic analysis. Am J Trop Med Hyg 1999, 61:677–680.PubMed
- Chambers TJ, Hahn CS, Galler R, Rice CM: Flavivirus genome organization, expression and replication. Annu Rev Microbiol 1990, 44:649.PubMedView Article
- Chen Y, Maguire T, Marks RM: Demonstration of Binding of Dengue Virus Envelope protein to Target Cells. J. Virol 1996, 70:8765–8772.PubMed
- Hung JJ, Hsieng MT, Young MJ, Kao CL, King CC, Chang W: An External Loop Region of Domain III of Dengue Virus Type 2 Envelope Protein Is Involved in Serotype-Specific Binding to Mosquito but Not Mammalian Cells. J Virol 2004, 78:378–388.PubMedView Article
- Konishi E, Mason PW: Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein. J. Virol 1993, 67:1672–16755.PubMed
- Allison SL, Stadler K, Mandl CW, Kunz C, Heinz FX: Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form. J. Virol 1995, 69:5816–5820.PubMed
- Courageot MP, Frenkiel MP, Dos Santos CD, Deubel V, Despres P: α-Glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum. J. Virol 2000, 74:564–572.PubMedView Article
- Konishi E, Yamaoka M, Win KS, Kurane I, Mason PW: Induction of protective immunity against Japanese encephalitis in mice by immunization with a plasmid encoding Japanese encephalitis virus premembrane and envelope genes. J Virol 1998, 72:4925–4930.PubMed
- Ming Zhang, Brian Gaschen, Wendy Blay, Brian Foley, Nancy Haigwood: Tracking global patterns of N-linked glycosylation site variation highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology 2004, 14:1229–1246.View Article
- Goffard A, Callens N, Bartosch B, Wychowski C, Cosset FL, Montpellier C: Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins. J Virol 2005, 79:8400–8409.PubMedView Article
- Goto A, Yoshii K, Obara M, Ueki T, Mizutani T, Kariwa H, Takashima I: Role of the N-linked glycans of the prM and E envelope proteins in tick-borne encephalitis virus particle secretion. Vaccine 2005, 23:3043–3052.PubMedView Article
- Wei HY, Jiang LF, Fang DY, Guo HY: Dengue virus type 2 infects human endothelial cells through binding of the viral envelope glycoprotein to cell surface polypeptides. J Gen Virol 2003, 84:3095–3098.PubMedView Article
- Jeong MK, Sang IY, Byung HS, Youn SH, Chan HL, Hyun WO, Young ML: A Single N-Linked Glycosylation Site in the Japanese Encephalitis Virus prM Protein Is Critical for Cell Type-Specific prM Protein Biogenesis, Virus Particle Release, and Pathogenicity in Mice. J Virol 2008, 82:7846–7862.View Article
- Konishi E, Ajiro N, Nukuzuma C: Comparison of protective efficacies of plasmid DNAs encoding Japanese encephalitis virus proteins that induce neutralizing antibody or cytotoxic T lymphocytes in mice. Vaccine 2003,21(25–26):3675–3683.PubMedView Article
- Kaur R, Sachdeva G, Vrati S: Plasmid DNA immunization against Japanese encephalitis virus: immunogenicity of membrane-anchored and secretory envelope protein. J Infect Dis 2002, 185:1–12.PubMedView Article
- Wu SC, Yu CH, Lin CW, Chu IM: The domain III fragment of Japanese encephalitis virus envelope protein: mouse immunogenicity and liposome adjuvanticity. Vaccine 2003, 21:2516–22.PubMedView Article
- Chambers TJ, Hahn CS, Galler R, Rice CM: Flavivirus genome organization, expression, and replication. Annual Review of Microbiology 1990, 44:649–688.PubMedView Article
- Libraty DH, Nisalak A, Endy TP, Suntayakorn S, Vaughn DW, Innis BL: Clinical and immunological risk factors for severe disease in Japanese encephalitis. Transactions of the Royal Society of Tropical Medicine and Hygiene 2002, 96:173–178.PubMedView Article
- Konishi E, Yamaoka M, Kurane I, Mason PW: Japanese encephalitis DNA vaccine candidates expressing premembrane and envelope genes induce virus-specific memory B cells and long-lasting antibodies in swine. Virology 2000, 268:49–55.PubMedView Article
- Igarashi A: Japanese encephalitis: virus, infection, and control. In Control of Virus Diseases. second edition. Edited by: Kurstak E. Marcel Dekker, New York, USA; 1992:309–342.
- Hasan UA, Abai AM, Harper DR, Wren BW, Morrow WJW: Nucleic acid immunisation: concepts and techniques associated with third generation vaccines. J Immunol Methods 1999, 229:1–22.PubMedView Article
- Kaur R, Vrati S: Development of a recombinant vaccine against Japanese encephalitis. J Neurovirol 2003, 9:421–431.PubMed
- Kodihalli S, Goto H, Kobasa DL, Krauss S, Kawaoka Y, Webster RG: DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice. J Virol 1999, 73:2094.PubMed
- Doe B, Steimer KS, Walker CM: Induction of HIV-1 envelope (gp120)- specific cytotoxic T lymphocyte responses in mice by recombinant CHO cell-derived gp120 is enhanced by enzymatic removal of N-linked glycans. Eur J Immunol 1994, 24:2369.PubMedView Article
- Min Liu, Zhang Xiao-Lian: Deletion of N-glycosylation sites of hepatitis C virus envelope protein E1 enhances specific cellular and humoral immune responses. Vaccine 2007, 25:6572–6580.View Article
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.