Recombinant tandem multi-linear neutralizing epitopes of human enterovirus 71 elicited protective immunity in mice
- Yue-Xiang Li1, 2Email author,
- Hui Zhao2Email author,
- Rui-Yuan Cao2,
- Yong-Qiang Deng2,
- Jian-Feng Han2,
- Shun-Ya Zhu2,
- Jie Ma2,
- Long Liu1, 2,
- E-De Qin1, 2 and
- Cheng-Feng Qin1, 2Email author
© Li et al.; licensee BioMed Central Ltd. 2014
Received: 16 January 2014
Accepted: 30 April 2014
Published: 6 May 2014
Human Enterovirus 71 (EV71) has emerged as the leading cause of viral encephalitis in children, especially in the Asia-Pacific regions. EV71 vaccine development is of high priority at present, and neutralization antibodies have been documented to play critical roles during in vitro and in vivo protection against EV71 infection.
In this study, a novel strategy to produce EV71 vaccine candidate based on recombinant multiple tandem linear neutralizing epitopes (mTLNE) was proposed. The three well identified EV71 linear neutralizing epitopes in capsid proteins, VP1-SP55, VP1-SP70 and VP2-SP28, were sequentially linked by a Gly-Ser linker ((G4S)3), and expressed in E.coli in fusion with the Trx and His tag at either terminal. The recombinant protein mTLNE was soluble and could be purified by standard affinity chromatography. Following three dosage of immunization in adult mice, EV71-specific IgG and neutralization antibodies were readily induced by recombinant mTLNE. IgG subtyping demonstrated that lgG1 antibodies dominated the mTLNE-induced humoral immune response. Especially, cytokine profiling in spleen cells from the mTLNE-immunized mice revealed high production of IL-4 and IL-6. Finally, in vivo challenge experiments showed that passive transfer with anti-mTLNE sera conferred full protection against lethal EV71 challenge in neonatal mice.
Our results demonstrated that this rational designed recombinant mTLNE might have the potential to be further developed as an EV71 vaccine in the future.
KeywordsEnterovirus 71 Vaccine Linear neutralizing epitopes
Human enterovirus 71 (EV71), a typical single-stranded, positive-sense RNA virus, belongs to the Enterovirus genus of the Picornaviridae family. In recent years, EV71 has emerged as the most important causative agent of Hand, Foot and Mouse disease (HFMD) affecting mostly young children, especially those younger than 5 years old. The clinical symptoms of EV71 infection include simple exanthema, serious aseptic meningitis, acute flaccid paralysis as well as brainstem encephalitis . Although present in most countries, the largest outbreaks of disease have been seen in the Asia-Pacific region over the past 15 years [2–6], and many areas have experienced cyclical epidemics that occur every 2–3 years [7–10]. EV71 infection has now been recognized as an important global public health issue.
Vaccination probably offers the best option for disease control, but there is no available licensed vaccine against EV71. Several vaccine candidates including formaldehyde-inactivated whole-virus vaccine, live-attenuated vaccine, virus-like particles (VLPs), DNA vaccine and subunit vaccine, have showed promise for clinical use [11–20]. Especially, inactivated EV71 vaccines manufactured in mainland China have undergone phase III clinical trials with ideal efficacy .
The genome of EV71 is about 7.4 kb in length, which first encodes a long polyprotein with a single open reading frame followed by a poly A tract. Then, the polyprotein is divided into three different precursor proteins (P1, P2 and P3). The P1 precursor protein is further cleaved to four structural proteins, including VP0 (the precursor of VP2 and VP4), VP3 and VP1, which make up the capsid; whereas P2 and P3 are cleaved to non-structural proteins that are involved in genome replication and translation . Among the capsid protein, VP1, VP2, and VP3 are external, and well exposed to host immune system. Neutralizing antibodies against EV71 have been demonstrated as the most important factors in limiting the severity of infection . Several linear neutralizing epitopes have been identified within the capsid proteins of EV71. Foo and colleagues have characterized two neutralizing epitopes in VP1, SP55 (amino acids 163–177) and SP70 (amino acids 208–222), both were capable of eliciting neutralizing antibodies and conferred protection against homologous and heterologous EV71 strains in neonatal BALB/c mice . Moreover, monoclonal antibody targeting the linear epitope on VP1 protein, spanning amino acids 215–219, provided full protection against EV71 challenge in vivo [25, 26]. Recently, screening with overlapping synthetic peptides covering the entire capsid protein of EV71 identified SP28 (amino acids 136–150 of VP2) as potential neutralizing epitope . Epitope-based vaccines containing well-defined protective epitopes are supposed to stimulate effective and specific protective immune responses while avoiding potential undesirable side-effect [28, 29]. Various epitope-based vaccine candidates against virus infection have been well demonstrated with ideal immunogenicity and protection [30–32], and several promising pre-clinical and clinical trials for vaccines that involve peptide-based strategies are currently being carried out [33, 34], yet only a few epitope-based EV71 vaccines have been described [24, 35, 36].
In this study, a novel EV71 epitope-based vaccine candidate was designed through tandem connecting the three known EV71 neutralizing epitopes with the Gly-Ser linker and expressed in E.coli. This recombinant multiple tandem linear neutralizing epitopes, named mTLNE, was evidenced to evoke humoral and cellular immune responses and passive transfer with anti-mTLNE sera conferred full protection against lethal EV71 challenge in mice.
Expression and characterization of recombinant mTLNE
Recombinant mTLNE induced EV71-specific humoral and cellular immune response in mice
ELISA titers against individual EV71 epitope
Passive transfer with anti-mTLNE confers full protection against lethal EV71 challenge in neonatal mice
Epitope-based vaccine provides an opportunity to rationally engineer specific epitopes to increase potency and breadth and avoid the potential side-effect of other unrelated epitopes that contribute few to protection [38, 39]. To data, only a few epitope-based vaccines against EV71 have been described. In our study, we propose a novel strategy to connect the three identified linear neutralizing peptides by a Gly-Ser linker, generating the recombinant epitope-based vaccine candidate mTLNE. Recombinant mTLNE was efficiently expressed in E. coli system and easily purified with affinity chromatography. The Gly-Ser linker was chosen to increase the flexibility of the recombinant protein, to minimize interference between adjacent epitopes, and to facilitate forming natural conformation . The designed mTLNE contained two epitopes in VP1 and one epitope in VP2, and antisera from mice immunized with mTLNE interacted simultaneously with both VP1 and VP2 proteins of EV71 (Figure 3A).
Further, immunization with mTLNE induced potent humoral immune responses as demonstrated by the elicitation of EV71-specific IgG antibodies (Figure 3B). Antibodies induced by mTLNE immunization were able to neutralize both the circulating strain AH08/06 and the prototype strain BrCr (Figure 3D), suggested that mTLNE might induce broad neutralization against homologous and heterologous genotypes of EV71. This is consistent with previous reports by Li et al. . The specific IgG antibody response in mice appeared somewhat divergent among different epitopes, with relative low titer against VP1-SP55 (Table 1). Such immunogenic inferiority of SP55 epitope has also been observed by others . These differences are likely a reflection of the nature of viral antigen.
In general, neutralizing antibodies are important index for protection against EV71 infection. In the present study, the geometric mean of neutralizing antibody titers induced by mTLNE was 50, which was higher than that of single synthetic peptide-SP70 . Compared with inactivated vaccines, the neutralizing titers elicited by mTLNE were relatively low due to lack of other antigens [17, 37]. However, the advantage of this epitope based vaccine approach should not be highlighted. Especially, antibody-dependent enhancement (ADE) of EV71 infection has been observed in both experimental and clinical setting [41, 43–45]. Recent findings from human intravenous immunoglobulin clarified the distinct function of each IgG subtype on neutralization and enhancement of EV71 infection , and IgG1 subtype is deemed to dominate neutralization while IgG3 associates with ADE. The robustness of IgG1 response, not IgG3, induced by mTLNE (Figure 4) indicated potential benefits for protection. Most importantly, our data clearly demonstrated that passive transfer of anti-mTLNE sera conferred full protection to neonatal mice against lethal EV71 challenge (Figure 6).
In addition to humoral immunity, mTLNE immunization induced cellular immune responses as evidenced by elevated production of specific cytokines (Figure 5). The production of IL-4 and IL-6 in mTLNE-immunized mouse splenocytes suggested a Th2 immune response. In mice, IL-4 and IL-6 generally switches activated B cells to the IgG1 subtype, which was confirmed by IgG subtypes assay and IgG1 subtype was prevalent in case of mTLNE group (Figure 4). These observations suggest that mTLNE contain mainly B-cell epitopes, which has also been observed by Foo et al. [24, 36]. Currently, only a few T-cell epitopes on the capsid proteins of EV71 have been identified [47, 48]. In the future work, a peptide-based vaccine containing multiple B-cell epitopes as well as T-cell epitopes can be expected, which might be an ideal vaccine that are capability of inducing a protective antibody response and a cytotoxic T-cell response important for killing infected host cells.
Overall, we propose a novel epitope-based vaccine approach by connecting linear neutralizing peptides, and primary immunogenicity and protection experiments in mice demonstrated the potential for further development as EV71 vaccine.
Materials and methods
Cell lines and viruses
Human Rhabdomyosarcoma RD cells and African green monkey kidney Vero cells were cultured in DMEM medium (Invitrogen) containing 10% fetal bovine serum (FBS) (Hyclone) at 37°C in the presence of 5% CO2 [46, 49]. EV71 prototype strain BrCr (GenBank accession no.U22521), belongs to genotype A, and genotype C4 strain AH08/06 (GenBank accession no. HQ611148.1) was isolated from an HFMD patient during an outbreak in 2008 in Anhui, China . The EV71 virus stocks were propagated in RD cells according standard protocol and titers were determined in Vero cells [35, 51].
Preparation of mouse and rabbit polyclonal antibody against EV71
The mouse polyclonal antibody against EV71 was prepared in our lab  and the rabbit polyclonal antibody against EV71 were prepared as followed. A rabbit (2.5-3 kg) was immunized subcutaneouly (s.c.) with heat-inactivated EV71 (2 × 107 PFU/ml) employing Freund’s complete adjuvant (Sigma), and boosted with the same dose antigen with Freund’s incomplete adjuvant (Sigma). Two weeks after the boost immunization, serum samples were titrated and stored frozen until use.
Overlap PCR amplification and plasmid construction
The cDNA fragments encoding three tandem EV71 neutralizing epitopes (VP1-SP55, VP1-SP70 and VP2-SP28) which connected by the Gly-Ser linker ((Gly4Ser)3) were amplified by overlap PCR. The primers and reaction conditions are available upon request. The PCR products were purified and inserted into the pET32a vector (Novagene) to generate the recombinant plasmid pET32a-mTLNE. The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing.
Protein expression and purification
The plasmid pET32a and pET32a-mTLNE was transformed in BL21 Chemically Competent cells (TIANGEN). The transformed BL21 cells were incubated with shaking at 37°C until OD600 reaches 0.6, then induced with 0.1 mM IPTG (Sigma) at 37°C for 6 h. The cells were harvested by centrifugation, and the pellet was re-suspended completely by mixing in phosphate-buffered saline (PBS). Following ultrasonication, recombinant mTLNE was purified through a Ni-NTA agarose (KWBIO, China) according to manufacturer’s instructions, and the concentration of was determined by a bicinchoninic acid Protein Assay Kit (Pierce).
SDS-PAGE and Western blotting assay
All samples were mixed with loading buffer and loaded onto a homogeneous 12% polyacrylamide gel. Western blotting analysis was performed using the corresponding antibodies described as above. In brief, the PVDF membranes were incubated with the corresponding antibody at room temperature for 1 h, followed by blocking in 10% skim milk overnight at 4°C. A horse-radish-peroxidase (HRP)-conjugated secondary antibody at a dilution of 1:5000 was used to detect the primary antibody. The membranes were subjected to three washes for 10 min each time in PBS containing 0.05% Tween 20 and developed with ECL Western substrate (Bio-Rad).
Enzyme-linked immunosorbent assay (ELISA)
The 96-well microtiter plates were coated with coating buffer (pH 9.6) containing 1 μg/ml of mTLNE overnight at 4°C followed by 10% skim milk blocking. The coated plate was then incubated with rabbit (1:400) or mouse (1:500) anti-EV71 antibody at 37°C for 1 h, and followed by incubating HRP-conjugated secondary antibodies at 37°C for 30 min. A total of 100 μl of TMB peroxidase substrate was added for incubation for 15 min at room temperature. Finally, the absorbance at 450 nm was recorded using an ELISA plate reader.
All animal experimental procedures were carried out in strict accordance with and approved by the Animal Experiment Committee of Beijing Institute of Microbiology and Epidemiology. Groups of 6-week-old female BALB/c mice (n = 10) were inoculated with 50 μg of mTLNE protein, or Trx protein (control group) by intraperitoneal (i.p.) injection with Freund’s complete adjuvant (Sigma). All mice were then boosted twice with the same dose in Freund’s incomplete adjuvant (Sigma) at a 2-week interval. Two weeks after each immunization, serum samples were prepared and stored frozen until use.
Antibody response assay
The titers of IgG antibody in mice sera were detected by IFA as previously described . In brief, confluent RD cells infected with EV71 were harvested and suspended in DMEM containing 10% FBS. The suspended cells were then inoculated onto slides and fixed with acetone for 30 min in -20°C. All serum samples were serially diluted (two fold) in PBS, and mice polyclonal antibodies (1:100 dilution) against EV71 were used as positive control. After incubation for 1 h at 37°C, the slides were washed three times in PBS. Then, Alexa Fluor®488 conjugated goat anti-mouse IgG (Invitrogen) in 0.02% Evans blue was added and incubated for 30 min at 37°C. Finally, positive cells were detected using a fluorescent microscope (Olympus). The IgG subtype profiles in mouse sera were determined with a commercially available mouse immunoglobulin isotyping kit (Invitrogen) according to the manufacturer’s instruction.
The IgG titers of the immunized serum to each epitope (VP1-SP55, VP1-SP70 and VP2-SP28) were determined by ELISA. The unconjugated synthetic peptides representing the amino acid sequence of VP1-SP55, VP1-SP70 or VP2-SP28 were synthesized by Shanghai Bootech Bioscience&Technology Co., Ltd (Shanghai, China). The 96-well microtiter plates were coated with 100 μl of coating buffer containing 15 μg/ml of unconjugated synthetic peptides. All sera samples serially diluted (two fold) in PBS were incubated in triplicate wells (100 μl/well) for 1 h at 37°C. Following procedures were made as described in Methods “ELISA”. IgG antibody titers were expressed by the highest serum dilution at which the absorbance at 450 nm is higher than cut off value.
The neutralizing antibody titers against different genotype of EV71 were measured by microneutralization assay in Vero cells as previously described . The neutralizing antibody titer was calculated using the Reed-Muench method .
The production of cytokine from stimulated splenocytes was detected by enzyme-linked immunospot (ELISPOT) analysis using BDTM ELISPOT Set. In brief, the 96-well plates was coated respectively with IFN-γ, IL-2, IL-4, IL-6 capture antibody overnight at 4°C. Spleen cells (106/well) from the immunized mice were added and cultured at 37°C for 36 h, with total proteins of heat-inactivated EV71 (100 PFU/well) or ConA (250 ng/well, Sigma). Following procedures were made according to the manufacturer’s instruction.
In vivo challenge experiments
The neonatal mice model of EV71 infection has been described . Groups of BALB/c neonatal mice (n ≥ 5) were intracranially (i.c.) inoculated with 10 LD50 of EV71 strain AH08/06, and 40 μl of anti-mTLNE or anti-Trx serum was i.p. administered at 4 h post infection. PBS was set as negative control. The mortality was then monitored for at least 21 days.
The survival curve of each group was compared by using the Log-rank test with GraphPad Prism 5.0, and the other data were analyzed using t-test, and p < 0.05 was considerate as significance.
All the experiments were performed in Dr. Cheng-Feng Qin’s lab. This work was supported by the National Natural Science Foundation of China (No. 81000721 and No. 31270195) and the Beijing Natural Science Foundation (No. 7112108 and No. 7122129). C.F. Qin was supported by the Beijing Nova Program of Science and Technology (No. 2010B041).
- Bible JM, Pantelidis P, Chan PK, Tong CY: Genetic evolution of enterovirus 71: epidemiological and pathological implications. Rev Med Virol 2007, 17:371–379.PubMedView Article
- Cardosa MJ, Krishnan S, Tio PH, Perera D, Wong SC: Isolation of subgenus B adenovirus during a fatal outbreak of enterovirus 71-associated hand, foot, and mouth disease in Sibu, Sarawak. Lancet 1999, 354:987–991.PubMedView Article
- Chan KP, Goh KT, Chong CY, Teo ES, Lau G, Ling AE: Epidemic hand, foot and mouth disease caused by human enterovirus 71, Singapore. Emerg Infect Dis 2003, 9:78–85.PubMed CentralPubMedView Article
- Ho M, Chen ER, Hsu KH, Twu SJ, Chen KT, Tsai SF, Wang JR, Shih SR: An epidemic of enterovirus 71 infection in Taiwan: Taiwan enterovirus epidemic working group. N Engl J Med 1999, 341:929–935.PubMedView Article
- Mao LX, Wu B, Bao WX, Han FA, Xu L, Ge QJ, Yang J, Yuan ZH, Miao CH, Huang XX, Zhang C, Xu H: Epidemiology of hand, foot, and mouth disease and genotype characterization of enterovirus 71 in Jiangsu, China. J Clin Virol 2010, 49:100–104.PubMedView Article
- Wu Y, Yeo A, Phoon MC, Tan EL, Poh CL, Quak SH, Chow VT: The largest outbreak of hand; foot and mouth disease in Singapore in 2008: the role of enterovirus 71 and coxsackievirus A strains. Int J Infect Dis 2010, 14:e1076-e1081.PubMedView Article
- Hosoya M, Kawasaki Y, Sato M, Honzumi K, Kato A, Hiroshima T, Ishiko H, Suzuki H: Genetic diversity of enterovirus 71 associated with hand, foot and mouth disease epidemics in Japan from 1983 to 2003. Pediatr Infect Dis J 2006, 25:691–694.PubMedView Article
- Mizuta K, Abiko C, Murata T, Matsuzaki Y, Itagaki T, Sanjoh K, Sakamoto M, Hongo S, Murayama S, Hayasaka K: Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003. J Clin Microbiol 2005, 43:6171–6175.PubMed CentralPubMedView Article
- Herrero LJ, Lee CS, Hurrelbrink RJ, Chua BH, Chua KB, McMinn PC: Molecular epidemiology of enterovirus 71 in peninsular Malaysia, 1997–2000. Arch Virol 2003, 148:1369–1385.PubMedView Article
- Chang LY: Enterovirus 71 in Taiwan. Pediatr Neonatol 2008, 49:103–112.PubMedView Article
- Wu CN, Lin YC, Fann C, Liao NS, Shih SR, Ho MS: Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine 2001, 20:895–904.PubMedView Article
- Chen HF, Chang MH, Chiang BL, Jeng ST: Oral immunization of mice using transgenic tomato fruit expressing VP1 protein from enterovirus 71. Vaccine 2006, 24:2944–2951.PubMedView Article
- Chiu CH, Chu C, He CC, Lin TY: Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71. Microbes Infect 2006, 8:1671–1678.PubMedView Article
- Arita M, Nagata N, Iwata N, Ami Y, Suzaki Y, Mizuta K, Iwasaki T, Sata T, Wakita T, Shimizu H: An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys. J Virol 2007, 81:9386–9395.PubMed CentralPubMedView Article
- Liu CC, Lian WC, Butler M, Wu SC: High immunogenic enterovirus 71 strain and its production using serum-free microcarrier vero cell culture. Vaccine 2007, 25:19–24.PubMedView Article
- Tung WS, Bakar SA, Sekawi Z, Rosli R: DNA vaccine constructs against enterovirus 71 elicit immune response in mice. Genet Vaccines Ther 2007, 5:6.PubMed CentralPubMedView Article
- Chung YC, Ho MS, Wu JC, Chen WJ, Huang JH, Chou ST, Hu YC: Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protects mice against lethal challenge. Vaccine 2008, 26:1855–1862.PubMedView Article
- Ong KC, Devi S, Cardosa MJ, Wong KT: Formaldehyde-inactivated whole-virus vaccine protects a murine model of enterovirus 71 encephalomyelitis against disease. J Virol 2010, 84:661–665.PubMed CentralPubMedView Article
- Cheng A, Fung CP, Liu CC, Lin YT, Tsai HY, Chang SC, Chou AH, Chang JY, Jiang RH, Hsieh YC, Su IJ, Chong PC, Hsieh SM: A Phase I, randomized, open-label study to evaluate the safety and immunogenicity of an enterovirus 71 vaccine. Vaccine 2013, 31:2471–2476.PubMedView Article
- Liang ZL, Mao QY, Wang YP, Zhu FC, Li JX, Yao X, Gao F, Wu X, Xu M, Wang JZ: Progress on the research and development of inactivated EV71 whole-virus vaccines. Hum Vaccin Immunother 2013,9(8):1701–1705.PubMed CentralPubMedView Article
- Zhu FC, Meng FY, Li JX, Li XL, Mao QY, Tao H, Zhang YT, Yao X, Chu K, Chen QH, Hu YM, Wu X, Liu P, Zhu LY, Gao F, Jin H, Chen YJ, Dong YY, Liang YC, Shi NM, Ge HM, Liu L, Chen SG, Ai X, Zhang ZY, Ji YG, Luo FJ, Chen XQ, Zhang Y, Zhu LW: Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 2013, 381:2024–2032.PubMedView Article
- Chan YF, Sam IC, AbuBakar S: Phylogenetic designation of enterovirus 71 genotypes and subgenotypes using complete genome sequences. Infect Genet Evol 2010, 10:404–412.PubMedView Article
- Xu J, Qian Y, Wang S, Serrano JM, Li W, Huang Z, Lu S: EV71: an emerging infectious disease vaccine target in the ear East? Vaccine 2010, 28:3516–3521.PubMedView Article
- Foo DG, Alonso S, Chow VT, Poh CL: Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide. Microbes Infect 2007, 9:1299–1306.PubMedView Article
- Li X, Mao C, Ma S, Wang X, Sun Z, Yi Y, Guo M, Shen X, Sun L, Bi S: Generation of neutralizing monoclonal antibodies against enterovirus 71 using synthetic peptides. Biochem Biophys Res Commun 2009, 390:1126–1128.PubMedView Article
- Lim XF, Jia Q, Khong WX, Yan B, Premanand B, Alonso S, Chow VT, Kwang J: Characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection. PLoS One 2012, 7:e29751.PubMed CentralPubMedView Article
- Liu CC, Chou AH, Lien SP, Lin HY, Liu SJ, Chang JY, Guo MS, Chow YH, Yang WS, Chang KH, Sia C, Chong P: Identification and characterization of a cross-neutralization epitope of enterovirus 71. Vaccine 2011, 29:4362–4372.PubMedView Article
- Dudek NL, Perlmutter P, Aguilar MI, Croft NP, Purcell AW: Epitope discovery and their use in peptide based vaccines. Curr Pharm Des 2010, 16:3149–3157.PubMedView Article
- Sette A, Fikes J: Epitope-based vaccines: an update on epitope identification, vaccine design and delivery. Curr Opin Immunol 2003, 15:461–470.PubMedView Article
- Dong XN, Chen Y, Wu Y, Chen YH: Candidate multi-peptide-vaccine against classical swine fever virus induced potent immunity with serological marker. Vaccine 2005, 23:3630–3633.PubMedView Article
- Ichihashi T, Yoshida R, Sugimoto C, Takada A, Kajino K: Cross-protective peptide vaccine against influenza A viruses developed in HLA-A*2402 human immunity model. PLoS One 2011, 6:e24626.PubMed CentralPubMedView Article
- Koopman G, Beenhakker N, Nieuwenhuis I, Doxiadis G, Mooij P, Drijfhout JW, Koestler J, Hanke T, Fagrouch Z, Verschoor EJ, Bontrop RE, Wagner R, Bogers WM, Melief CJ: DNA/long peptide vaccination against conserved regions of SIV induces partial protection against SIVmac251 challenge. AIDS 2013, 27:2842–2851.View Article
- Purcell AW, McCluskey J, Rossjohn J: More than one reason to rethink the use of peptides in vaccine design. Nat Rev Drug Discov 2007, 6:404–414.PubMedView Article
- El-Awady MK, El Gendy M, Waked I, Tabll AA, El Abd Y, Bader EI D, EI Shenawy R, Allam A, Abdelhafez TH, Dawood RM: Immunogenicity and safety of HCV E1E2 peptide vaccine in chronically HCV-infected patients who did not respond to interferon based therapy. [http://dx.doi.org/10.1016/j.vaccine.2013.07.074] Vaccine 2013.
- Liu JN, Wang W, Duo JY, Hao Y, Ma CM, Li WB, Lin SZ, Gao XZ, Liu XL, Xu YF, Xu WB, Qin C, Zhang LF: Combined peptides of human enterovirus 71 protect against virus infection in mice. Vaccine 2010, 28:7444–7451.PubMedView Article
- Tian X, Su X, Li X, Li H, Li T, Zhou Z, Zhong T, Zhou R: Protection against enterovirus 71 with neutralizing epitope incorporation within adenovirus type 3 hexon. PLoS One 2012, 7:e41381.PubMed CentralPubMedView Article
- Lin YC, Wu CN, Shih SR, Ho MS: Characterization of a vero cell-adapted virulent strain of enterovirus 71 suitable for use as a vaccine candidate. Vaccine 2002, 20:2485–2493.PubMedView Article
- Sette A, Keogh E, Ishioka G, Sidney J, Tangri S, Livingston B, Mckinney D, Newman M, Chesnut R, Fikes J: Epitope identification and vaccine design for cancer immunotherapy. Curr Opin Investig Drugs 2002, 3:132–139.PubMed
- Sette A, Newman M, Livingston B, McKinney D, Sidney J, Ishioka G, Tangri S, Alexander J, Fikes J, Chesnut R: Optimizing vaccine design for cellular processing, MHC binding and TCR recognition. Tissue Antigens 2002, 59:443–451.PubMedView Article
- Livingston BD, Newman M, Crimi C, McKinney D, Chesnut R, Sette A: Optimization of epitope processing enhances immunogenicity of multiepitope DNA vaccines. Vaccine 2001, 19:4652–4660.PubMedView Article
- Li HY, Han JF, Qin CF, Chen R: Virus-like particles for enterovirus 71 produced from saccharomyces cerevisiae potently elicits protective immune responses in mice. Vaccine 2013, 31:3281–3287.PubMedView Article
- Foo DG, Alonso S, Phoon MC, Ramachandran NP, Chow VT, Poh CL: Identification of neutralizing linear epitopes from the VP1 capsid protein of enterovirus 71 using synthetic peptides. Virus Res 2007, 125:61–68.PubMedView Article
- Wang SM, Chen IC, Su LY, Huang KJ, Lei HY, Liu CC: Enterovirus 71 infection of monocytes with antibody-dependent enhancement. Clin Vaccine Immunol 2010, 17:1517–1523.PubMed CentralPubMedView Article
- Han JF, Cao RY, Deng YQ, Tian X, Jiang T, Qin ED, Qin CF: Antibody dependent enhancement infection of enterovirus 71 in vitro and in vivo. Virol J 2011, 8:106.PubMed CentralPubMedView Article
- Chen IC, Wang SM, Yu CK, Liu CC: Subneutralizing antibodies to enterovirus 71 induce antibody-dependent enhancement of infection in newborn mice. Med Microbiol Immunol 2013, 202:259–265.PubMedView Article
- Cao RY, Dong DY, Liu RJ, Han JF, Wang GC, Zhao H, Li XF, Deng YQ, Zhu SY, Wang XY, Lin F, Zhang FJ, Chen W, Qin ED, Qin CF: Human IgG subclasses against enterovirus Type 71: neutralization versus antibody dependent enhancement of infection. PLoS One 2013, 8:e64024.PubMed CentralPubMedView Article
- Foo DG, Macary PA, Alonso S, Poh CL: Identification of human CD4 T-cell epitopes on the VP1 capsid protein of enterovirus 71. Viral Immunol 2008, 21:215–224.PubMedView Article
- Cello J, Strannegard O, Svennerholm B: A study of the cellular immune response to enteroviruses in humans: identification of cross-reactive T cell epitopes on the structural proteins of enteroviruses. J Gen Virol 1996,77(Pt 9):2097–2108.PubMedView Article
- Zhao H, Li HY, Han JF, Deng YQ, Li YX, Zhu SY, He YL, Qin ED, Chen R, Qin CF: Virus-like particles produced in Saccharomyces cerevisiae elicit protective immunity against coxsackievirus A16 in mice. Appl Microbiol Biotechnol 2013, 97:10445–10452.PubMedView Article
- Han JF, Cao RY, Tian X, Yu M, Qin ED, Qin CF: Producing infectious enterovirus type 71 in a rapid strategy. Virol J 2010, 7:116.PubMed CentralPubMedView Article
- REED LJ, MUENCH H: A simple method of estimating fifty per cent endpoints. Am J Epidemiol 1938, 27:493–497.
- Chen S, Yu M, Jiang T, Deng Y, Qin C, Qin E: Induction of tetravalent protective immunity against four dengue serotypes by the tandem domain III of the envelope protein. DNA Cell Biol 2007, 26:361–367.PubMedView Article
- Cao RY, Han JF, Jiang T, Tian X, Yu M, Deng YQ, Qin ED, Qin CF: In vitro and in vivo characterization of a new enterovirus type 71-specific human intravenous immunoglobulin manufactured from selected plasma donors. J Clin Virol 2011, 51:246–249.PubMedView Article
This article is published under license to BioMed Central Ltd. 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 credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.