Human Vaccines & Immunotherapeutics 8:2, 208–215; February 2012;
G
2012 Landes Bioscience
Immunogenicity of a multi-epitope DNA vaccine against hantavirus Chen Zhao,1,2 Ying Sun,1,2 Yujie Zhao,1,3 Si Wang,1,2 Tongtong Yu,1,2 Feng Du,1,2 X. Frank Yang4 and Enjie Luo1,2,* 1
China Medical University; Shenyang, Liaoning, China; 2Department of Pathogen Biology; 3Biochip Center; the Key Laboratory of Cytobiology; 4 Department of Microbiology and Immunology, Indiana University School of Medicine; Indianapolis, IN USA
Keywords: hantavirus, glycoprotein, chemical gene synthesis, multi-epitope DNA vaccine, HTNV, SEOV, PUUV
Hemorrhagic fever with renal syndrome (HFRS) is a severe epidemic disease caused by hantaviruses including Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV) and Puumala virus. Three of the four HFRS hantaviruses, HTNV, SEOV, and PUUV are found in China. Currently, there is no effective strategy available to reduce infection risk. In this study, we constructed a multi-epitope chimeric DNA vaccine that encodes expressing 25 glycoprotein epitopes from SEOV, HTNV and PUUV (designated as SHP chimeric gene). Vaccination of BALb/c mice with SHP multi-epitope chimeric DNA vaccine led to a dramatic augmentation of humoral and cellular responses. The SHP vaccine DNA was detected in many organs but not for more than 60 d. There was no risk of mutation due to integration. Thus, the SHP multi-epitope chimeric DNA vaccine is a potential effective and safe DNA vaccine against infection by SEOV, HTNV, and PUUV.
© 2012 Landes Bioscience. Hantaviruses are enveloped viruses with three segments of antisense single-stranded RNA designated as large (L), medium (M), and small (S). The genome encodes four proteins: a nucleocapsid protein (N), two glycoproteins (Gc and Gn), and an RNAdependent RNA polymerase (L).1 Protective immunity correlates with neutralizing antibodies to Gc and Gn, and passive transfer of sera containing neutralizing antibodies provides protection against infection in animals.2 Hemorrhagic fever with renal syndrome (HFRS) is caused by the Hantaan (HTNV), Dobrava/Belgrade (DOBV), Seoul (SEOV), and Puumala (PUUV) hantaviruses.3 HTNV, SEOV and PUUV are found in China.4,5 HTNV and SEOV are the classical pathogens of HFRS in China, and PUUV is recently found in the northeast of China. PUUV poses a large infection risk, and more importantly, a mixed epidemic risk with HTNV and SEOV can occur in some areas of China. However, there is no vaccine currently available against the mixed infection of HTNV, SEOV, and PUUV hantaviruses. To protect people from infection by hantavirus, effective vaccines are needed. DNA vaccines for HV is a effective method against the infection risk of hantaviruses, DNA vaccine circumvent some problems associated with cell culture- or rodent brain-derived vaccines.2 Several DNA vaccines had been studied to protect against HTNV,6,7 SEOV8 and PUUV.9 One caveat of these studies is that these vaccines are not cross protective and therefore, are not effective against mixed infection. In this regard, multiepitope-based DNA vaccines could overcome this weakness. Multi-epitope DNA vaccines can simultaneously elicit strong humoral and cellular immune responses.10 Hooper11 constructed a DNA vaccine that expressed the HTNV M gene products,
Gc and Gn, and conferred cross-protection against infection with HTNV, SEOV, and DOBV but not PUUV in hamsters. However, there have been no reports on cross-protective vaccines against the infection risk of HTNV, SEOV and PUUV to-date. To construct an effective DNA vaccine, the vaccine gene is the key. Conventional DNA vaccines need to cultivate viruses in vitro to extract the DNA samples. Hantaviruses are highpathogenicity viruses, there are highly infection risks to cultivate viruses in vitro and it is time-consuming. Thus, chemical synthesis of DNA sequences offers a highly effective technique to elucidate gene functions and analyze protein–nucleic acid interactions.12 Chemical gene synthesis can synthesize any DNA sequence, which avoids cultivation of viruses. In many cases, chemical synthesis may be the only choice because template DNAs are not readily available. In addition, chemical gene synthesis may be preferable to avoid tedious and costly site-directed mutagenesis and subcloning,12 which allows researchers to spend less time on constructing DNA molecule but more time on designing custom DNA molecules and characterizing their efficacy. In this study, we constructed a multi-epitope chimeric DNA vaccine to against infection of HTNV, SEOV and PUUV. This synthetic gene encodes 25 dominant B- and T-cell epitopes corresponding to regions in the glycoproteins of HTNV, SEOV and PUUV. We then investigated the cellular and humoral immunity induced by this multi-epitope DNA vaccine in mice.
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Materials and Methods Experimental animals, viruses and cells. Specific-pathogen-free (SPF) BALb/c mice were purchased from Vital River Laboratories
*Correspondence to: Enjie Luo; Email:
[email protected] Submitted: 08/16/11; Revised: 10/06/11; Accepted: 10/12/11 http://dx.doi.org/10.4161/hv.18389
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(Beijing, China). HTNV strains 7611813 and SEOV strain SR-1114 were propagated in Vero E6 cells (We have no PUUV). Vero-E6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at pH 7.2 and were kept at 37°C with 5% CO2. Selection of HV vaccine candidate epitopes. The glycoprotein’s sequences of HTNV, SEOV and PUUV were obtained from GenBank (GenBank ID: NP_941978 and AAB19434 and AAC37848.1). Antigen epitope of the glycoproteins sequences were analyzed by BcePred Prediction Software of Predicting Antigenic Peptides and T-Cell Epitope Prediction Tools of IEDB Analysis Resource, and its secondary structure analyzed by DNASTAR software. Twenty-five epitopes, which can induce B-cell or T-cell immunoreactions were selected for constructing multi-epitope DNA vaccine. Design and construction of the multi-epitope containing plasmids. Twenty-five selected epitopes of HTNV, SEOV and PUUV glycoproteins were engineered together and separated with residues AAY spacer. This chimeric gene encoding multiple epitopes was optimized for mammalian codon usage according to the pervious study.15 The DNA construct also included CpG motif at the N- and C-terminus to enhance the immune response. We designated this chimeric gene as SHP gene. The DNA molecule of SHP gene was generated using chemical gene synthesis (Shanghai Xuguan Biotechnological Development Co., Ltd.). The synthetic DNA was then incorporated into expression vector pcDNA3.1(+) eukaryotic plasmid and pGEX-6p-1 prokaryotic plasmid. The final plasmid was designated as pcDNA3.1SHP and pGEX-6p-1-SHP and the plasmid DNAs were dissolved in 1 X PBS buffer, pH 7.0.with final concentration of 1 mg/ml SHP protein expression and purification. The pGEX-6p-1SHP plasmid was transformed into E. coli BL21, cultivated in LB/AMP (final concentration 50 mg/mL) liquid medium, and induced with IPTG (final concentration 1.0 mM) for 4 h to express SHP. After IPTG induction, the supernatant of the samples were harvested using centrifugation for 10 min at 10,000 rpm. Then we used GST affinity chromatography to purify the SHP protein as Lee did16 and identified by western-blotting. Transfected Vero-E6 cells with pcDNA3.1-SHP. Six-well tissue culture plates were seeded with Vero-E6 cells (2 ¾ 106/well). Monolayers of 80–90% confluent cells were transfected with pcDNA3.1-SHP using LipofectamineTM 2000 (Tiangen Biotech, Beijing, China). The expression of the SHP protein was analyzed by indirect immunofluorescence. Convalescent sera of HFRS patients were used as primary antibodies. The secondary antibody was FITC-conjugated–goat-anti-human IgG (Sigma). Immunization of BALb/c mice with the SHP multi-epitope DNA vaccine. pcDNA3.1-SHP amplified in E.coli BL21 was extracted using the EndoFree Plasmid Kit (Tiangen Biotech, Beijing, China). For DNA immunizations, 6 to 8 week-age BALb/c mice were randomly divided into three groups (n = 40 each). The mice were immunized intramuscularly with either 100 mg of pcDNA3.1-SHP (group 1), 100 mg of pcDNA3.1CpG (group 2), 100 mg of pcDNA3.1 (+) (group 3), or 100 ml PBS (group 4). All groups were boosted with an equivalent dose
on the 14th and 28th days after the initial inoculation. Blood samples were collected by retro-orbital bleeding. Detection of anti-HV serum IgG. ELISA plates were coated with 10 mg/ml purified SHP protein. Mouse sera were collected on the 0th, 7th, 17th, 31st, 60th and 90th days after the initial inoculation and serially diluted in triplicate. HRP-coupled goat anti-mouse IgG (Sigma) was used as the secondary antibody. The titers were defined as the reciprocal of the positive highest serum dilution. Detection of neutralization antibodies. This test was performed in 96-well tissue culture plates which were coated with SEOV- or HTNV-infected Vero-E6 cell lysates, respectively. Mouse sera diluted in blocking solution were added to the wells. Then the sera and viruses were mixed it up and applied to VeroE6 cell monolayers. Virus-sera mixtures were incubated at 37°C for 8–10 d and lysed with three consecutive freeze-thaw cycles. HTNV and SEOV antigen in the lysate was detected by ELISA at 490 nm. Detection of splenic lymphocyte proliferation. Twenty-eight days after the final inoculation, mice were sacrificed and splenocytes were collected. Splenocytes were seeded in 96-well microtiter plates (1 ¾ 106/well) and stimulated with SHP protein (5 mg/mL) for 72 h, then pulsed with 20 mL of MTT (Sigma) solution (5 mg/mL) for 4 h and 150 mL of dimethyl sulfoxide (DMSO; Sigma) per well, and evaluated in a multiskan spectrum reader at 550 nm. Concanavalin A (ConA; Sigma) was used as a positive control (final concentration 5.0 mg/mL). The results were expressed as the stimulation index (SI). SI = stimulated culture OD550 value/unstimulated culture OD550 value. Detection of cytokines INF-c, IL-4, and IL-10. On day 0, 7, 17, 31, 60 and 90 after the final inoculation, mice were sacrificed and splenocytes were collected. Single-cell suspensions were obtained by filtration and then seeded in flat-bottomed 96-well microtiter plates (1 ¾ 106/well) with 200 mL DMEM culture medium (Gibco). Cytokine production was measured by ELISA (450 nm, Thermo). Each serum test, including negative and positive control sera, was assayed in triplicate. Detection of the tissue distribution of plasmid DNA. Blood, muscle from the injection site, liver, kidney and heart tissues were obtained on day 1, 7, 31, 60 and 90 after the initial inoculation. DNA was isolated, and PCR was performed with 2 mg DNA as follows: 35 cycles of 94°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min. The three pairs of primers used to detect the beginning, middle, and end of the SHP gene were 5'-TCCCTGGTACTGCTGTCTATT-3' and 5'-AGGTC GGGCATTCGGTCA-3', resulting in a 337 bp product; and 5'-TTCTGTGCCCGGTGCGCAAA-3' and 5'-GCGGG TCAGGGTCACGCTTT-3', resulting in a 386 bp product; 5'-GAAAACGCGCATGGCGTGGG-3' and 5'-TCGCGG TTTGCCACGGGTAC-3', resulting in a 301 bp product. Detection of DNA vaccine integration into host DNA. DNA was isolated from blood, muscle at the injection site, liver, kidney and heart tissues on day 1, 7, 31, 60 and 90 after the initial inoculation. To remove free extrachromosomal plasmid from the isolated DNA, we performed 0.8% agarose gel electrophoresis. The DNA extracts were analyzed by PCR as above.
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Statistical analysis. Data were presented as means and standard deviation. (GraphPad Prism version 4.0) Difference between two groups of mice was compared using wilcoxon-matched pairs test. A probability of less than 0.05 was taken as significant. Results Construction of multi-epitope DNA vaccine. To constructed a multi-epitope chimeric DNA vaccine which against infection of HTNV, SEOV, and PUUV, we performed bioinformatic analysis. Twenty-five epitopes from HTNV, SEOV and PUUV glycoproteins were engineered into a synthetic gene, with residues AAY spacers between each epitope peptide (Table 1). The construct included CpG motif at the N- and C-terminus of the chimeric gene to enhance immune response. The full length of the constructed multi-epitope chimeric gene, designated as “SHP,” is 1,542 bp in length. (Fig. 1A). The constructed plasmid containing correct SHP insert, pcDNA3.1-SHP, was then confirmed by restriction digestion analyses (Fig. 1B and C) and sequencing analysis (data not shown). This recombinant plasmid pcDNA3.1-SHP was then used as SHP multi-epitope chimeric DNA vaccine.
SHP protein expression and purification. To purify the SHP protein we transformed the pGEX-6p-1-SHP plasmid into E. coli BL21. After IPTG induction (final concentration 1.0 mM), the SHP protein expressed in E. coli BL21. After affinity chromatography purification, the eluted soluble SHP protein was collected and identified by western-blotting. The specific 78 kDa band of SHP protein was consistent with the predicted protein in size. (Fig. 2A and B) Expression of multi-epitope DNA vaccine in Vero-E6 cells. To determine the expression of chimeric epitope, pcDNA3.1SHP DNA was transfected into Vero-E6 cells. Indirect immunofluorescence using convalescent sera from HFRS patients as primary antibodies demonstrated cytoplasmic expression of SHP protein in Vero-E6 cells (Fig. 3). This result indicated that pcDNA3.1-SHP expressed chimeric epitopes efficiently in eukaryotic cells. Cytokines IL-4, INF-c, and IL-10 induced by SHP DNA vaccine. To assess the immune response of constructed DNA vaccine, we immunized mice with pcDNA3.1-SHP DNA. At day 0, 7, 17, 31, 60 and 90 after injection, we examined cytokine expression in mice. As shown in Figure 4A–C, levels of cytokines INF-c, IL-4, and IL-10 were induced in the SHP DNA
© 2012 Landes Bioscience. Table 1. Character of selected epitopes Epitope F1 F2
Origin
Position (aa)
Epitope aa sequences
Epitope type
H
4~31
GNWIVLIVLCVFLLFSLVLLSILCPVRK
B
Do not distribute. S
35~49
GNWIVLIVLCAFLIF
B
P
51~61
GNWIVLVVLCI
B
H
65~83
NDCFVSRHVKVCIIGTVSK
B
S
89~107
NDCFVSRHVKICIIGTVSK
B
F6
H
113~151
SGVGFTLTCLVSLTECPTFLTSIKACDKAICYGAESVTL
B
F7
H/S/P
160~172
LKTSFHCYGACTK
B
F3 F4 F5
F8
H
174~181
YSRRVCVQ
B
F9
H
188~221
NPSDCPGVGTGCTACGLYLDQLKPVGSAYKIITI
B
F10
H
228~252
STFRCCHGEDCSQIGLHAAAPHLDK
B
F11
H
259~269
QTIGVDVHALG
B
F12
H
272~280
QGDTLLFFG
B
F13
H
287~300
GAPQCGIKCWFVKS
B
F14
H/P
308~321
GLIFKHWCTSTCQF
B
F15
H
326~333
KGFLCPEF
B
F16
H
338~346
DHINILVTK
B
F17
H
353~363
GENPCKIGLQT
B
F18
H/S
370~377
HGVGSVPM
B
F19
H
379~391
EYPWHTAKCHYER
T
F20
P
399~411
KYQYPWHTAKCHF
T
F21
S
416~426
YYPWHTARCHF
T
F22
P
435~449
KYSYPWQTAKCFYEK
T
F23
P
458~472
GYAYPWQTAKCFFEK
T
F24
H/S
483~490
SGYKKVMA
B
F25
H/S/P
495~504
TDLELDFSLT
T
Note: H, HTNV; S, SEOV; P, PUUV.
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Figure 1. Construction and identification of plasmid pcDNA3.1-SHP. (A) Design of SHP multi-epitope chimeric gene; (B) Construction of plasmid pcDNA3.1-SHP. (C) Identification of plasmid pcDNA3.1-SHP. Lane 1, DL15000; lane 2, plasmid pcDNA3.1-SHP; lane 3, plasmid pcDNA3.1-SHP digested by PstI.
vaccine group, which are much higher levels than the pcDNA3.1 (+) and PBS groups (p , 0.01). HV-specific antibody production induced by SHP multiepitope DNA vaccine. To assess the HV-specific antibody titer of
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the constructed DNA vaccine, we detected immunized mouse sera collected on various time points. ELISA plates were coated with 10 mg/ml purified SHP protein. The ELISA results showed that the SHP multi-epitope chimeric DNA vaccine induced a
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significant antibody reponse specific to SHP protein in mice one week after a booster immunization. The antibody titer of the SHP DNA vaccine-immunized group was higher than the pcDNA3.1 (+) and PBS groups (p , 0.01) (Fig. 4D). Neutralization antibodies induced by SHP multiepitope DNA vaccine. To assess the protect effection of constructed DNA vaccine, we detected the neutralization antibodies titer. Neutralization test results showed that the immunized mice of pcDNA3.1-SHP group could elicit neutralizing antibodies with different titers. We diluted the sera of immunized mice from 1:10 to 1:320. As shown in Table 2, the levels of protective neutralizing titers against HTNV- and SEOV-infected Vero-E6 cell were 1:10 to 1:20 and 1:10 to 1:40, respectively. Proliferation of splenic lymphocytes induced by the SHP DNA vaccine. To assess cellular immunity induced by the constructed SHP DNA vaccine, we examined the proliferation of splenic lymphocytes. Specific proliferative responses were observed in splenocyte cultures from mice immunized with the SHP DNA vaccine. The SI of the specific proliferative responses were much higher than in the pcDNA3.1(+) and PBS control groups (p , 0.01) (Fig. 4E). Distribution of the SHP DNA vaccine in vivo. To determine the distribution of the chimeric DNA vaccine Figure 2. Purification of recombinant SHP protein. (A) Chromatographic profile in mice, we analyzed different tissues of immunized of GST-SHP protein eluted from a GST affinity column; (B) western-blotting analysis of the SHP protein collected from the GST affinity column. mice by PCR. On day 1, 7 and 31 after intramuscular injection, pcDNA3.1-SHP plasmids were detected in all collected tissues. However, on the 60th day after injection, pcDNA3.1-SHP plasmid was detected only in muscle from the injection site, liver and kidney, but not in the heart and blood. No pcDNA3.1-SHP plasmid was detected in any tissue on the 90th day after injection (Table 3). DNA vaccine integration into host DNA. To detect whether the chimeric DNA vaccine can be integrated into host DNA of immunized mice, we isolated DNA samples from different tissues of immunized mice and analyzed by PCR. The potential for vaccine DNA integration into host genomic DNA was examined by PCR. All purified genomic DNA samples from day 1, 7, 31, 60 and 90 after injection were negative for SHP DNA sequences.
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Discussion
Figure 3. Indirect immunofluorescence detection of the SHP recombinant protein in Vero-E6 cells. (A) Negative control; (B) Vero-E6 Cells transfected with plasmid pcDNA3.1-SHP showed positive results. Convalescent sera from HFRS patients were used as primary antibodies.
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Multi-epitope DNA vaccines are based on both classical DNA and polypeptide vaccines. They are constructed as a multi-epitopeencoded gene with each epitope separated by a spacers.17,18 Multi-epitope DNA vaccines more faithfully mimic antigen processing and presentation during natural infection. In addition, multi-epitope DNA vaccines induce more potent immunoreactions than whole protein vaccines. Since the epitopes are derived from multiple antigens and packaged into a relatively small delivery vehicle, the vaccine can induce powerful cross-reactive responses toward multiple antigens and elicit strong humoral and cellular immune responses.10 Thus, multi-epitope DNA vaccines are effective vaccines against those rapid mutated viruses
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Figure 4. ELISA detection of antibody levels in peripheral blood and cytokine and splenocytes proliferation levels stimulated by SHP recombinant protein in vitro. (A) INF-c levels of cultured splenocytes stimulated by SHP recombinant protein in vitro.; (B) IL-4 levels of cultured splenocytes stimulated by SHP recombinant protein in vitro.; (C) IL-10 levels of cultured splenocytes stimulated by SHP recombinant protein in vitro; (D)Peripheral blood anti-HV IgG antibody levels in immunized mice. (E) Proliferation of splenocytes stimulated by SHP recombinant protein in vitro.
and multiple blood serums viruses, such as SARS,19 H5N1 influenza virus20 and other viruses. Novel multi-epitope and chimeric genes can be valuable vaccine targets. Construction of highly complex artificial vaccine
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target proteins is often not possible using the natural sequences. In some cases, synthetic target antigens may have higher efficacy in inducing an immune response.21,22 Synthetic genes could significantly impact the study of immunoreactivity against a large
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Table 2. The detection of neutralization titers in the sera of immunized mice No
Neutralization Titers HTNV
SEOV
pcDNA3.1-SHP pcDNA3.1 PBS pcDNA3.1-SHP pcDNA3.1 PBS 1
1:20
-
-
1:10
-
-
2
1:10
-
-
1:20
-
-
3
1:20
-
-
1:40
-
-
4
1:20
-
-
1:20
-
-
Table 3. Biodistribution of the plasmid pcDNA3.1-HSP in mice Days after intramuscular inoculation 1st day
7th day
Blood
+
+
31st day 60th day 90th day +
-
-
Muscle of the injected site
+
+
+
+
-
neutralization antibodies and the levels of specific antibodies and neutralization antibodies in SHP multi-epitope chimeric DNA vaccine group were higher than that of pcDNA3.1(+) group and PBS group. Th1 cells preferentially produce IL-2 and IFN-c, which promote cellular immune responses against intracellular pathogens and play a key role in vaccine efficacy, whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13, which promote humoral immunity by promoting B-cell growth and differentiation and play important roles in disease recovery and virus clearance.10,28-30In this study, antigen-stimulated splenocytes from vaccinated mice produced cytokines INF-c, IL-4, and IL-10. The levels of IL-4, and IL-10 developed in SHP multi-epitope chimeric DNA vaccine group was higher than that of pcDNA3.1 (+) group and PBS group, respectively, which suggested that the DNA vaccine induced remarkable humoral response. The level of INF-c and the in vitro proliferation of splenocytes stimulated by SHP recombinant protein demonstrates that the DNA vaccine induced intensive cellular immunoreactions. The pcDNA3.1-SHP plasmid disseminated widely throughout the body, as expected.31,32 The pcDNA3.1-SHP plasmid was maintained longer in the muscle of the injection site, liver, and kidney than in heart and blood, but the plasmid dissipated from all sites between 60 and 90 d. Foreign DNA can integrate into the host genome when it is injected into a host. This increases the likelihood of malignant transformation, genomic instability, and cell growth dysregulation.33 The WHO guidelines on nucleic acid vaccines include the following theoretical risks: risk of integration of the plasmid DNA, tolerance due to long-term presentation of the antigen, adverse reactions due to the co-administration of cytokine DNA and autoimmune reactions due to anti-DNA antibody induction.34 Thus, we performed PCR to detect integrated plasmid in genomic DNA. We show that the pcDNA3.1-SHP DNA vaccine did not integrate into the host genome. Therefore, the risk of mutation due to integration is negligible. In conclusion, we constructed a SHP multi-epitope chimeric DNA vaccine. The DNA vaccine elicited strong humoral and cellular immune responses and it has no risk of mutation due to integration in mice. The novel immunogenic multi-epitope DNA vaccine constructed by chemical gene synthesis revealed in this study provides a new candidate target for HV vaccine development. Current study is underway to determine whether the SHP multi-epitope chimeric DNA vaccine elicited effective protection against challenge with HTNV, SEOV and PUUV in mice.
© 2012 Landes Bioscience. Spleen
+
+
+
+
-
Kidney
+
+
+
+
-
Heart
+
+
+
-
-
diversity of viruses’ envelope proteins. A panel of diverse viral surface antigens could be easily generated and studied by immunoassays offering potential new insights for vaccine targets and development.21 Bioinformatics can be used to engineer artificial proteins to match the highly complex antigenic strain variations to induce greater immune response. In this study, we constructed a multi-epitope chimeric DNA vaccine to against infection by HTNV, SEOV and PUUV. We selected 25 dominant epitopes which can induce B-cell or T-cell immunoreaction corresponding to regions in the glycoproteins of HTNV, SEOV and PUUV by computer bioinformatics analyses. Then we constructed the SHP multi-epitope chimeric DNA vaccine by means of chemical gene synthesis. DNA vaccine is an effective measure to against various pathogens and tumor antigens, but it seems to be more difficult to induce high effectively immune responses in large animals other than in mice,23although recent studies have shown some promise in horse,24dog25 and pig.26 One key factor is most likely due to poor intracellular uptake of the DNA. The relation between the injected dose and injected method in relation to the size of the treated animal and injection site must be considered. In this study, we immunized BALB/c mice (6–8 week old) intramuscularly three times at 2 week intervals, with 100 mg of plasmid into each set of quadriceps muscles as previously reported by Bharadwaj et al.27 After inoculated BALb/c mice with the SHP multi-epitope chimeric DNA vaccine, a remarkable humoral response and cellular immunoreactions were detected. In response to antigen stimulation, the SHP multi-epitope chimeric DNA vaccine elicted
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
We gratefully acknowledge the financial support of the Shenyang Bureau of Science of Technology (1071166–9-00). We also acknowledge BioMed Proofreading for native English-speaking experts’ edition.
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