The Linear Multi-epitope Peptide Containing The B-and T-cell Epitope Of DEN-2 EDⅢ Inducing B And T Mediated Immunity

Dengue virus (DENV), which is a member of the Flaviviridae family that includes several other medically important virus, comprises 4 serotypes (DEN-1,-2,-3 and-4). DENV is single-stranded positive-strand RNA virus which the genome length of dengue virus is 11kb and the single open reading frame encodes three structural protein (PrM/M, E and C) and seven nonstructural protein (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5).Dengue virus are responsible for dengue fever (DF), life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). These diseases spread throughout most of tropical and subtropical areas of the world. There are about 50-100 million cases of DF, and 250-500 thousand cases of DHF each year. Although urgently needed, a licensed vaccine for dengue is not yet available.Recent advances in molecular biology and vaccinology has given rise to new hopes with the development of live attenuated vaccines, viral-vector vaccines, subunit protein, DNA vaccine. But, a safe and effective dengue vaccine has been not available.The main disturbance lies in the following four directions:(1) The undefined etiopathogenesis for dengue fever and dengue shock syndrome. Three hypothesis, antibody-dependent enhancement (ADE) effect hypothesis, virulence theory and immunopathology doctrine, were not accredited uniformly. Especially how B-and T-cell immunological responses take effect was still a puzzle; (2) Infected animal model was not available; (3) Effective vaccines should offer allround protect for four serotypes; (4) The long period, low product and the potential side effect limited the application of routine vaccines.Nowadays, multi-epitope peptide vaccine become a new approach of dengue vaccine. Since Strohmaier found the amino acid sequence of 146-154 aa and 200-213 aa in hand-foot-and-mouth disease virus contains the antigenic sites, a newtype vaccine epitope-based vaccine was found. Epitope-based vaccine has several advantages, such as safty, high degree of specificity, easy and safe to use. With the development of the molecule immunology, molecule biology and biotechnology informatics, antigenic epitope was found more easier. Recently, epitope-based vaccine is the new direction for virus. Several multi-epitope vaccines have entered into the clinical trials including HIV multi-epitope vaccine, HCV multi-epitope vaccine.An ideal vaccine can not only stimulate the B-cell to produce the specific antibody, but also stired CTL to remove cells infected virus and activated CD4+ T lymphocyte to assist the humoral immunoresponse. The envelope glycoprotein E can induce production of protective immunity, which exists as 90 "head-to-tail" homodimers on the virion surface. The E monomer can be divided into three structural domains:DⅠ, DⅡ, and DⅢ. Antibodies to both DII and DⅢhave been shown to neutralize virus. But anti-DIII antibodies tend to be more virus type-specific, and be powerful neutralizing antibodies.In this study, the EDⅢwas researched again. We have found the strongest immunogenicity and type-specific epitopes in the EDIII by the bioinformatics and sequence analysis software. They may be T- and B- cell peitope, RHVLGRLITVNPIVT E345～359, and EPGQLKLNWFKKGSS E383～397. In the study of subunit vaccine, Consogno predicted hybrid T-cell epitope when studied the tumor multi-epitope vaccine. An important outcome the pan-DR epitope (PADRE) peptides were ascertained, which are artificial general Th cell epitope containing 13 amino acid residue.In our research, we used the netservers, databases and DNAstar software to analyze Physico-chemical property, secondary structure, flexibility, accessibility and hydrophilicity of the linear multi-epitope peptide, which consisted of PADRE, T- and B- cell epitope. The P1 (AKFVAAWTLKAAAGGRHVLGRLITVNPIVTGGEPGQL NWFKKGSS,96.5%), and the control peptide P2 (AKFVAAWTLKAAAGG KKSKAINVLGGIGESHFKGLVLI,76.7%) were synthesized. We inoculated C57BL/6j mice with peptide and observed its effect.Part 1:Bioinformatic analysis of the multi-epitope peptideAccording to the amino acid sequence of P1, online server http://www.expasy.org/tools/protparam was applied to analyze the molecular weight, isoionic point and stability analysis. Molecular weight (MW) of peptide is 4474.28 Dalton, isoelectric point (PI) is 11.17, T50 is 4.4h.Using the method of Chou-Fasman and Garnier-Robson of the protean software from DNA star package analyzed the amino acid segments for a helix,βsheet,βturn and Coil.Blast software from net server http://blast.ncbi.nlm.nih.gov was applied to predicted the type-specificity. And the network server http://www.cbs.dtu.dk/ services/SignalP was used to analysis of the singal peptide and the cleavage sites. The multi-epitope peptide (PI) has antigenicity, type-specificity and singal peptide.Part 2:Humoral and cell immunity induced by peptideGroups of 4-6 week-old female C57BL/6j mice (ten mice/group) were inoculated with 100μg of P1 and P2 diluted in 100μL of PBS, 100μL of PBS as negtive control. Mice were injected on day 0 and boosted on weeks 2 and 4. The mice were bled on week 0,2,4 and 6. And sera were stored at-20℃. Sera were tested for anti-peptide antibodies by ELISA. On week 2,4 and 6, the antibody titer of group P1 and P2 is 200±141.42,25600±18101,95085±19351 and 8.57±3.78,62±46,814±589, respectively. But the group PBS can not induced humoral immunity. SPSS 13.0 was applied and analysis of ANOVA for the repeated measures was adopted. The group inoculated P1 got higher antibody titer when compared with the P2 (F=109.75, P<0.001).Furthermore, Vero cells were seeded in 24 cell culture plate and infected with 100TCID50 DEN-2 NGC.96 h post infection, cells were washed and fixed, then incubated with 1:50 dilution of sera (anti-P1, P2 and PBS serum, rabbit anti-DEN-2 serum and normal serum) for 16 h at 4℃. Cells were detected with fluorescein-conjugated goat anti-mouse IgG or anti-rabbit IgG, then were observed with a fluorescent microscope. The antiserum of P1 and the rabbit anti-DEN-2 were be able to bind DEN-2 NGC in the Vero cells, which was infected with DEN-2 NGC. After staining, scattered small foci of immunofluorescence were observed at the cytolemma or cytoplasm. But, foci of immunofluorescence was not observed in groups of normal serum, the antiserum of P2 and PBS.Two weeks after the last immunization, the mice splenic lymphocyte were isolated. Cells were seeded at a concentration of 2.5×105 cells/well in 96-well cell culture plate. Splenic lymphocytes were stimulated with 25μg/mL of P1 or P2, 5μL/mL of PBS and 5μg/mL of PHA for 48 hours, then 10μL of CCK-8 was added in the culture for 5 hours. The reaction absorbance at 450nm was measured.The P1 group got higher SI (SI= [the absorbance value of the sample pore-the absorbance value of the blank pore]/[the absorbance value of negative pore-the absorbance value of the blank pore]) (2.368±0.488) when compared with unrelated peptide P2 (1.656±0.312), PHA group (1.56±0.320) and PBS group (0.862±0.075), the difference is statistically significant (P<0.05).After inoculating C57BL/6j mice as described previously, spleen cells were harvested and stimulated with 25μg/mL of PI or P2, and 5μL/mL of PBS for 6 hours. Intracellular cytokine staining (ICS) method was applied to quantify Thl (CD3+CD8-IFN-r+) and Th2 (CD3+CD8-IL-4+). The ratio of Thl in group P1 (3.332±1.17) is higher than P2 (0.685±0.262) and PBS (0.6075±0.276), the difference is statistically significant (P<0.05). But, the ratio of Th2 in groups are not different (F=1.282, P=0.35).Part 3:Protective immunity induced by multi-epitope peptideTwo weeks after the last immunization, mice were infected with DEN-2 NGC (2×109 copies/mouse) through peritoneal injection. Blood samples were collected at days 1,3 and 5 after infection. And the real-time PCR was used to quantify RNA of DENV in the blood. The amount of DENV RNA in the group of P1 was 5.56±3.302 (×103 copies/mL) at the first day; The amount of RNA in the group of P2, PBS and the blank group (on day 1,3 and 5) was 8.44±6.89,75±24.595,2.86±0.948; 8.79±7.63, 16.84±7.23,2.7±0.734; 13.26±8.92,15898±7601.44,33.4±31.848 (×103copies/mL), respectively. Analysis of ANOVA for the repeated measures was adopted. The amount of DENV RNA of groups are different, and the difference is statistically significant (F=21.92, P<0.001).Sera from mice immunized with different antigen (P1, P2 and PBS) were assayed for neutralizing antibodies against Den-2 virus using cell culture. Tissue culture infective dose (TCID) inhibition assays in Vero cells showed neutralization tirer of 1:64 for sera from mice immunized with P1, whereas sera from mice immunized with P2 and PBS had not neutralizing antibodies.ConclusionI. Bioinformatic analysis showed properties of the peptide P1, such as hydrophilicity, secondary structure, singal peptide and the cleavage sites; Ⅱ. The peptide of P1 is complete antigen (immunogenicity and antigenicity), which can induce IgG in the inoculated mice. And the antiserum can bind the virus in the vero cells which were infected with DEN-2 NGC. Moreover, P1 can provoke proliferation of splenic lymphocyte and induce ThO to drift to Th1;Ⅲ. Mice inoculated with P1 can challenge with DEN-2 NGC infection. And the antiserum against P1 is able to neutralize the DEN-2 NGC.