The Research On Recombinant Epitope Peptide Vaccine Of SARS-CoV S Protein

In November of 2002, the first epidemic of severe acute respiratory syndrome (SARS), with acute nature, the highly contagious and high mortality, imposed tremendous psychological and economic burden on the public. The etiological agent of this atypical respiratory disease has been identified as a novel coronavirus, SARS-CoV. Belonging to Coronaviridae family, the SARS-CoV is enveloped, single-strand RNA virus that replicates in the host cell. The genomic organization of the virus is similar to that of other coronaviruses with a general order of replicase, spike glycoprotein (S), envelope (E), membrane protein (M), and nucleocapsid (N) from 5′to 3′direction. The spike protein, the largest type-I transmembrane glycoproteins of SARS-CoV, could be functionally divided into N-terminal S1 and C-terminal S2 subunits without cleavage. The S1 subunit is the knob region of the spike, involved in viral attachment with the cellular receptors and contains epitopes that stimulate the production of SARS-CoV neutralizing antibody to protect animals upon viral challenge. The S2 subunit is the stem region of the spike and its coiled-coiled and transmembrane regions are involved in host cell entry and cell-to-cell fusion. Mutations in the S glycoprotein have been shown to alter virulence and cellular tropism. Accordingly, the S protein is a major interest as a target of antiviral drug as well as for development vaccine. So, we hoped to target S protein to analyze antigenic fragments for vaccine development. So, for development effective vaccine of SARS-CoV, we predicted the antigenic regions with putative B cell epitopes of SARS-CoV S protein by bioinformatics methods. Then 168aa-216aa(S2)and 726aa-843aa(S6)were chosed to expresse as recombinant PS2 and CS6 proteins in E.coli and recombinant proteins were purify by Ni affinity chromatography. Recombinant PS2 proteins could elicit high level IgG1 antibody, which could bind to S1 peptide and inactivated SARS-CoV. Besides, recombinant PS2 proteins could induce the proliferation of lymphocytes and B cells in PS2 proteins-immunized mice in recall responses as well as the production of S1 peptide specific antibody and IL-6. Recombinant PS2 proteins could also induce the proliferative and activated of PBMC and B cells of human. These findings suggest that recombinant PS2 proteins could elicit neutralizing antibody against SARS-CoV, which will have implications for developing a novel and effective vaccine of SARS-CoV. The main results: 1. The predictions of B cell epitopes fragments of SARS-CoV S protein and vaccines designTo choose the B cell epitopes in SARS-CoV for developing a vaccine, we first analyzed the hydrophilicity, antigenic index and secondary structure in SARS-CoV S protein, which could indicate the antigenicity fragments in S protein. Based on the analysis, we predicted eight candidate fragments (S1-S8) with better antigenicity. The predicted fragments were further analyzed by average antigenicity index, which indicated the B cell epitopes and possibility. As had suggested, the epitopes contained in S2 and S6 have higher AI value, so two fragments 168aa-216aa(S2)and 726aa-843aa(S6)were selected to express for the antigenicity analysis. 2. Expression, purification and identification of PS2 and CS6 proteins To produce the recombinant PS2 and CS6 proteins, we synthesized the genes of S2 and S6 in several rounds of PCR reactions, respectively. For strengthening the antigenicity, PS2 gene, six copies of S2, was constructed and between the single copy of PS2 protein, there was a Gly-Gly-Asp-Glu-Asp-Glu-Gly-Gly linker predicted to enhance the visability of each copy. As for S6, it had been confirmed difficultly to expressed in E.coli, so chaperonin 10 was fused to assisting S6 protein folding and enhancing expression productions. The codons of the PS2 and CS6 gene were optimized to expressed in E. coli and the recombinant proteins were purified by Ni chromatography. The LPS remnant in the purified protein was confirmed to be less than 50EU/mg. Because S2 and S6 were located in S1 and S2 subunits,respectively, we used the expressed S1 or S2 peptides and the induced antibodies to identify the recombinant PS2 and CS6 proteins. In Western blots assay, the recombinant PS2 and CS6 proteins were transferred onto nitrocellulose membranes and the S1 or S2 peptides immunized guinea pigs serum were used as detection antibody. As shown in results, the recombinant PS2 and CS6 proteins could be specifically recognized by the antisera induced by S1 or S2 peptides, respectively, but not by the antisera of PBS injected guinea pigs. The results showed the similar conformation between candidate antigenic fragments and full length S1 or S2 peptides. 3. Antigenicity analyse of recombinant PS2 and CS6 proteins Firstly, we detected the antibodies level induced by recombinant PS2 and CS6 proteins by ELISA. The results showed the induced antibody level was high enough to bind with recombinant PS2 and CS6 proteins. Secondly, we detected whether the antisera induced by recombinant PS2 and CS6 proteins could recognize conformational S1 or S2 peptides by immunodot blot. S1 and S2 peptides were spotted onto nitrocellulose membranes, then were probed with murine anti-PS2 or CS6 polyclonal antibodies or PBS injected sera. The results showed that the induced antibodies by recombinant PS2 proteins could specifically recognize S1 peptide and the induced antibodies by recombinant CS6 proteins could specifically recognize S2 peptide, while antibodies from PBS injected mice could recognize neither S1 nor S2 peptides. These results indicated that the expressedrecombinant PS2 and CS6 proteins kept its antigenicity to bind to S1 or S2 peptide, which will lead to recognize SARS-CoV. 4. The recognizing effect of induced antibody by PS2 protein on inactivated SARS-CoV Accordingly, we continued to test whether the recombinant PS2 and CS6 proteins could induce the SARS-CoV recognizing antibodies. The mice were immunized with recombinant PS2 or chaperonin 10-S6 proteins at 50 or 100 μg for three times at two-week interval. The sera of immunized mice were collected at two-week interval and assayed for specific IgG antibodies using an inactivated SARS-CoV coated ELISA detection Kit. The data showed that recombinant PS2 proteins could stimulate mice to produce the IgG antibodies recognizing SARS-CoV, which could be detected in two weeks, then reached a peak at sixth week and maintained a plateau from the sixth to the tenth weeks after first immunization. Whereas, recombinant CS6 proteins couldn't induce mice to produce detectable antibodies recognizing SARS-CoV particles, indicating that recombinant CS6 proteins, though confirmed to induce anti-S2 proteins antibodies, couldn't induce the production of SARS-CoV recognizing antibodies. The recognizing antibody to protective epitopes is of particular importance and will lead to the clearance of virus. Therefore, we selected the recombinant PS2 protein as a candidate antigen vaccine against SARS-CoV and further studied its potential efficacy to induce humoral immune responses. 5. Identiification of antibodies types in the sera of immunized miceTo determine the types of antibodies induced, the sera were firstly prepared from the PS2-immunized mice at the 6th week after the first immunization, then, IgG,IgM and IgA antibody profiles were detected The results showed that PS2 proteins could obviously stimulate the production of IgG antibodies, but not IgM and IgA, compared with PBS control. Furthermore, IgG antibodies were tested for IgG1 and IgG2a isotypes in purpose to identify the Th1/Th2 balance in the immune response of PS2 proteins. Indirect ELISA was used to analyze the subtypes of IgG antibody. As described, the coating-antigen S1 proteins were reacted with antiserum of PS2 proteins, then probed with HRP-conjugated goat anti-mice IgG1/ IgG2a. The results showed a significantly higher level of S1-specific IgG1 antibody than IgG2a in the sera of 50 or 100 μg PS2 protein immunized mice, indicating Th2 cells were activated in the immune response elicited by PS2 proteins, which attributed to humoral immune responses. 6. Detection of IgG antibodies in the culture supernatants of immunized murine splencytes To detect the specific antibodies elicited in vitro, culture supernatant were collected from splencytes of immunized mice for indirect ELISA test. Significantly, the higher level of S1 specific IgG antibodies could be tested in PS2 protein stimulation wells than medium incubation wells, which both showed the dose-dependent relations. While, S1 specific IgG antibodies could not be tested in the culture supernatant from PBS injected mice even with PS2 proteinstimulation in vitro. The results demonstrated that PS2 protein could elicit the production of anti-S1 antibodies that may be useful for neutralizing SARS-CoV. 7. Identiification of cytokine in the sera of immunized mice Secretion of distinct cytokines implied the types of an antigen-specific immune response, such as humoral or cellular immune response. Accordingly, for identifying the PS2 protein specific immune response, we detected two representative cytokines, IL-6 and IFN-γin the sera of PS2-immunized mice. As the result showed, IL-6 was obviously induced as high as 320 pg/ml in the sera of 100 μg protein immunized mice and 160 pg/ml in 50 μg immunized group. In contrast, IFN-γlevel was as low as the PBS control group. Therefore, we concluded that predominantly Th2-associated pattern immune response was triggered by recombinant PS2 protein. 8. The proliferative induction effect of PS2 proteins on lymphocytes and B cells in mice 8.1 The proliferative induction effect of PS2 proteins on lymphocytes in mice In order to evaluatehe proliferative induction effect of recombinant PS2 protein, [3H]-thymidine incorporation assay were performed to investigate the proliferation of mice splenocytes to PS2 stimulation in vitro. The results show that mice in 50 μg of PS2 immune group exhibited the similar proliferative responses to 100 μg immune group when incubated with medium, compared with PBS control group.When spleen cells were stimulated with 10 μg/ml of PS2 protein for 3 days in vitro, the stronger PS2-specific proliferative responses was investigated in 50 μg immune group than that of 100 μg immune group. As expected, PS2-specific proliferative responses did not show in PBS group. The results showed recombinant PS2 protein immunized spleen cells could proliferate spontaneously in vitro and with the stimulation of recombinant PS2 protein, lymphocyte proliferation were more stronger in recall response. 8.2 The proliferative induction effect of PS2 proteins on B cells in mice In order to define the population of proliferative splenocytes following the recombinant PS2 protein stimulation, we have developed an alternative assay that allows the detection and identification of the proliferating cells in vitro, based on dilution of CFSE with which the cells are prelabelled. The results of FACS presented the obvious proliferation effect of B cells. Similar proliferation effect occurred at 24, 48, 72 h of two dose groups, which could be clearly distinguished from the PBS control groups. So the CFSE and FACS assay in vitro could perfectly confirm the preponderant proliferation of B cells, resulted from PS2 stimulation, which are likely to contain B cell epitopes and contribute to antibodies production. 9. The proliferative and activated induction effect of PS2 proteins on PBMC and B cells of human 9.1 The proliferative induction effect of PS2 proteins on PBMC of humanFor further confirming the proliferation effect of PS2 proteins in human, PBMC were isolated from health blood by density centrifugation over Ficoll-Paque. 6×105 cells per well in IMDM media were cultured with different doses of PS2 protein for 3 days, then detected by [3H]-thymidine incorporation assay. These data reveal that lymphocytes in human may have the receptors to specifically recognize PS2 protein, which are essential to produce neutralizing antibodies. 9.2 The activated induction effect of PS2 proteins on B cells of human Lymphoproliferative assays and activation in PBMC had been the conventional methods to assess the immunostimulatory properties of Ag. Here, we also selected the early activation molecular—CD69, as the marker to assess the B cells activation by flow cytometry. 1×107 PBMC per well were cultured in 24-well plates with medium alone or with 10,20 or 30 μg /ml of PS2 proteins at 37 °C in 5%CO2 for 36 h. As measured, PS2 protein was an effective up-regulator to CD69 molecule gated on CD19 positive human PBMC in a dose-dependent manner, while, PBMC incubated with medium only did not show the up-regulation effect of CD69 molecule. The up-regulated CD69 molecule suggested the activation of B cells with the stimulation of recombinant PS2 proteins. So we can conclude that recombinant PS2 antigen is an efficient stimulator that lead to the activation of human B cells. In summary, the predicted PS2 protein could induce specific...