| Hepatitis C virus (HCV), a member of the Flaviviridae family, is a major pathogenthat causes chronic liver disease. Approximately3%of the worldwide population isinfected with HCV and an estimated350,000people have died from HCV-relatedend-stage liver disease and related complications per year. Therefore, HCV infection hasbecome a major public health issue that requires global attention. Currently, pegylatedinterferon (IFN)-α with ribavirin remains the standard treatment method for chronic HCV.However, these medications are not only expensive with multiple side effects, but alsoeffective for only50%of patients infected with HCV. HCV infected patients can not oftentolerate and adhere to the treatment. Therefore, it is crucial to explore the prevention andtreatment of HCV infection in different ways. Development of an effective vaccine is oneof the best ways to reduce the incidence of HCV infection and HCV-related liver disease.Some studies have demonstrated that the effective cellular immune response is veryimportant for the control of HCV infection. Therefore，more and more attentions havebeen paid to how to induce effective cellular immune responses with HCV vaccine, andthe latest studies have showed that the vaccines with multiple HCV T cell epitopes,which mainly locate in the Core, NS3, NS4B, NS5A and NS5B proteins, has been one of the novel directions in HCV vaccine development. In this study, firstly we synthesizedeight multi-epitope polypeptide vaccines and evaluated the immunologic effects of thesevaccines which contained the2conservative CTL epitopes of HCV, NS4B and NS5A,and a universal Th epitope in Core protein with different combinations and sequences.Secondly, we reconstructed a prokaryotic expression vector which contained the genes ofthe above multi-epitope peptide with the best immunologic effects, EDA and truncatingHCV NS3. Then, we expressed them through IPTG induction in E. coli and purified themin their active form using affinity chromatography. The immunological characteristics ofthe target proteins were then evaluated by using immunized HHD-2mice.【Methods and results】1. Design and construction of HCV multi-epitope peptide vaccine and theirimmunological effects in HHD-2miceHCV NS4B (1793-1801) and NS5A (1991-1999), the two CTL epitopes, areHLA-A2-restricted, which cover about50percent of the population in China. These twoCTL epitopes were chosen after bioinformatics analysis. During the construction of thismulti-epitope polypeptide vaccine, we optimized the selected multi-epitopes in two phases:1) a lysine-lysine (KK) sequence was inserted as an intervening sequence between theepitopes; and2) the common Th epitope was selected. Three epitopes were designed aseight polypeptides in order to select the optimal multi-epitope polypeptide vaccine.The eight polypeptides were listed below:VL-44（NS5A1991~1999-KK-NS4B1793~1801-KK-Core23~44）SL-44（NS4B1793~1801-KK-NS5A1991~1999-KK-Core23~44）VAL-44（NS5A1991~1999-KK-Core23~44-KK-NS4B1793~1801）SWL-44（NS4B1793~1801-KK-Core23~44-KK-NS5A1991~1999）KWL-44（Core23~44-KK-NS4B1793~1801-KK-NS5A1991~1999）KAL-44（Core23~44-KK-NS5A1991~1999-KK-NS4B1793~1801）VL-20（NS5A1991~1999-KK-NS4B1793~1801）SL-20（NS4B1793~1801-KK-NS5A1991~1999）The eight polypeptides were synthesized using a solid-phase polypeptide synthesizer, and then sequenced through mass spectrographic analysis. The purity of each polypeptidewas95%or above.Using HHD-2mice as immune subjects, β2-microglobulin and MHC were knockedout. Mice were then transfected with the αl and α2domains of the human HLA-A2geneand the human β2microglobulin gene. The mice were immunized using syntheticpolypeptides and then were used to evaluate the resulting immunological effects.Fifty-four HHD-2mice aged6-8weeks were selected and divided into9groups thatreceived one of the eight polypeptides and PBS groups. There were six mice in each group.Multi-epitope polypeptides (200μl, final concentration0.5μg/μl) were completelydissolved in Freund’s adjuvant and injected subcutaneously into mice. Complete Freund’sadjuvant was used for the first injection, and incomplete Freund’s adjuvant was used forthe booster injections. Mice were immunized in PBS control group with same method.The cellular immune response was detected with different methods for immunizedmice.1) VAL-44SWL-44, KWL-44polypeptide can stimulate the splenocytesproliferation of immunized mice significantly (P<0.05). VAL-44had the maximal effecton the splenocytes proliferation.2) We determined the percentage of CD4+and CD8+T-cells using flow cytometry. VAL-44polypeptide could increase the percentage of CD4+and CD8+T cells. They were all significantly higher compared with contol group（P＜0.05）.3) We assayed the secretion of IL-2, IL-4and IFN-γ by immunized murine spleniccells using the ELISpot method. The number of IL-2secreted mice splenocytes wasrelatively higher in3polypeptide immunization groups: VAL-44, SWL-44and KWL-44groups. They were all significantly higher than that in contol group（P＜0.05）. Thenumber of corresponding antigens stimulated IFN-γ-secreted splenocytes reached morethan30/106cells in4polypeptide immunization groups: VAL-44, SWL-44, KWL-44andVL-20groups. They were all significantly higher than that in contol group（P＜0.05）.VAL-44had the maximal effect on the secretion of IFN-γ and IL-2by splenic cells.4) TheCTL results showed the killing effect of the polypeptide was higher in the VAL-44,SWL-44, KWL-44and VL-20groups at an effector-target ratio of50:1compared to thePBS control group (P <0.05). The killing effect was the most significant in the VAL-44 group.After HHD-2mice were immunized using the designed HCV multi-epitopepolypeptides, four polypeptides in the VAL-44, SWL-44, KWL-44and VL-20groupswere preliminarily verified to have advantages in four respects: stimulating theproliferation of splenic cells, activating CD4+and CD8+T cells, inducing the productionof cytokines, and good CTL effect in vitro. The affinity and stability of the epitopes weredetermined using T2cells. Their relative affinity was tested using competitive inhibition.These results showed that the VAL-44, VL-20and HLA-A2polypeptides had the highestaffinity. The synthetic peptides and HLA-A*0201-stability test showed that VAL-44andVL-20peptides have good levels of stability and the DC50was more than6h.These data show that VAL-44can induce the best immune response among the8polypeptide vaccines which contain the2conservative CTL epitopes and a universal Thepitope.2. Construction of the recombinant protein containing DC activation molecule EDA,ΔNS3and VAL-44and its immunological effectsIn this part, in order to enhance the immunogenicity and immune response effect ofthe multi-epitope peptide VAL-44, we added the DC cell activation molecule EDA toenhance the efficiency of VAL-44presenting, and added the truncated HCV NS3gene(ΔNS3) to enhance the immunogenicity. The EDA-ΔNS3gene was inserted into theupstream of the multi-epitope peptide VAL-44in a prokaryotic expression vector tofurther produce the EDA-ΔNS3-VAL-44recombinant protein vaccine. Total fourprokaryotic expression vectors, the pET-32a-EDA-ΔNS3-VAL-44, the pET-32a-EDA, thepET-32a-EDA-ΔNS3and the pET-32a-ΔNS3, were reconstructed. After digestion,sequencing and correct identification, these four proteins were expressed through IPTGinduction in E. coli. SDS-PAGE analysis showed that the expressed proteins had the samemolecular weight as expected, and Western blot analysis showed that the expressedproteins were the target proteins. The four proteins were purified in their active form usingaffinity chromatography.Overall,36HHD-2mice aged6-8weeks were selected and divided into six groups (n =6in each group): EDA-ΔNS3-VAL-44, EDA-ΔNS3, ΔNS3, EDA, and EDA-ΔNS3-VAL-44DNA combined protein and PBS. The mice were immunized using each of thedifferent recombinant proteins as described above. Complete Freund’s adjuvant was usedfor the first injection, and incomplete Freund’s adjuvant was used for the booster injections.The single immunizing dose of the purified recombinant protein was100μg, and with50μg poly (I: C), emulsified sufficiently with Freund’s adjuvant. The recombinant proteinwas injected subcutaneously into the mice, and the DNA vaccine (75μg) was injectedintramuscularly. The mice were given two booster injections after2weeks.The serum antibodies were tested using the tail vein blood at2,4and6weeks afterthe initial immunization, and mice were sacrificed to test the cellular immune response at6weeks. The test results for humoral immune response levels showed that the antibodytiter was maximal in the recombinant protein EDA-NS3-VAL-44group (1:12,800at4weeks after the initial vaccination) and the IgG2a/IgG1ratio was significantly higher thanthe controls (P <0.05). This suggested that the immune process was dependent on IgG2a.Mice were sacrificed two weeks after the last immunization. The spleens wereremoved and purified in lymphocyte separation medium. Different methods were used todetect the levels of cellular immune responses.1) Splenocytes proliferation assays showedthat the EDA-NS3-VAL-44recombinant protein group significantly stimulatedlymphocyte proliferation（P＜0.05).2) Flow cytometry showed that EDA-NS3-VAL-44recombinant protein group caused significant proliferation of CD4+and CD8+Tlymphocytes compared with other groups (P <0.05).3) The levels of IL-2, IL-4and IFN-γsecretion by splenic cells in immunized mice were increased in the EDA-NS3-VAL-44,EDA-NS3, EDA-NS3-VAL-44DNA recombinant protein groups according to theELISpot method, and EDA-ΔNS3-VAL-44recombinant protein caused most significantincrease of three cytokines in the immunized mice. Moreover, the secreted level ofTh1-type cytokines was significantly higher than Th2-type cytokines.4) The CTL resultsshowed that the killing effect in the EDA-NS3-VAL-44group was higher than the otherexperimental groups (P <0.05). Each recombinant protein as well as theEDA-ΔNS3-VAL-44DNA/recombinant protein can induced T-cell-mediated specific cytotoxicity in mice (P <0.05).In order to evaluate the CTL effects of recombinant proteins in vivo, we injected apIRES2-dsRED-EDA-NS3-VAL-44plasmid using a hydrodynamic method and assayedthe relative hepatic expression levels of EDA-NS3-VAL-44mRNA using real-timequantitative PCR. The relative expression levels in the EDA-NS3-VAL-44group werethe lowest compared with other groups (P <0.05), which suggested that the CTL effectsgenerated by the vaccine induced significant killing of the target liver cells.【Conclusion】In conclusion, the VAL-44polypeptide is the optimal option for vaccine developmentin designed and synthesized eight HCV multi-epitope peptides. VAL-44(the amino acidsequence: VLSDFKVWL-KK-KFPGGGQIVGGVYLLPRRGPRL-KK-SMMAFSAAL)induced the strongest cellular immune response in mice. The EDA-NS3-VAL-44prokaryotic expression protein induced good humoral and cellular immune responses inimmunized HHD-2mice, and the recombinant expression protein had more enhancedimmunological effects than the individual proteins. The EDA-NS3-VAL-44recombinantproteins could induce the killing of HCV-infected liver cells through an initial vaccinationand boosted vaccination program. The results support this novel EDA-NS3-VAL-44recombinant protein will be as a potential candidate HCV preventive/therapeutic vaccine. |
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