Multi-epitope Of Wt1 Antigen Gene Vaccine Design And Immunogenicity Study

ObjectiveThe WT1 gene was isolated as the gene responsible for a childhood renal reoplasm. In healthy adults , WT1 expression is limited to a set of tissues. However, the majority of acute leukemias and many solid tumors is ove rexpress WT1. And the xpression levels is an adverse prognostic factor in these malignant disease. The finding that blockade of WT1 function by WT1-antisense oligomers in primary leukemic cells significantly slows cell growth in vitro additionally suggests that WT1 is of critical importance to the tumor phenotype and that tumor escape by simple WT1 down-modulation or loss is unlikely to occur. These features of WT1 in terms of its biological functions and expression patterns indicate that WT1 could be an ideal cancer antigens for therapeutic vaccination.Up to date, WT1 antigen has been studied in clinical trials , and has showed a certain curative effect. However the efficiency of clinical trials is not satisfied because most of researchers use a single CTL epitope vaccination. The drawbacks inclued: inadequate activation of the innate immune system, T-cell epitope restriction to a particular MHC haplotype and possible immunoselection of epitope-loss variants.The WT1 polyepitope gene vaccines can make up the above drawbacks: (a)acompared to a single CTL epitope vaccine, the injection of multiple epitopes can overcome the potential loss of expression of a given -single epitope in cancer cells. (b) the vaccination with multiple epitopes would allow to enroll patients with different HLA moleular. (c) the vaccination with Th epitopes would not only elicit CTL responses, but also lyse the specific target cells. (d) The DNA vaccine can be easily design and construction.The gene adjuvant is an effective way to enhance genetic vaccine immunogenicity. It has been reported the adjuvant effect of mHSP70 is a effective adjuvant. The C-terminal stimulatory domain of mHSP70359-610 has the immuno adjuvant function, which can rapidly and directly induce the secretion of multiple cytokines from macrophages and dendritic cells by interacting with CD40. p407-426 is a stimulatory domain of HSP359-610. It could be more effectly induce DC mature than HSP359-610 , but with a better performance by reducing the self-immune responses caused by mHSP70. Therefore we chose the C-terminal stimulatory domain of mtHSP70.Based on the principles mentioned above, we constructed a WT1 polyepitope gene vaccines of which parts encode multiple CTL and Th epitopes, then we got the fusion genetic vaccine consisted of the mHSP70_407-426 and WT1 polyepitope gene. which was used to verify whether it has the activity to induce the immune responses and inhibit the growth of WT1-expressing tumor. Methods1. Design and synthesis of WT1 polyepitope cassettes Based on collecting published WT1 epitopes, WT1 cassettes of good immunogenicity with CTL and Th epitopes were designed. Then the cassettes were optimized by computer-based modeling . Ulitimately, the cassettes were synthesized.2. Design and construction of WT1 polyepitope genetic vaccine2.1 Construction of eukaryotic expression plasmidThe synthetic nucleotide sequence was cloned into pUC57-T easy vector. After digestion, it was cloned the eukaryotic vector to construct the plasmid pcDNA-WT1. For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplificated by polymerase chain reaction from pGEM-mHSP70 vector and cloned into pcDNA3.1(+). Then WT1 polyepitopewas fused to the N-terminal of pcDNA-mHSP70_407-426 to make the multi-epitope fusion gene vaccine pcDNA-WT1-HSP70_407-426.2.2 Transfecion of genetic vaccine to 293T /Hela cellsHela and 293T cells were transfected with empty pcDNA,pcDNA- HSP70_407-426,pcDNA-WT1 and pcDNA-WT1-HSP70_407-426 vector by liposome transfection.2.3 RT-PCR and western blotTransfect HEK-293T cells with genetic vaccine and identify the expressed product by RT-PCR. Transfect Hela cells with the constrcuted DNA vaccine pcDNA-WT1-HSP407-426 and identify the expressed product by Western blot.3 Study of immunogenicity of WT1 polyepitope genetic vaccine3.1 Vaccination of miceThe immunogenicity of the vaccine was assessed using male 4-6 week-old C57BL/6mice, divided into 5 groups. The mice were immunized with the pcDNA-WT1 and with pcDNA-WT1-HSP70_407-426, pcDNA-HSP70_407-426, empty pcDNA vector and PBS as controls. Each mouse was inoculated into each tibialis muscle with with 0.25 % bupivacaine and then after 48 hour with 50ug plasmid every 2 weeks intramuscularly for three times. After 10 days of the last time of inoculation, we isolated the lymphocytes of the mice's spleen.3.2 The immunogenicity of the genetic vaccineT lymphocyte proliferation by MTT, T lymphocyte subpopulations by FCM, peptide-specific T cells by ELISPOT assay and cytotoxicity by LDH release assay evaluated the genetic vaccine immunity resposes.3.3 Effect of genetic vaccine on therapy of established tumorsMale C57BL/6 mice, 4 to 6 weeks old, were selected and divided into 5 groups. Before the start of the treatments , five groups mice were inoculated with 2×106 FBL3 tumor cells. At the 7 days after the inculation, mice were immunized with the pcDNA-WT1, with pcDNA-WT1-HSP70_407-426, pcDNA- HSP70_407-426, empty pcDNA vector and PBS as controls. The muscle DNA administrations were repeated at 14 days after the first treatment, and during this time the tumor sizes in the mice were recorded every five days.3.4 Induction of HLA-A*0201-restricted CTLPBMC were isolated from the blood of HLA-A*0201+ healthy donors .The fusion genetic vaccine pcDNA-WT1-HSP70_407-426 were transfected into PBMC to stimulate autologous lymphocytes in vitro. The effector fuction of inudced CTL were analyzed in cytotoxicity assays.Results1 Design and synthesis of WT1 polyepitope cassettes1.1 Screening of WT1 epitopesThe selection WT1 epitopes comply with two basic principle. First it has advantage in induction of CTL or Th immune response. Second it can bind to more MHC molec-ular. So the selected CTL epitopes included WT1.37(VLDFAPPGA;HLA*0201),WT1.187(SLGEQQYSV;HLA*0201/HLA*0206) and WT1.235(CMTWNQMNL; HLA*0201/HLA*2402); Th epitopes included WT1.121A(SGQAYMFPNAPYLPSCLES;HLA-DRB1*0401) and WT1.332(KRYFKLSHLQMHSRKH,HLA-DRB1*0405/HLA-DRB1*1501/HLA-DRB1*1502/HLA-DPB1*0901).And the WT1.122A concealed a CTL epitoes WT1.126(underline,HLA-A*0201/H2Db). In order to break through the MHC restriction better,one universal Th Pan-DR epitope (PADRE, KFVAAWTLKAAA) was also selected.1.2 Design and optimization of the cassettesIn order to make every epitope has its independent function, the portion of the gene encoding CTL epitopes were designed using computer-based modeling(PAPro and NetChop )to optimize proteasome mediated epitope processing through the introduction of amino acid spacer sequences, such as AAY,KK,KAA,KAAA,AAA and NAAA. The portion of the gene encoding the HTL epitopes was designed with GPGPY amino acid spacers between sequential HTL epitopes . The IgGκchain leader sequence was used as signal peptide, and inserted Kozak sequence at the 5'-terminal of signal peptide as the ribosome-binding site. Codon optimization of nucleotide sequence by DyNAVacS. Ultimately the cassettes by software proved that the design was consistent with that expected in theorpy .2. Design and construction of WT1 polyepitope genetic vaccine2.1 Construction of eukaryotic expression plasmidRestriction endonuclease digestion analysis and PCR result of the recombinant vector pcDNA-WT1,pcDNA -HSP70_407-426 and pcDNA-WT1-HSP70_407-426 showed that were indentical with aim gene fragments, and DNA sequence analysis showed WT1 and HSP70 was right in the recombinant plasmid . It is concluded that all vector were successfully designed.2.2 Transfecion of genetic vaccine to 293T /Hela cellsCompared with non-tracsfected cell, EGFP expression was only observed in cotrans -fected cells 293T/Hela that showed pcDNA-WT1,pcDNA-HSP70_407-426 and pcDNA-WT1 -HSP70_407-426 could be expressed in eukaryotics.2.3 RT-PCR and western blotAfter 48h of being transfected with 293T and Hela cell, RT-PCR showed correct expression of target gene in HEK-293T cells and western blot showed correct expression of WT1 protein in Hela cells by pcDNA-WT1-HSP70_407-426 transfection.3 Study of immunogenicity of WT1 polyepitope genetic vaccine3.1 The immunogenicity of the genetic vaccineThe result of T lymphocyte proliferation by MTT indicated that immunized mice by pcDNA-WT1-HSP70_407-426 and pcDNA-WT1-HSP70_407-426 had significantly higher levels of stimulation index compare to PBS,pcDNA and pcDNA-HSP70_407-426 controls(p<0.01). Flow cytometry results showed that immunized mice by pcDNA-WT1 and pcDNA-WT1-HSP70_407-426 had significantly higher levels of CD4+/CD8+ compare to three controls(p<0.01). The immunized mice also have significantly more T cells and higher CD4+/CD8+ values than controls(p<0.01).The result of IFN-γdetection by ELISOPT indicated that there are more numbers of IFN-γ-secreting CD8+T cells induced by peptide mixtures in pcDNA-WT1 and pcDNA-WT1-HSP70_407-426 than that of three controls(p<0.01). Furthermore, pcDNA-WT1 immunized mice induced by four CTL peptides individually, all of them can detect specific IFN-γ-secreting. LDH release assay experiments showed that the immunized mice had higher CTL killing-effects than three controls. While according to above-mentioned experiment, there was no significant different between pcDNA-WT1 and pcDNA-WT1-HSP70_407-426 group(p>0.01). Comparing with PBS, pcDNA and pcDNA-HSP70_407-426 group, the frequency of WT1^-specific CTL was more and cytotoxicity to FBL3 cells were higher in pcDNA-WT1 and pcDNA-WT1^-HSP70_407-426 group(p<0.01). However, there had no significant difference between pcDNA-WT1 and pcDNA-WT1^-HSP70_407-426(p>0.01), that indicate pcDNA-WT1^-HSP70_407-426 can not enhance the potential of pcDNA-WT1 vaccine.3.3 Effect of genetic vaccine on therapy of established tumorsThe tumor volume was significantly lower in the pcDNA-WT1 and pcDNA-WT1^-HSP70_407-426 than three controls(p<0.01).And the survival time was longer in the pcDNA-WT1 and pcDNA-WT1^-HSP70_407-426 than controls(p<0.01).3.4 Induction of HLA-A*0201-restricted CTLThe human pcDNA-WT1^-HSP70_407-426 transfected PBMC through making HLA-A*0201 restrticted CTL assay. CTLs could recognize and lyse NB4(HLA-A*0201+WT1⁺). Whereas control target U937(HLA-A*0201+WT1^-), or K562(HLA-A*0201-WT1⁺ were not killed by the CTLs. it appears that tumour cell lysis was WT1 specific and HLA-A0201-restricted.Conclusions1 The expression cassette for multiple-epitope antigen of WT1 was successfully designed.2 The genetic vaccine pcDNA-WT1 and fusion vaccines pcDNA-WT1^-HSP70_407-426 were successfully constructed.3 The WT1 polyepitopegenetic vaccines could elicit WT1^-specsific cellular immunity and have therapeutic effects against the WT1^-expressing tumors.4 The WT1 poly-epitope genetic vaccines had potential therapeutic effects against WT1^-expressing tumors.5 The linkage of mHSP70_407-426 to WT1 can not enhance the potengcy of WT1 vaccines.6 Every CTL epitopes could have its independent function.7 The pcDNA-WT1^-HSP70_407-426-transfected HLA-A0201+ PBMC are remarkably effective in stimulating potent HLA-A*0201-restricted CTL.