| Backgrounds: Papillomavirus vaccine, Mucosal immunity and DNA vaccine: Worldwide, cervical cancer is the second most common malignant disease among women, Invasive cervical cancer develops in approximately 500,000 per year worldwide, and resulted in app. 200,000 deaths per year, with 80% of cases arising in less developed countries. The median age at diagnosis is 47 years and nearly half of cases are diagnosed before the age of 35. The primary cause in development of cervical cancer is human papillomavirus. More than 90% of squamous cervical cancers contain HPV DNA, the virus is acquired mainly through sexual activity. HPV types 16, 58, 18, 58, 31 and 33 cause most invasive cancers. Other factors associated with the development of cervical cancer include sexual activity starting age (<16 years), a high total number of sexual partners (more than 4), and history of genital warts. The prosperous vaccines against the infection of HPV are the Papillomavirus-like particle vaccines and VLP-based chimeric vaccines. Papillomavirus contains two late genes (L1 and L2 ORFs) and 7-8 early genes, L1 protein alone, expressed in prokaryotic cells and eukaryotic cells, can self-assemble into papillomavirus-like particles (VLP), VLP is morphologically and structurally indistinguishable from naive virion, except lack of the oncogenic viral genome. VLP can induce specific neutralization antibodies against type-specific papillomavirus infection, VLP also can bind and result in mouse RBC cell hemoagglutation, and binds and enter mammalian cells through papillomavirus receptor.Approximately 70-80% of all Ig-producing cells in human body are found in mucosal tissues, the remaining 20~30% are found in the bone marrow (the mose important source of plasma IgG and IgA), and in the spleen and lymph nodes. In mucosae, some 80% of total Ig-producing plasma cells secrete IgA, precursors of these cells originate in the IgA-inducing sites, such as GALT, PP, BALT and nosal mucosa cells derived from mucosal inductive sites home to the mucosal tissues and glands, Oral and intranasal immunization elicits a more humoral response in saliva and female vaginal washes. Because antibodies of IgG isotype are dominant in both male and female genital tract secretion and are largely of plasma origin, so the humoral immune responses manifested mainly by IgG antibodies can be stimulated in genital tract secretions by systemic immunization. In human cervical mucus, there are higher levels of IgG than of IgA, this is contrasts with other typical external secretions, such as saliva, tears, milk, and intestinal fluids, in which sIgA is the dominant isotype. IgG and IgA- producing cells are dominant and almost all IgA-producing cells contain J chain, a maker of synthesis of pIgA. The mechanism involved inthe appearance of IgG in cervicovaginal secretions has not been elucidated. Ig produced locally and transported from blood by uterine tissues provided humoral immunity in vaginal canal. The problem is that about 60-70% of women who have CIN or who are infected with HPV are serum positive for anti-HPV16 IgG antibody, these observation also suggested that about 30-40% of women with cervical cancer do not have HPV antibodies, perhaps HPV-specific antibodies are induced locally in many women infected with HPV, but the serum titer is too low to be detected. slgA were present in 13.1~27% of infected cases w/o visible pathology; Mucosal IgG response was observed in 6.5-27.5% of patients w/o visible pathology; in the other words, there are more than 70% of cervical cancer patients do not have mucosal IgG and IgA antibodies secretions. SO it is expected that the appearance of IgG and IgA in cervical mucus may play a much important role in preventing the entry of the papillomavirus infectibns.DNA vaccine encompasses vaccination using naked DNA or DNA delivered by a carrier where the antigen is expressed under the control of a eukaryotic promoter, however efficient delivery often requires the use of specialized equipment, such as gene gun-type technology for intra-dermal delivery of DNA coated particles, following vaccination, it is thought that primed APCs need to migrate to the spleen for the induction of immune responses, it is desirable to deliver DNA vaccines directly to APCs, which can be achieved through the exploiting of attenuated intracellular bacteria. Invasive intracellular bacteria are able to transfer eukaryotic expression plasmids into mammalian host cells in vitro and in vivo. This can be used to induce immune responses toward protein antigens encoded by the plasmid or to complement genetic defects. For the sake of enhancing the immune responses strength of the DNA vaccines, high levels expression of antigens and long-time mRNA stability have the capacity of inducing strong immune responses, Now there are several different dual-promoter plasmids, but they contain both the eukaryotic promoters only, we think this can not meet the requirements of the intracellular bacteria-based DNA vaccines strategy, because the intracellular bacteria have the capacity of the DNA vaccine delivery vehicle, places of the DNA plasmids replication and the antigen expression, So the intracellular bacteria-based DNA vaccine expression plasmids should have both the eukaryotic and prokaryotic promoters, so the antigen can be expressed in both bacteria and host APCs. Of the many E.coli phage promoters studied, only a few satisfy the these requirements, for example: PL from lambda DNA, Lac and trp from E. Coli, and the hybrid trp-lac (tac) promoter and T7 RNA polymerase promoter. But all of these promoters rely on either a temperature shift or the addition of a chemical to induce their activity. Chemical additions are often expensive and require subsequent removal at the purification stages. Now there are several in vivo inducible promoter, including oxygen-regulated nirB, an promoter from E. coli, which can drive expression of nitrite reductase gene nirB, and nirD, nirC and cysG, which is regulatedby low stress of the oxygen through the FNR and NARL factors, in order to meet the requirements in vivo using, a modified version of nirB promoter was designed by deleting the nitrite-response element, so the modified version was activated by the low stress of oxygen in micro-condition, nirB promoter should belong to strong promoter, because it can provide 0.6-6.5 fold higher levels of the antigen genes. The Vitreoscilla sp haemoglobin (VHb) and pKatG promoters were regulated by oxygen, proU promoter derived from E.coli is induced by the Osmolarity, And the ssaH and spiC promoters are turned on in 5a//wone//a-containingvacuoles of antigen-presenting cells such as macrophages. The ssaH and pagC promoters were repressed when the bacteria were grown in media containing high concentrations of Mg2* ions. The pagC promoter is activated at a relatively low concentration of Mg2+, a condition which is found within Sa/moneZ/a-containingvacuoles in macrophages, the systemic and mucosal immune responses to the heterologous antigens delivered by the Salmonella vaccine strains were optimal with the macrophage-inducible pagC and spiC promoters. Based on the current developments at the in vivo inducible promoters, nirB should be an idea promoter for designing dual-promoter DNA vaccine expression plasmids, SO in this study, The Human Cytomegalovirus immediate-early promoter and nirB in vivo inducible promoter were selected to construct dual-promoter pCMVnir.Construction and evaluate of the dual-promoter DNA vaccine vector pCMVnir: Based on the developments on the plasmids with multiple promoters, in order to make the gene cloning works easier, pTriEx-4 was constructed with 3 different promoters, human cytomegalovirus(HCMV) immediate early enhancer and promoter region, T7 RNA polymerase gene promoter, and baculovirus P10 promoter, and other expression plasmids with eukaryotic promoter and prokaryotic promoter, but all these plasmids are not suitable for DNA vaccine construction, because the chemical compounds-inducible prokaryotic promoters can not be used in vivo, because these promoters are activated by addition of chemical induction compounds or temperature shifting, even attenuated intracellular bacteria were administered through oral or nasal pathway, the recombinant bacteria were cleared quickly only several hours postinoculation of the vaccine. Based on the documents on the nirB promoter, a new version of nirB promoter was designed by modifying the transcriptional elements and deleting the nitrite-dependable activation element, nirB promoter is derived from E.coli, its activation depends on the addition of nitrite and low-level of oxygen tension. The synthesized nirB was modified by deleting the nitrite element. 2 oligos, around 100 bases, were synthesized commercially by addition of two different Enzyme sites and Kozak elements, which can enhance the antigen expression from the CMV ie promoter, after annealing, and inserted it into the pTriEx-4 plasmid by replacing its original T7 and baculovirus 10 promoters, so new plasmid pCMVnir contains 2 different promoters, humanCMV ie promoter and anaerobically-inducible promoter nirB, ampicillin resistant gene, Kozak consensus element, His-Tag purification tags for purifying antigens with His-beads, eukaryotic and prokaryotic transcriptjonal terminators, polyA signal and multiple cloning sites(MCS) for inserting other antigen genes. The pCMVnir was confirmed by DNA sequencing. In order to study the ability of these promoters, two different reporter gene EGFP-N1 and Ds-Red were inserted into the pCMVnir by two restriction enzymes BamHI and Not I, separately, pCN-EGFP and pCN-Ds-Red two reporter plasmids were used to study the activity of the CMV and nirB promoters. The competent Attenuated Salmonella strain SL3261 were transfected with these plasmids by using normal CaC12 method, Salmonella/pCN-EGFP and Salmonella/pCN-DsRed, were cultured in LB broth containing ampicillin 100 ug/ml as the following steps: inoculating one colony in 3 ml LB containing ampicillin (lOOug/ml) overnight with vigorous shaking at 37°C, then dilute 1 in 100, culture 4 hours with vigorous shaking at 37 "C, then covered the medium with mineral oil, culture overnight without shaking at 30'C . Interestingly, the fluorescent bacteria were visible by naked eyes, the bacterial pellets turned to be light red and green, it indicated that the nirB can express the reporter genes EGFP and DsRed at high level at the anaerobical microenvironment. When the bacteria were cultured on the LB plates containing ampicillin(100 ug/ml), the colonies become light red and green, the expression of the reporter genes were confirmed by the flow cyytometer detection. In order to study the HCMV ie promoter, The cervical cancer cell line HeLa and human prostate cancer cell line were transient transfected with purified plasmds pCN-EGFP and pCN-DsRed with Fugene transfection reagents, 48-72 hours posttransfection, the expression of the fluorescent proteins were checked under the fluorescent microscopy, as the data indicated the CMV ie promoter was activated in mammalian cells. Above all, the pCMVnir promoter can express antigens in vivo, it is suitable for developing intracellular bacteria-based DNA vaccine, should have the capacity to enhance the immunogenicity of DNA vaccine, because it can express the antigen genes in both intracellular bacteria and human cells.Construction and evaluation of the Dual-promoter DNA vaccine expression vector pCMVnir-16L1E7 of Papillomavirus type 16. Papillomavirus contains two late genes ( LI and L2 ORFs) and 7~8 early genes, LI protein alone, expressed in prokaryotic cells and eukaryotic cells, can self-aasemble into papillomavirus-like particles (VLP), VLP are morphologically and structurally indistinguishable from nai've virion, except lack of the oncogenic viral genome. VLP can induce specific neutralization antibodies against type-specific papillomavirus infection. VLP also can binds and resulted in mouse RBC cell hemoagglutation, and binds and enter mammalian cellsthrough papillomavirus receptor. Most papillomavirus candidate vaccines are VLP-based, for.8-example purified VLP vaccine isolated from Insect/baculovirus expression systems, In order to provide more antigenicity of the VLP antigen, LI gene was often fused with other short peptides, including E6, E7, E2 and other tumour-associated epitopes. pUC-16LlE7 plasmid was gift plasmid from Dr. Martin Muller, which contains LI and E7 genes, E7 (l~50aa) was inserted into the LI ORF at the site of 301aa, so the fusion L1-E7 gene express Ll(l~301aa)-E7(l~50aa) fusion protein. The LI gene is isolated from CA (wildtype), which does not contains the mutation at 202 aa, which was confirmed to provide much more VLP formation efficacy than LI gene isolated from human cervical cancer cell lines. SO in this study, we constructed a Salmonella-based dual-promoter DNA vaccine expression plasmid pCMVnirl611E7 of Human papillomavirus type 16. The L1E7 fusion gene was amplified by Polymerase chain Reaction, The up- and down- primers were modified by addition of different restriction Enzymes recognition sites at the 5' ends. The PCR products were inserted into pMD T-vector and then the L1E7 gene was transferred into the pTriEx-4 plasmid, which is the commercial vector from Novagen Co., The up-primer specifically binds LI gene and down-primer binds E7 gene with addition of a stop code at the end of E7(l~50aa). The annealed nirB promoter was digested with proper restriction Enzymes and then inserted into the pTriExl6LlE7 to replace its original T71ac and P10 promoters, so pCMVnirl6LlE7 was obtained, which contains the Human cytomegalovirus immediate-early promoter and in vivo inducible bacterial promoter NirB. The pCMVnirl6LlE7 was transformed into competent E.coli, to do Enzymes digestion and DNA sequencing, as the data indicated that sequences of the CMV ie promoter and nirB promoter was correct and pCMVnirl611E7 was ready for the further research as a vaccine candidate of papillomavirus. Then the pCMVnirl6LlE7 was transformed into the competent attenuated Salmonella SL3261 by normal CaC12 method, and the L1E7 was induced as the following protocol: cultivated the SL3261/pCMVnirl6LlE7 overnight at 37 degree with vigorous shaking and then diluted the cultivant 1:100, and then cultivated as above for 4 hours and at final, the bacteria were cultivated at 30 degree statically overnight in microanaerobic conditions by addition of the paraffin liquid, then harvested the bacterial pellet and the L1E7 was confirmed by the Immunoblotting with HPV16 LI specific McAb, which was able to recognize the linear epitopes of the papillomavirus LI protein. As the data showed, the antibody can bind L1E7 protein, SL3261/pCMVnirl6LlE7 can express the L1E7 successfully. SO the Salmonella-based Dual-promoter DNA vaccine of papillomavirus was able to do the further research. On the other hand, the pCMVnir promoter is idea dual-promoter vector for expressing foreign proteins, including papillomavirus L1E7 gene, Enhanced Green Fluorescence protein gene EGFP) and Red Fluorescence protein gene (DsRedN2). Mutant forms of aequorea victoria fluorescent protein (GFP) with blue, cyan, green and yellow emission are commonly used as reporter proteins for monitor ing gene expression in-Q.eukaryotic and prokaryotic cells. One of the popular reporter protein is the enhanced GFP mutant (EGFP), the EGFP has excitation and emission maxima at 489 and 508 nm, respectively. Although many GFP mutanta are commercially available, until recently, there were none with emission peak longer than 529 nm. A red fluorescent protein is desirable because a longer wavelength results in diminished nonspecific background antofluorescence. The recently cloned red fluorescent protein drFP583 (pDsRed) form discosoma coral, is the first red fluorescent protein, encoded the wild-type DsRedprotein, which has excitation and emission peak at 558 and 583 nm, respectively. Although Flow Cytometry is considerable mainly to be a technique for the analysis of mammalian cells, it is alos a powerful tool for microbiologists to gather information, even though bacterial cells are in the lower end of the detection capabilities of most Flow Cytometers, it is usually possible to analysize bacteria with standard flow cytometers. (Maksimow M, et al. Simultaneous detection of bacteria expressioning gfp and dsred genes with a flow cytometer. Cytometry 2002;47:243-247).Construction of HPV16 vaccine expression plasmids: pNir!6LlE7, pNir!6LlE7-CTA2B and pCMV-16L!E7 with the in vivo-inducible (IVD nirB promoter and CMVie promoter separately: About papillomavirus vaccine researches, Now there are several different candidate vaccines, but most of them are VLP-based with different expression systems, including baculovirus/insect system and recombinant replication virus expression systems, for example Adenovirus type5 and Poxvirus, recombinant bacterial vaccines expressing papillomavirus LI gene and DNA vaccines, because the last two expression system are cost-effective and easy to produce and store, they are suitable for the developing countries, which account up the most cervical cancer populations. On the other hand, The LI protein isolated from bacteria (E.coli and Salmonella) is able to self-assemble into virus-like particles and pentric of VLP, which can induce neutralizing antibodies against papillomavirus infections. So in this study, several different papillomavirus vaccine expression plasmids were constructed, including recombinant bacterial vaccine vectors: pNir-16L1E7, pNir-16LlE7-CTA2B, which were under the control of in-vivo bacterial promoter nirB, pNir-16LlE7-CTA2B carried the L1E7 and CTA2B fusion gene, which can express L1E7CTA2 fusion and CTB protein separately, after expression in bacteria, these two pepitides can form a protein complex: L1E7CTA2-5B and normal DNA vaccine of papillomavirus, pCMV-16LlE7, which was driven by the Human Cytomegalovirus immediate-early promoter, and the pCMVnir-16L1E7, which was transcript by the HCMV ie promoter and in-vivo inducible promoter nirB. In the other hand, these vaccines expression plasmids are control group of the Dual-promoter expression vector pCMVnir-16LlE7. pTriExl6LlE7 was constructed by inserting the HPV16 L1E7 gene into pTriEx-4 plasmid, the primers for amplifying the HPPV16L1E7 gene frompUC16LlE7 was designed by adding the Nco I and Xho I restriction enzymes sites at the ends of the up- and down- primers, after PCR amplifying, the L1E7 gene fragments were cloned into the pMD-T vector, a T-A cloning vector from Takara co., and then transferred the L1E7 DNA fragments from pMD-T vector to pTriEx-4 plasmid, so the L1E7. genes were under the control of the HCMV ie promoter, bacterial IPTG-inducible T7 promoter and baculovirus P10 promoter. pET-LlE7-CTA2B, is derived from pUAB024 containing the mucosal immune adjuvant cholera CTA2 subunit and B subunit( CTA2B) under the single T7 promoter, pUAB024 is a gift plasmid from Dr. Michael at University of Alabama at Birmingham, New primers were designed for amplification of the HPV16 L1E7 DNA fragments from the first ATG start code to the 50th amino acid without stop code, the L1E7 DNA fragments were inserted into upstream of the CTA2B at pET-CTA2B by the immediate plasmid pMD-T, so the T7 promoter of pET can express the L1E7-CTA2B as a fusion pattern, so the L1E7 protein was fused with the mucosal adjuvant CTA2B, it was expected to boost the immune reactions compared with L1E7 protein alone. pNirLlE7-CTA2B was derived from the above plasmid, new primers was designed to amplify the L1E7-CTA2B DNA fragments by modifying the primer with two restriction enzyme sites Apal and Nhel, and constructed the pMD-16LlE7-CTA2B, and then transferred to pTET15 plasmid by digestion the plasmid with Apal and Nhel restriction enzymes. pTET15 is a gift plasmid from Dunstan SJ at Impereal College in UK, the pTET15 contains the nir B promoter, which s a modified version of native nirB promoter of E. coli nitrite reductase gene, at pNir-16L1E7-CTA2B plasmid, the L1E7-CTA2B was under the control of nir B promoter, it s suitable for expression of the antigen genes in vivo. pNir-16LlE7 is a control plasmid for confirming the CTA2B immune enhancing ability, the pNirl6LlE7 was constructed by the following protocols, the pNirl6LlE7-CTA2B was digested with Xhol and Nhel restriction enzymes, the cut plasmid was filled-in with larger fragment of Klenow polymerase, and then the DNA fragments were ligated with T4 DNA ligase, after ligating a new in-frame stop code appeared just downstream of L1E7 DNA fragment, so the pNirl6LlE7 can express the L1E7 protein properly. These plasmids are good controls for studying the advantages of the pCMVnirl6LlE7 DNA vaccine. pCMV-16L1E7 was constructed by inserting the HPV16 L1E7 gene into pcDNA3.1( a commercial vector from Clontech), the L1E7 gene was amplified by the primers with Bglll restriction enzyme recognition sites at both ends of the primers, these primers can bind the L1ORF both ends, so it can amplify the L1E7 gene completely, because there is stop code in the end of E7(l~50aa), so it can only encode Ll(l~301aa)-E7(l~50aa), because the L1E7 gene was under the control of HCMV ie promoter, so the plasmid pcDNA-16LlE7 was renamed as pCMV-16L1E7.Construction and expression of recombinant Adenovirus of HPV16 L1E7 fusion gene rAd5-16L!E7 in 293 cells: Human papillomaviruses can not be produced in vitro at large quantity now, the complete virion and the capsid protein were not detected in the proliferating cells and the cervical cancer cells, HPV also has tumorgeniocity, These properties of HPVs severely hampered the vaccine developing of human papillomavirus. Current researches indicated that Papillomavirus-like particle, especially chimeric papillomavirus-like particle (cVLP) is a promising prophylactic vaccine candidate to prevent and treat human papillomavirus infection and HPV associated epithelial cancer. The adenovirus expression system is the ideal gene transfer vector with many advantages at present. In order to develop an attenuated recombinant adenoviral vaccine against HPV infection and human cervical cancer, the recombinant adenoviral vaccine of HPV should express HPVL1/E7 at high level. The L1E7 gene was cut from pUC-1611E7 plasmid and transferred into adenoviral shuttle plasmid pCA14, and co-transfecteded 293 cells, harvest the recombinant adenovirus of HPV16. The expression level and the chimeric virus-like particle were detected using ELISA immunoblot assay and the electron microscopy. Result: The fusion L1-E7 protein was expressed at the high level and can self assembled into the chimeric virus-like particle, which is morphologically indistinguishable from the naitive virions and display the conformational epitopes. We constructed the L1E7 gene, developed the L1E7 protein as the antigen for ELISA assay, pQE-16LlE7 was transformed into E.coli M15, can express the L1E7 fusion protein in E.coli at high level, the L1E7 takes up above 25% of total cell protein, the data indicated that the fusion L1E7 can be candidate gene for further research, the expressed L1E7 protein can be the antigen for ELISA assay.The colonization kinetics and stability of recombinant Salmonella and pCMVnir!6LlE7 in vivo by inoculating through oral, nosal and transcutaneous pathways: According to the documents published, the problem of the intracellular bacteria-based DNA vaccine is that the plasmids and attenuated bacteria are not stable, The plasmid stability maybe depends on both the type of the promoter and the individual inserted genes. There are controversial data about the immunogenicity of salmonella-based DNA vaccine, Jalil found that the immunogenicity of the attenuated salmonella vaccine strains does not correlate with either the number of persisting bacteria after immunization or the levels of in vitro expression of antigen carried. But we think the longer the bacteria/plasmid persist in vivo, the stronger the immune response the vaccine induced. We checked the stability of the Salmonella SL3261/pCMVnirl6LlE7 system. The Balb/c mice were inoculated the anaerobically-induced SL3261/pCMVnirl6LlE7 (108 pfu) through the oral pathway, the nosal pathway and transcutaneous pathway (TEI), because thenasal and oral pathways are well known, but the TCI is a new immunization pathway, so we tried this way, The skin immune system has recently been recognized as a highly attractive target for vaccine delivery. This is in part driven by characterization of skin immune mechanisms and in part by renewed interest in needle-free vaecine delivery strategies. Transcutaneous immunization (TCI) is a novel, needle-free approach to immunization using a patch or similar means to deliver vaccine antigens with accompanying adjuvants via the skin, and it is in clinical evaluation. The immunostimulation achieved by the use of adjuvants in the skin appears to take advantage of the potent antigen-presenting cells (APCs) in the epidermis, Langerhans cells (LCs), which in combination with topical use of the most powerful adjuvants lead to strong systemic immune responses to vaccines. And after the inoculation, the mice were sacrified at week 3, week 4, week 5 and week 6, the live, spleen, lymphoid nodes arid Peyer's Patch were harvested and homogenized with PBS buffer, and spread onto the LB plates with ampicillin (lOOug/ml) and cultivated overnight, then the number of the colonies was counted. After that randomly picked up several colonies from each plate and cultivated in LB broth containing ampicillin (lOOug/ml) and the plasmids were extracted by Qiagen miniprep plasmid kit and the DNA was run on the Agar gel and was confirmed by digesting with proper restriction enzymes. As the data indicated, the salmonella S13261/pCMVnirl6LlE7 can last in vivo for up to 6 weeks, the recombinant salmonella can enter and grow in vivo, even inoculated through the different pathways. The number of the salmonella colonies reached the peak at around week 4 and week 5, and after that the recombinant Salmonella declined quickly, maybe because the salmonella strains are attenuated and can be cleared because of lack of enough nutrition. The plasmids can be extracted from the recombinant salmonella isolated from mice organs and tissues, there was no significant difference between the bacterial from in vivo and in vitro cultivated bacteria. The data indicated that the nirB promoter in pCMVnir plasmid can regulated by the in vivo mechanisms and the high expression of the antigens from the nirB promoter does not result in the instability of the pCMVnir and recombinant SL3261, unlike other bacterial plasmids.The mucosal immune reaction induced by the Salmonella-based papillomavirus vaccines: pCMVnir-16L1E7, pNir-16LlE7/CTA2B and pCMV-16LlE7: in order to evaluate the advantages of the Salmonella-based papillomavirus pCMVnirl611E7 vaccine, the HPV16L1E7 were regulated under the control of the NirB and/or CMVie promoters, pNirl6LlE7 could express HPV16L1E7 under the nirB promoter only, the pCMV-16LlE7 could express HPV16L1E7 under the control of CMVie promoter only and pCMVnirl6LlE7 could express HPV16L1E7 under the control of CMVie and nirB promoters. The attenuated salmonella SL3261 was transformed with pNirl6L!E7, pCMV-16L!E7 and pCMVnir!6LlE7 separately,... |
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