EDA-E7 activated DCs induces specific cytotoxic T lymphocyte immune responses against HPV expressing cervical cancer in human setting

EDA-E7 activated DCs induces specific cytotoxic T lymphocyte immune responses against HPV expressing cervical cancer in human setting | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article EDA-E7 activated DCs induces specific cytotoxic T lymphocyte immune responses against HPV expressing cervical cancer in human setting Juan Feng, Yongliang Liu, Na Zhuang, Zixuan Chai, Limei Liu, Cheng Qian, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1782711/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cervical cancer is a major cause of cancer death in women worldwide. Human papillomavirus (HPV) infection especially genotypes 16 and 18 is the main factor induces cervical lesions. Targeting HPV viral oncoproteins E6 and E7 is a new strategy for cervical cancer immunotherapy and has been associated with resolution of HPV-induced lesions. How to efficiently induce T cell target killing of HPV infected cervical cancer is of great potential benefit for cervical cancer treatment. Methods Fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-E7) has been shown to efficiently induce dendritic cells maturation and trigger specific antitumor CD8 + T cells response in mouse. In this study, we constructed EDA-E7 fusion protein of human origin and tested its function in dendritic cell maturation as well as specific antitumor T cell response. Results We found that EDA-E7 could be efficiently captured by human PBMC derived dendritic cells (DCs) in vitro and induce DCs maturation. Importantly, this effect can work in synergy with the TLR ligand anti-CD40 agonist, polyinosinic-polycytidylic acid [poly (I:C)], R848 and CpG2216. EDA-E7 matured DCs could activate T cells and trigger anti-tumor response in vitro . Single RNA sequencing and T cell target killing assay confirmed the activation of T cells by EDA-E7 matured DCs. Conclusions Therapeutic vaccination with EDA-E7 fusion protein is effective for human cervical carcinoma treatment. Cervical cancer Human papillomavirus (HPV) extra domain A (EDA) Cancer immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Cervical cancer is a major cause of cancer death in women worldwide. Consistent evidence has indicated that human papillomavirus infection (HPV) is the main factor that induces cervical lesions and cervical cancer [ 3 – 5 ]. It is worth noting that while the detection rate of HPV in cervical cancer tissue is as high as 99%, the HPV genotypes 16 and 18 are 73.8% and 16.4% in Southwest of China respectively [ 6 , 7 ]. Traditional treatment methods such as surgery, radiotherapy, chemotherapy, etc. are still the preferred treatment at present for cervical cancer. However, the effect is not satisfactory for advanced stage, metastasis and recurrent cervical cancer [ 8 ]. The HPV viral oncoproteins E6 and E7 are considered tumor-specific targets for immunotherapy, making them as the most effective vaccine for cervical cancer treatment. With the development of molecular cell biology and immunology, basic study and clinical trials have been carried out to evaluate the efficiency and safety of immunotherapy in cervical cancer. In 2018, the National Comprehensive Cancer Network (NCCN) recommended Pembrolizumab, one of the immune checkpoint inhibitors targeting PD-1, as a new treatment for unsatisfactory advanced, metastatic, and recurrent cervical cancer [ 9 ]. However, the inhibitor is effective only for patients with high expression of PD-L1, while has no benefit for patients who do not express or express low level of PD-L1 [ 10 ]. Therefore, tumor vaccine to stimulate HPV antigen specific T cells for the treatment of cervical cancer was back to our attention. Ferrara et al reported a clinical trial for autologous DCs stimulated by recombinant HPV16E7 or HPV18E7 protein in the treatment of patients with advanced, metastatic, and recurrent cervical cancer, and found that the recombinant HPV E7 induced antitumor T cell responses in a portion of late stage cervical cancer patients [ 11 ]. Due to their outstanding antigen presenting ability, DCs are the key mediator of T cell immune response. They capture and process antigens in the context of major histocompatibility complex (MHC) to naïve T cells, and trigger a specific adaptive immune response. However, the ex vivo DCs-based vaccine is difficult to standardize, therefore, in vivo induction of DCs with HPV antigen as vaccine for cervical cancer has great potential for clinical application. Juan Jose´ Lasarte et, al reported that the spliced exon encoding the type III repeat extra domain A (EDA) from fibronectin, which is produced in response to tissue injury and works as a damage-associated molecular pattern molecule [ 12 ], is able to target antigens to DCs while inducing maturation through TLR4 ligation [ 13 – 15 ]. Moreover, they amplified mouse origin EDA and constructed recombinant fusion protein EDA-E7 ( HPV16E7), then evaluated the immune response in mouse condition and found that EDA-E7 could efficiently induce specific immune rejection of HPV16E7 infected TC-1 tumors[ 1 ]. In the present work, we cloned human origin EDA and successfully constructed and purified EDA-HPV16E7 (EDA-E7), a fusion protein containing human EDA and part of HPV16E7. Further, EDA-E7 and toll-like receptor (TLR) agonist were applied to induce human DCs cell maturation in vitro . We found specific activation of human T cells by EDA-E7 matured DCs, and T cells mediated cell lysis of HPV16E7 infected cervical cancer cell were also observed. Our research will fill the gap between bench study and clinical application in human for the treatment of HPV infected cervical cancer using EDA-E7 vaccine. Methods Cell culture Siha, 293(HEK-293) and THP-1 were from Shanghai Cell Collection (Shanghai, China). Siha and 293 were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Gibco). THP-1 were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. DCs culture: Human PBMC were obtained by density gradient centrifugation using Ficoll (Cytiva). PBMC were suspended in RPMI 1640 (Gibco) basic medium with concentration of 2 ×10 6 ／m L. Seed PBMC in 24 well plate and culture in 37℃, 5%CO 2 for 90 minutes. To obtain DCs, the attached mononuclear cells were cultured for additional 5 days in RPMI 1640 (Gibco) supplemented with 5% human serum, 100 U/ml penicillin and 100 µg/ml streptomycin (P/S), 2 mM L-glutamine, 800 IU/ml GM-CSF (Peprotech) and 200 U/ml IL-4 (Peprotech). For DCs maturation, 500uM EDA-E7, 500uM EDA-E7+10ug/ml poly (I:C) (Invivogen), 500uM EDA-E7+2uM CPG (CPG2216, Invivogen), 500uM EDA-E7+1ug/ml R848 (Invivogen) or 500uM EDA-E7+100ng/ml anti-CD40 (abcam) were added into DCs culture medium on day6 after DC stimulation. For lymphocyte purification, we collected the unattached cell after incubating the PBMC in 24 well plate for 90 minutes as described above, then purify T cells using human Pan T Cell Isolation Kit Miltenyi Biotec) and cultured them in lymphocyte serum-free medium (Dayou, cat#:6111021). For T cell activation, on day7 of DCs culture, 1:1 (DCs:T cells) naïve pan T cells were added into DCs cells with addition of 200U/ml IL-2, 30ng/ml IL-21, 5ng/ml IL-15 and 5ng/ml IL7, then culture in 37℃, 5%CO 2 for 10 days before analysis. Human recombinant EDA-E7 fusion protein preparation RNA from Siha was isolated with RNA isolation kit (Omega) and reverse transcript into cDNA using PrimeScript RT reagent kit from Takara. We depleted PRb binding dormain of HPVE7 and link E7 1-29 first amino acids (aa) to the N terminal of EDA and 43-98 aa to the C terminus of EDA to make target gene: E7(1-29)aa+EDA+E7(43-98)aa (EDA+E7), while control protein without EDA as: E7(1-29)aa+E7(43-98)aa (E7). To construct the fusion protein, we used Over-lap PCR method. PCR primers are shown in Table 1. Wavy line indicates the overlap sequence, Straight line indicates Ndel or NotI enzyme restriction site. To construct E7(1-29)aa+EDA target DNA, primer 1 and 2 were used and Siha cDNA was applied as template to amplify E7(1-29)aa target sequence; while primer 3 and 4 were used and 293 cells cDNA were used as template to amplify human EDA target sequence. Purify E7(1-29)aa and EDA target DNA and link them to get E7(1-29)aa +EDA target DNA. E7(1-29)aa +EDA from previous step were further amplified using primer 1 and 4. E7(43-98)aa were amplified with primer 5 and 6, then linked to E7(1-29)aa +EDA. To get E7(1-29)aa + E7(43-98)aa DNA, product of primer 1 and 7 with template of Siha cDNA, and product of primer 6 and 8 with template of EDA expression plasmid were linked together via the overlap sequence. For the construction of pET20b expressing EDA-E7 or E7, plasmid pET20b (Kindly provided by Prof. Jesu´s Prieto from Centro de Investigacio´n Me´dica Aplicada CIMA, Pamplona, Spain) and previously obtained target DNA were digested with restriction enzyme NdeI and NotI, and then ligated to construct pET20b-EDA-E7 and pET20b-E7. To obtain EDA-E7 or E7 recombinant protein, pET20b-EDA-HPVE7 and pET20b-E7 were transfected into BL21 (DE3) E-coli; incubate in 37℃, 260r/min shaking culture. Add 0.5mM IPTG (isopropylthio-β-galactoside, purchased from Thermo Fisher Scientific) to the culture when the BL21 OD600 reached 0.5, then culture another 4h before protein purification. SDS-PAGE and coomassie brilliant blue staining were applied to confirm the target protein size as well as purity. Recombinant protein were further purified with affinity chromatography as described previously [1]. As the recombinant exist mainly in inclusion body, protein renaturation using gentle removal of urea were applied as described previously[2]. DCs endocytosis assay E7 and EDA-E7 were labeled with LinKine™ FITC Labeling Kit (Abbkine), then added into DCs culture as described previously. DCs endocytosis were measured through GFP signal by fluorescent microscope and flow cytometry after 24 hours incubation. Flow cytometry For surface staining, cells were blocked with 2% normal rabbit serum and subsequently stained with fluorochrome-conjugated antibodies in FACS buffer (PBS+2%FBS+P/S) at 4°C for 30 minutes and analyzed with cytometer(Beckman coulter cytoflex). DCs activation were analyzed using anti-HLA-DR(Biolegend), anti –HLA-ABC (BD Biosciences), anti-CD80(Biolegend), anti-CD83(Biolegend), anti-CD86(Biolegend). For T cells activation analysis, DCs activated T cells were collected by centrifuge suspended pan T cells in 1000rpm for 5 min, then stained with anti-CD3(Biolegend), anti-CD4(Biolegend), anti-CD8(Biolegend), anti-CD107a(Biolegend), anti-4-1BB (Biolegend) and anti-OX40 (Biolegend) for flow analyze. Intracellular staining for DCs IL12, TNFα: activated DCs were washed, fixed and permeablilized using BD Cytofix/Cytoperm kit at 4°C for 20 minutes. The cells were then stained with anti-IL-12(Biolegend) and anti-TNFα (Biolegend) in permeabilization solution following the protocol provided by the kit. Data were acquired on Beckman coulter cytoflex. For caspase-3 staining, target cell Siha were washed after coculture with T cells to remove T cells, then trypsin (Gibco) digested to single cells. Fix and permeabilized as described previously using BD cytofix/Cytoperm kit, and stain anti-cleaved caspase-3 (BD Bioscience) before analysis with cytometer. Monocyte activation analysis THP-1 cells were cultured in 12 well plate and treated with different doses of EDA-E7 and cultured in 37℃, 5% CO 2 for 10 minutes. Cells were collected and washed with PBS, then lysed with RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Roche). Purified and degenerated proteins were loaded onto 12% SDS-PAGE gels followed by electrophoretic transfer to nitrocellulose membranes. Primary antibodies for p65, P-p65 were purchased from abcam; anti-β-actin antibody were from CST company. T-cell receptor (TCR) coupled single cell RNA sequencing Collect T cells on day7 of T cells activation by EDA-E7 matured DCs for TCR coupled single cell RNA sequencing. Sequencing was completed by Beijing Genomics Institute (Beijing, China). To identify clonotypes, we used a 10×Genomics Cell Ranger pipeline with alignment and annotation according to the manufacturer’s instruction. TCR were aligned to GRCh38 reference genome. In-frame TCR alpha-beta pairs were considered as dominant TCR of a single cell. Statistical analysis Data are presented as mean±SEM. Statistical comparisons between groups were analyzed by a Student test. A p value < 0.05 was considered statistically significant. Results Recombinant fusion protein EDA-E7 activates TLR4 signaling pathway and stimulate DCs maturation EDA from fibronectin could activate TLR-4 signaling pathway of dendritic cells (DCs) and fusion protein EDA-OVA, EDA-E7 could stimulate specific CTL killing of OVA expression tumor cells and HPV-E7 infected tumor cell accordingly in mouse [13]. Nevertheless, whether EDA-E7 could be used for activation of DCs and trigger antigen specific Cytotoxic T lymphocyte (CTL) killing in human setting is not known. To address this question, we amplified human EDA from human 293 cells [1], and constructed recombinant fusion protein of EDA-HPV16E7 (EDA-E7) expressing plasmid with his tag as shown in Fig1A. The protein sequence of EDA-E7 and control E7 was shown in FigS1A. EDA-E7 and control protein E7 were purified using anti-histidine antibodies. Protein purity was confirmed by SDS-PAGE stained with coomassie brilliant blue (FigS1B). As human monocyte cell line THP-1 cells express TLR4, we checked whether EDA-E7 could activate THP-1 TLR4 signaling pathway. Signaling through canonical TLR4 leads to phosphorylation of p65, one component of the NF-KB complex [16]. Therefore, we applied western blotting to check the phosphorylation of p65, and found that p65 phosphorylation was upregulated upon EDA-E7 treatment in a dose dependent manner (Fig1A and FigS2). To study whether EDA-E7 could activate DCs. We firstly checked whether EDA-E7 recombinant protein could be captured by DCs. E7 and EDA-E7 were labeled with LinKine™ FITC Labeling Kit (Abbkine), then co-cultured with DCs, which were purified from human PBMC. FITC positive DCs were observed in both E7 and EDA-E7 treated group as shown in Fig1B. Flow cytometry confirmed the binding of EDA-E7 to DCs with higher efficiency than E7 alone (Fig1B). Maturation of DCs upregulates the expression of cell surface MHC genes, co-stimulatory molecules as well as pro-inflammatory cytokines such as TNFα and IL12[17]. Therefore, to functionally demonstrate the role of EDA-E7 on DCs activation, we evaluated the activation marker of DCs cells with flow cytometry for HLA-DR, HLA-ABC as well as co-stimulatory molecules CD80, CD83 and CD86. The results showed that EDA-E7 upregulate both the MHC proteins and the costimulatory molecules with much better efficiency than E7 alone (Fig1C). 500uM EDA-E7 has the best effect to activate DCs in vitro Even though previous data in THP-1 cell showed a dose dependent manner for EDA-E7 on TLR4 pathway activation from 0uM to 0.1uM, we asked whether higher dose of EDA-E7 works more efficient for DCs activation in vitro. We tried 100uM, 200uM, 500uM, 800uM and 1000uM for DCs activation and used flow cytometry for MHC molecule and costimulatory molecule as activation marker. Mean flow index data showed that from 0uM to 500uM, activation effect of EDA-E7 on DCs increased with dosage. However, after 500uM, higher concentration of EDA-E7 does not achieve better activation (Fig2). We conclude that 500uM EDA-E7 is the best concentration for DCs activation in vitro . Therefore, we will use 500uM EDA-E7 in this study unless otherwise mentioned. Combined use of TLR activator upregulated EDA-E7 effect on DCs activation Previous studies have shown that TLR activators anti-CD40 agonist, poly (I:C), R848 and CpG2216 has the potential to stimulate the activation of DCs [1]. We asked whether combined use of anti-CD40 agonist, poly (I:C), R848 and CpG2216 with EDA-E7 would work in synergy to stimulate DCs maturation. We treated DCs with EDA-E7, EDA-E7+anti-CD40, EDA-E7+poly (I:C), EDA-E7+R848, EDA-E7+CpG2216 and E7 as control. Flow cytometry was applied to evaluate the activation markers of DCs including antigen presenting molecule as well as costimulatory molecules. From the results we found that EDA-E7+anti-CD40 does not show better effect compared with EDA-E7, however, TLR4 activator poly (I:C), R848 and CpG2216 indeed upregulated the efficiency of EDA-E7 to stimulate DCs (Fig3A,3B). In addition to antigen presenting molecule as well as costimulatory molecules, we also checked the expression of pro-inflammatory cytokine IL-12 and TNFα in the DCs with flow cytometry. In accordance with the expression of antigen presenting molecule and costimulatory pathway, IL12 and TNFα expression by DCs were also elevated after stimulation with EDA-E7. Importantly, this effect was further upregulated by combination use of TLR4 activators poly (I:C), E7+R848, CpG2216. Interestingly, when combined use of EDA-E7 with anti-CD40 agonist, TNFα also showed elevated expression even though no significant upregulation of IL12, MHC molecules or costimulatory molecules (Fig3C,3D). EDA-E7 matured DCs could activate T cells in vitro As a specialized antigen-presenting cell, DCs are the key mediator of T cell immune response. They capture, ingest and process related antigens, then present the antigen to naive T cells and trigger a specific immune response. We thus asked whether EDA-E7 activated DCs could activate T cells in vitro . We treat DCs with E7, EDA-E7 or EDA-E7 in combination with anti-CD40, poly (I:C), E7+R848, CpG2216, then co-culture the DCs with human T cells purified from PBMC. OX40 and 4-1BB were used as CD4+T cell activation markers while CD107 and 4-1BB were used as CD8+T cell activation markers as reported previously[18]. We firstly analyzed the proliferation of T cells, which could indicate the activation of T cells. From the data, we found that even we seed the same number of naïve T cells before activation, cell number increased after co-culture with EDA-E7stimulated DCs compared with E7 stimulated group on day 10 post activation. Moreover, combined use of EDA-E7 with TLR activators increased the proliferation of T cells compared to EDA-E7 used alone (Fig4A). On day10 post activation, we collected the T cells for the analysis of activation. Using flow cytometry we found that compared to E7 stimulated DCs, activation efficiency of EDA-E7 matured DCs on T cells was indeed upregulated. Poly (I:C) treated group further increased CD4+T cells activation percentage from 11.8% to 13.4%; while anti-CD40, poly(I:C) and R848 treated group increased CD8+T cells activation percentage from 2.78% to 3.91, 5.78 and 8.78 respectively. These data indicates that EDA-E7 treated DCs activate T cells more efficiently than E7, and combination of EDA-E7 with the TLR3 ligand poly (I:C), which promotes T cells proliferation and survival through the production of type I IFN[19, 20], has the best efficiency to further improve T cell activation (Fig4B, 4C ). TCR coupled single cell RNA sequencing revealed TCR enrichment and cytotoxic property of T cells after co-culture with EDA-E7 activated DCs To determine that whether there is clonal selection and amplification of T cells after DCs stimulation, we analyzed the results from TCR coupled single cell sequencing for total T cells after stimulation with EDA-E7 matured DCs. From Fig5A we could see that each cluster was composed of different combinatorial subsets of the clonotypes. Clonal expansion was observed with clonal sizes ranging from 1 to 765 (Fig5B). CD8+T cells had more clonal cells than CD4+T cells and naïve CD4 T and CD8 T displayed very limited clonal expansion (data not shown). Cytotoxic clonetype1 which expressed high level of granzyme A, B, IFNG et, al showed higher expansion than other clonotypes (Fig5B). Pseudotime analysis indicate clonotype1 emerged as the earliest T cells clonotype activated by DCs (Fig5C). Top 10 frequency expanded clonotypes all showed high expression of GZMA, GZMB,IFNG,TNF,LAMP1 (Fig5D). To analyze the function of clonotypes, we used GO enrichment analysis to identify pathways that have been enriched in the T cells after stimulation. The results indicated biological process (BP) especially immune response related pathway were enriched in the T cells. For cellular components (CC) analysis we found extracellular components ranked most significantly upregulated, indicating immune related cytokines may be elevated in the activated T cells. Molecular function (MF) analysis found that cytokines activity was upregulated most significantly which is in accordance with BP and CC results (Fig5E). T cells activated by EDA-E7 matured DCs efficiently kills HPV16E7 infected SiHa Since we have shown that EDA-E7 could stimulate DCs, and DCs would present E7 antigen to activate naive T cells. We asked whether the activated T cells could specifically target HPV16E7 infected cancer cells. EDA-E7 stimulated DCs were co-cultured with naïve T cells, then activated T cells were purified and co-cultured with HPV infected cervical cancer cell line SiHa. We tried effector T cells to target cell ratio (E:T) as 1:1, 5:1, 10:1 and analyzed the lysis efficiency at 12h and 24h post co-culture. As target cells SiHa were labeled with luciferase, we could use luciferase signal to determine the lysis percentage. From the data we can see that as early as 12h, T cells start to lysis the target cells, and EDA-E7 stimulated T cells has significantly better efficiency compared with E7 alone control group for all E:T ratio groups (Fig6A). We then used flow cytometry for caspase-3, which is the marker for apoptotic cells to further confirm the anti-tumor effect of the T cells and also found that EDA-E7 matured DCs activated T cells has better lytic efficiency compared to E7 stimulated alone (Fig6B). Discussion Cervical cancer is one of the main malignant tumors that endanger the health of women worldwide. The fact that 99% of cervical cancer was positive for HPV while type 16 HPV in Southwest of China is as high as 73.8% [ 7 , 8 ] makes the vaccination of cervical cancer via HPV possible. Compared to checkpoint inhibitor immunotherapy such as anti-PD-1 or anti-PD-L1 therapy, which relies largely on the expression of PD-L1 in cancer cells, vaccination with HPV antigen seems more promising. Indeed, preventive vaccine against HPV has already shown great potential to prevent 90% occurance of cervical cancer [ 21 ]. However, tumor immunosuppressive microenvironment including the recruitment of regulatory T cells and myeloid derived suppressor cells makes the immune response not efficient for therapeutic vaccination for established tumor. In addition, in vitro generation of HPV vaccine is expensive, time consuming as well as difficult to standardize each batch of product. Therefore, how to generate efficient therapeutic vaccine for HPV positive cervical cancer is of great interest. The spliced exon encoding the type III repeat extra domain A(EDA) from fibronectin could target antigens to DCs and induce maturation through TLR4 [ 15 ]. Furthermore, mouse derived EDA-E7 recombinant fused protein has been shown to induce maturation of DCs and was able to eradicate well-established tumors expressing HPVE7 protein in mouse system [ 1 ]. In this study, we generated human derived EDA-HPVE7 fused protein and confirmed that this recombinant protein maintains the pro-inflammatory property of the EDA domain as well as to induce the maturation of DCs through binding of HPV16E7. From Fig. 1 B we can see that EDA-E7 could bind to DCs, upregulates antigen presenting molecules and costimulatory molecules. 500uM concentration of EDA-E7 was found to achieve the best activation of DCs (Fig. 2 ). TLR agonist was reported to work in synergy with EDA-E7 to eradicate established tumor through induction of pro-inflammatory cytokines such as TNFα and IL12. In this study, we also found that the recombinant protein EDA-E7 and TLR agonist could work in synergy to promote the secretion of cytokines from DCs to achieve functional maturity. Since it is difficult to use humanized mouse model to mimic human immune system for eradicating established tumor in vivo , we used in vitro experiment to evaluate whether the DCs could induce antigen specific T cells. From T cell activation marker as well as target killing experiments, we can conclude that naïve T cells were indeed activated after incubation with EDA-E7 and TLR agonist matured DCs. TCR coupled single cell RNA sequencing indicated TCR clonal selection and amplification of T cells. In vitro T cell cytotoxic experiment indicated that T cell incubated with EDA-E7 matured DCs could successfully lyse HPV infective cervical cancer cell (Fig. 6 ). In future, we will continue to use humanized mouse models to verify the effect of EDA-E7 in inducing antigen specific T cells and eradication efficiency of HPV infected cervical cancer in vivo . In conclusion, we synthesized a human origin fusion protein EDA-E7, which could induce maturation of human DCs and activate anti-HPV infected cervical cancer immune responses in vitro . Moreover, combined use with TLR agonist such as poly (I:C) will achieve better maturation of DCs and T cell activation. Our study would fill the gap between bench study and clinical application in human for the treatment of HPV infected cervical cancer using EDA-E7 vaccine. Conclusion In this study, we successfully synthesized fusion protein EDA-E7 from human fibronectin and human HPVE7, and found that EDA-E7 could be efficiently captured by human PBMC derived dendritic cells (DCs) in vitro and induce DCs maturation. Importantly, this effect can work in synergy with the TLR ligand anti-CD40 agonist, polyinosinic-polycytidylic acid [poly (I:C)], R848 and CpG2216. EDA-E7 matured DCs could activate T cells and trigger anti-tumor response in vitro . Single RNA sequencing and T cell target killing assay confirmed the activation of T cells by EDA-E7 matured DCs. These results demonstrated that therapeutic vaccination with EDA-E7 fusion protein is effective in human cervical carcinoma treatment. Abbreviations HPV Euman papillomavirus infection HPVE7 HPV oncoprotein E7 EDA Extra domain A EDA-E7 Extra domain A- HPVE7 fusion protein MHC Major histocompatibility complex DCs Dendritic cells Poly (I C):Polyinosinic-polycytidylic acid TLR Toll-like receptor TCR T-cell receptor Declarations Ethics approval and consent to participate This study was approved by Chongqing University Cancer Hospital ethics committee for the use of human PBMC. Consent for publication Not applicable Availability of data and materials The data and related materials that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests The authors indicate no potential competing of interests. Funding This work was supported by National Natural Science Foundation of China (91959206 and 82120108019),and the funder of these grants is Prof. Cheng Qian, whose contribution to this study includes designing of the research, correction of the manuscript. General project of Chongqing Natural Science Foundation (cstc2020jcyj-msxmX0086), the funder of this grant if Ms. Juan Feng and her contribution to this study includes performing the experiments and data analysis. Postdoctoral Science Fund project of Chongqing Natural Science Foundation (cstc2020jcyj-bshX0009), funder of this grant is Dr. Na Zhuang, her contribution to this study is performing the experiments and data analysis. Fundamental Research Funds for the Central Universities (2021CDJYGRH-013), funder of this grant is Prof. Jiatao Li, whose contribution to this study includes data interpretation, research design and manuscript writing. Guidance program of Chongqing Research Institutes (cstc2019jxj1130015), funder of this grant if Prof.Limei Liu, and her contribution to this study is data analysis. Authors’ contributions J.F, Y.L, N.Z, L.L performed experiments and analyzed the data; Z.C analyzed the data; J.L interpreted the data and wrote the manuscript; C.Q, J.S designed the research, interpreted the data and wrote the manuscript. Acknowledgements We thank all members of the oncology laboratory for their technical help throughout this study. We thank Prof. Jesus Prieto and Juan Jose Lasarte from Centro de Investigacio´n Me´dica Aplicada CIMA, Pamplona, Spain, for providing EDA-E7 expressing plasmid pET20b. References Mansilla C, et al. Eradication of large tumors expressing human papillomavirus E7 protein by therapeutic vaccination with E7 fused to the extra domain a from fibronectin. Int J Cancer. 2012;131(3):641–51. Maeda Y, et al. Effective renaturation of reduced lysozyme by gentle removal of urea. 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Human papillomavirus vaccine against cervical cancer: Opportunity and challenge. Cancer Lett. 2020;471:88–102. Tables Table 1 is available in the Supplementary Files section. Supplementary Files figureS1.tif Construction of EDA-E7 and control E7 fusion proteinA. Protein sequence of fusion protein EDA-E7 and E7;B. SDS-PAGE coomassie brilliant blue staining for EDA-E7 and E7. figureS2.tif Full lengths of blots for western blotting for p65, P-p65 upon stimulation with different dose of EDA-E7 protein. Table1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1782711","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":120082331,"identity":"89e80c24-a815-426f-9ab0-3c2fbf92296b","order_by":0,"name":"Juan Feng","email":"","orcid":"","institution":"Chongqing University Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Feng","suffix":""},{"id":120082332,"identity":"a685b197-e4a1-473b-ae86-9ed6cba68645","order_by":1,"name":"Yongliang Liu","email":"","orcid":"","institution":"Chongqing University Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yongliang","middleName":"","lastName":"Liu","suffix":""},{"id":120082333,"identity":"4b243e29-90f3-46d7-bbf8-371f85f02bb6","order_by":2,"name":"Na Zhuang","email":"","orcid":"","institution":"Chongqing University Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Zhuang","suffix":""},{"id":120082334,"identity":"1d474801-8656-4a8f-b5e7-c77a679070c4","order_by":3,"name":"Zixuan Chai","email":"","orcid":"","institution":"Chongqing University Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zixuan","middleName":"","lastName":"Chai","suffix":""},{"id":120082335,"identity":"7c443274-239b-4912-96da-097d93b0c976","order_by":4,"name":"Limei Liu","email":"","orcid":"","institution":"Chongqing University Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Limei","middleName":"","lastName":"Liu","suffix":""},{"id":120082336,"identity":"fbd62dee-94d0-47a8-938c-20779b55092b","order_by":5,"name":"Cheng Qian","email":"","orcid":"","institution":"Chongqing University Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Qian","suffix":""},{"id":120082337,"identity":"ebd752bd-19fc-4368-b21f-574ecd12c72f","order_by":6,"name":"Jiatao Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACxmYILcPG3tj48AORWhgbgDQPG8/hZmMJYi0Ca2GQSG8T4CFGPXM78/MHPyoYePgkH7YxSDDYyek2EHQYm2Fjzxmgw6QT2x4UMCQbmx0gqIXBsIG3Dayl3UCC4UDiNsJa2D82/gVpkTzYJsFDnBYew2awLRKMxGspnC0D8gtPIjCQDYjwi2H/8Q0f31QwyMm3H3/48EOFnRxhLQ1g6j+Ua0BAOQjIE6FmFIyCUTAKRjoAAF6bObGlznpwAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9479-1397","institution":"Chongqing University Cancer Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jiatao","middleName":"","lastName":"Li","suffix":""},{"id":120082338,"identity":"7072c266-84d7-4a06-a0cc-3546ceb46057","order_by":7,"name":"Juanjuan Shan","email":"","orcid":"","institution":"Chongqing University Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Juanjuan","middleName":"","lastName":"Shan","suffix":""}],"badges":[],"createdAt":"2022-06-22 03:35:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1782711/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1782711/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24047301,"identity":"9a1040d7-e0df-402d-bb9e-4bc7ecaba946","added_by":"auto","created_at":"2022-07-19 16:28:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":331900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecombinant fusion protein EDA-E7 activates DCs through TLR4 \u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA. Schematic diagram showing pET-20b-EDA-E7-His plasmid design, and western blotting for p65, P-p65 upon stimulation with different dose of EDA-E7 protein.\u003c/p\u003e\u003cp\u003eB. Fluorescent image and flow cytometry for endocytosis assay of E7,E7-EDA by DC cell. \u003c/p\u003e\u003cp\u003eC. Flow cytometry for DC HLA-DR HLA-ABC, CD80,CD83,CD86 expression on DCs after stimulation with EDA-E7, E7. ISO: isotype control.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1782711/v1/b7a765aa2643fe262e114bf3.png"},{"id":24048409,"identity":"16d18ac9-470d-44a5-bee3-d0745247fc7d","added_by":"auto","created_at":"2022-07-19 16:33:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":102461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDA-E7 concentration trial for DCs activation\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA. Flow cytometry for antigen presenting molecular expression on DCs after stimulation with different dose of EDA-E7.\u003c/p\u003e\u003cp\u003eB. Flow cytometry for costimulation molecule expression on DCs after EDA-E7 stimulation.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1782711/v1/43e63f0a9588ed0a1bdee635.png"},{"id":24047304,"identity":"a66805d1-559b-4f1a-aa40-b7cc5c571672","added_by":"auto","created_at":"2022-07-19 16:28:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":624419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTLR activator upregulated EDA-E7 effect on DCs activation\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA. Flow cytometry for DCs antigen presenting molecule expression after stimulation with EDA-E7 and TLR activators;\u003c/p\u003e\u003cp\u003eB. Flow cytometry for costimulatory molecule expression after stimulation with EDA-E7 and TLR activators;\u003c/p\u003e\u003cp\u003eC. Flow cytometry for DCs proinflammatory cytokines IL-12 and TNF α after stimulation.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1782711/v1/4c5627450da483357929deea.png"},{"id":24048410,"identity":"da1244cc-3517-4591-a8be-e0e0928c0eb7","added_by":"auto","created_at":"2022-07-19 16:33:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":597679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombined use of E7-EDA with TLR upregulated DC activation of CD4+T and CD8+T cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA. T cell growth curve after treatment with DCs which has been matured by EDA-E7 or combination with TLR activators.\u003c/p\u003e\u003cp\u003eB. Flow cytometry for CD4+T cells activated by DCs.\u003c/p\u003e\u003cp\u003eC. Flow cytometry for CD8+T cells activated by DCs.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1782711/v1/453a8005648497a579075bb6.png"},{"id":24047303,"identity":"f2443a49-a9e8-4a64-99d2-9f5299892fa1","added_by":"auto","created_at":"2022-07-19 16:28:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":612066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTCR coupled single cell RNA sequencing for DCs activated T cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA. Umap for TCR coupled single cell RNA sequencing showing top 20 clonetypes after activation by DCs.\u003c/p\u003e\u003cp\u003eB. Graph for top 10 clonotypes frequency.\u003c/p\u003e\u003cp\u003eC. Pseudotime data for top 10 clonotypes.\u003c/p\u003e\u003cp\u003eD. Representative inflammatory cytokines expression in top 10 clonotypes.\u003c/p\u003e\u003cp\u003eE. GO analysis showing the enriched pathways in biological process(BP), cellular component(CC) and molecular function(MF).\u0026nbsp;\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1782711/v1/f01eefb89665205dc786310d.png"},{"id":24048828,"identity":"752d990b-2d4d-402d-a97b-11d6b5a8e7b4","added_by":"auto","created_at":"2022-07-19 16:38:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":248726,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeted killing of HPV infected cancer cells by the activated T cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA. Luciferase lysis assay for T cell target killing of SiHa cells at 12h and 24 h after co-culture with different E:T ratios.\u003c/p\u003e\u003cp\u003eB. Apoptotic marker cleaved caspase3 flow cytometry for SiHa cells after co-culture with DCs activated T cells.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1782711/v1/0a9a79ba6b38243adf492996.png"},{"id":29220392,"identity":"059ec7c5-d421-418f-a889-8edd2835564a","added_by":"auto","created_at":"2022-11-18 04:30:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2339603,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1782711/v1/ff37bdd9-b281-4c9b-9b6f-15df3d31e79e.pdf"},{"id":24049254,"identity":"fc291faf-656c-43d9-9dde-86f91eed7466","added_by":"auto","created_at":"2022-07-19 16:43:35","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1525516,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of EDA-E7 and control E7 fusion protein\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA. Protein sequence of fusion protein EDA-E7 and E7;\u003c/p\u003e\u003cp\u003eB. SDS-PAGE coomassie brilliant blue staining for EDA-E7 and E7.\u003c/p\u003e","description":"","filename":"figureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-1782711/v1/3d2b6a3b16e04b0164b27ea8.tif"},{"id":24047309,"identity":"368690a7-42de-4c27-9fc6-c1bc0ab43bcb","added_by":"auto","created_at":"2022-07-19 16:28:35","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11811048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFull lengths of blots for western blotting for p65, P-p65 upon stimulation with different dose of EDA-E7 protein.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-1782711/v1/cf4ae51436897f58f6dd610c.tif"},{"id":24047302,"identity":"e8f3af34-3d08-44ef-8da6-acd17f23afe9","added_by":"auto","created_at":"2022-07-19 16:28:35","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12621,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-1782711/v1/8d349e952bddb090d281457f.docx"}],"financialInterests":"","formattedTitle":"EDA-E7 activated DCs induces specific cytotoxic T lymphocyte immune responses against HPV expressing cervical cancer in human setting","fulltext":[{"header":"Background","content":"\u003cp\u003eCervical cancer is a major cause of cancer death in women worldwide. Consistent evidence has indicated that human papillomavirus infection (HPV) is the main factor that induces cervical lesions and cervical cancer [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It is worth noting that while the detection rate of HPV in cervical cancer tissue is as high as 99%, the HPV genotypes 16 and 18 are 73.8% and 16.4% in Southwest of China respectively [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Traditional treatment methods such as surgery, radiotherapy, chemotherapy, etc. are still the preferred treatment at present for cervical cancer. However, the effect is not satisfactory for advanced stage, metastasis and recurrent cervical cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The HPV viral oncoproteins E6 and E7 are considered tumor-specific targets for immunotherapy, making them as the most effective vaccine for cervical cancer treatment.\u003c/p\u003e \u003cp\u003eWith the development of molecular cell biology and immunology, basic study and clinical trials have been carried out to evaluate the efficiency and safety of immunotherapy in cervical cancer. In 2018, the National Comprehensive Cancer Network (NCCN) recommended Pembrolizumab, one of the immune checkpoint inhibitors targeting PD-1, as a new treatment for unsatisfactory advanced, metastatic, and recurrent cervical cancer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the inhibitor is effective only for patients with high expression of PD-L1, while has no benefit for patients who do not express or express low level of PD-L1 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, tumor vaccine to stimulate HPV antigen specific T cells for the treatment of cervical cancer was back to our attention. Ferrara et al reported a clinical trial for autologous DCs stimulated by recombinant HPV16E7 or HPV18E7 protein in the treatment of patients with advanced, metastatic, and recurrent cervical cancer, and found that the recombinant HPV E7 induced antitumor T cell responses in a portion of late stage cervical cancer patients [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to their outstanding antigen presenting ability, DCs are the key mediator of T cell immune response. They capture and process antigens in the context of major histocompatibility complex (MHC) to na\u0026iuml;ve T cells, and trigger a specific adaptive immune response. However, the \u003cem\u003eex vivo\u003c/em\u003e DCs-based vaccine is difficult to standardize, therefore, in vivo induction of DCs with HPV antigen as vaccine for cervical cancer has great potential for clinical application. Juan Jose\u0026acute; Lasarte et, al reported that the spliced exon encoding the type III repeat extra domain A (EDA) from fibronectin, which is produced in response to tissue injury and works as a damage-associated molecular pattern molecule [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], is able to target antigens to DCs while inducing maturation through TLR4 ligation [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, they amplified mouse origin EDA and constructed recombinant fusion protein EDA-E7 ( HPV16E7), then evaluated the immune response in mouse condition and found that EDA-E7 could efficiently induce specific immune rejection of HPV16E7 infected TC-1 tumors[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In the present work, we cloned human origin EDA and successfully constructed and purified EDA-HPV16E7 (EDA-E7), a fusion protein containing human EDA and part of HPV16E7. Further, EDA-E7 and toll-like receptor (TLR) agonist were applied to induce human DCs cell maturation \u003cem\u003ein vitro\u003c/em\u003e. We found specific activation of human T cells by EDA-E7 matured DCs, and T cells mediated cell lysis of HPV16E7 infected cervical cancer cell were also observed. Our research will fill the gap between bench study and clinical application in human for the treatment of HPV infected cervical cancer using EDA-E7 vaccine.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSiha, 293(HEK-293) and THP-1 were from Shanghai Cell Collection (Shanghai, China). Siha and 293 were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Gibco). THP-1 were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin.\u003c/p\u003e\n\u003cp\u003eDCs culture: Human PBMC were obtained by density gradient centrifugation using Ficoll (Cytiva). PBMC were suspended in RPMI 1640 (Gibco) basic medium with concentration of 2 \u0026times;10\u003csup\u003e6\u003c/sup\u003e／m L. Seed PBMC in 24 well plate and culture in 37℃, 5%CO\u003csub\u003e2\u003c/sub\u003e for 90 minutes. To obtain DCs, the attached mononuclear cells were cultured for additional 5 days in RPMI 1640 (Gibco) supplemented with 5% human serum, 100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin (P/S), 2 mM L-glutamine, 800 IU/ml GM-CSF (Peprotech) and 200 U/ml IL-4 (Peprotech). For DCs maturation, 500uM EDA-E7, 500uM EDA-E7+10ug/ml poly (I:C) (Invivogen), 500uM EDA-E7+2uM CPG (CPG2216, Invivogen), 500uM EDA-E7+1ug/ml R848 (Invivogen) or 500uM EDA-E7+100ng/ml anti-CD40 (abcam) were added into DCs culture medium on day6 after DC stimulation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor lymphocyte purification, we collected the unattached cell after incubating the PBMC in 24 well plate for 90 minutes as described above, then purify T cells using human Pan T Cell Isolation Kit Miltenyi Biotec) and cultured them in lymphocyte serum-free medium (Dayou, cat#:6111021). For T cell activation, on day7 of DCs culture, 1:1 (DCs:T cells) na\u0026iuml;ve pan T cells were added into DCs cells with addition of 200U/ml IL-2, 30ng/ml IL-21, 5ng/ml IL-15 and 5ng/ml IL7, then culture in 37℃, 5%CO\u003csub\u003e2\u003c/sub\u003e for 10 days before analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman recombinant EDA-E7 fusion protein preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA from Siha was isolated with RNA isolation kit (Omega) and reverse transcript into cDNA using PrimeScript RT reagent kit from Takara. We depleted PRb binding dormain of HPVE7 and link E7 1-29 first amino acids (aa) to the N terminal of EDA and 43-98 aa to the C terminus of EDA to make target gene: E7(1-29)aa+EDA+E7(43-98)aa (EDA+E7), while control protein without EDA as: E7(1-29)aa+E7(43-98)aa (E7). To construct the fusion protein, we used Over-lap PCR method. PCR primers are shown in Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWavy line indicates the overlap sequence, Straight line indicates Ndel or NotI enzyme restriction site.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo construct E7(1-29)aa+EDA target DNA, primer 1 and 2 were used and Siha cDNA was applied as template to amplify E7(1-29)aa target sequence; while primer 3 and 4 were used and 293 cells cDNA were used as template to amplify human EDA target sequence. Purify E7(1-29)aa and EDA target DNA and link them to get E7(1-29)aa +EDA target DNA. E7(1-29)aa +EDA from previous step were further amplified using primer 1 and 4. E7(43-98)aa were amplified with primer 5 and 6, then linked to E7(1-29)aa +EDA. To get E7(1-29)aa + E7(43-98)aa DNA, product of primer 1 and 7 with template of Siha cDNA, and product of primer 6 and 8 with template of EDA expression plasmid were linked together via the overlap sequence. For the construction of pET20b expressing EDA-E7 or E7, plasmid pET20b (Kindly provided by Prof. Jesu\u0026acute;s Prieto from Centro de Investigacio\u0026acute;n Me\u0026acute;dica Aplicada CIMA, Pamplona, Spain) and previously obtained target DNA were digested with restriction enzyme NdeI and NotI, and then ligated to construct pET20b-EDA-E7 and pET20b-E7.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo obtain EDA-E7 or E7 recombinant protein, pET20b-EDA-HPVE7 and pET20b-E7 were transfected into BL21 (DE3) E-coli; incubate in 37℃, 260r/min shaking culture. Add 0.5mM IPTG (isopropylthio-\u0026beta;-galactoside, purchased from Thermo Fisher Scientific) to the culture when the BL21 OD600 reached 0.5, then culture another 4h before protein purification. SDS-PAGE and coomassie brilliant blue staining were applied to confirm the target protein size as well as purity. Recombinant protein were further purified with affinity chromatography as described previously [1]. As the recombinant exist mainly in inclusion body, protein renaturation using gentle removal of urea were applied as described previously[2]. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDCs endocytosis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE7 and EDA-E7 were labeled with LinKine\u0026trade; FITC Labeling Kit (Abbkine), then added into DCs culture as described previously. DCs endocytosis were measured through GFP signal by fluorescent microscope and flow cytometry after 24 hours incubation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor surface staining, cells were blocked with 2% normal rabbit serum and subsequently stained with fluorochrome-conjugated antibodies in FACS buffer (PBS+2%FBS+P/S) at\u0026nbsp;4\u0026deg;C for 30 minutes and analyzed with cytometer(Beckman coulter cytoflex).\u0026nbsp;DCs activation were analyzed using anti-HLA-DR(Biolegend), anti \u0026ndash;HLA-ABC (BD Biosciences), anti-CD80(Biolegend), anti-CD83(Biolegend), anti-CD86(Biolegend). For T cells activation analysis, DCs activated T cells were collected by centrifuge suspended pan T cells in 1000rpm for 5 min, then stained with anti-CD3(Biolegend), anti-CD4(Biolegend), anti-CD8(Biolegend), anti-CD107a(Biolegend), anti-4-1BB (Biolegend) and anti-OX40 (Biolegend) for flow analyze.\u003c/p\u003e\n\u003cp\u003eIntracellular staining for DCs IL12, TNF\u0026alpha;: activated DCs were\u0026nbsp;washed, fixed and permeablilized using BD Cytofix/Cytoperm kit at 4\u0026deg;C\u0026nbsp;for 20 minutes. The cells were then stained with anti-IL-12(Biolegend) and anti-TNF\u0026alpha; (Biolegend) in permeabilization solution following the protocol provided by the kit. Data were acquired on Beckman coulter cytoflex.\u003c/p\u003e\n\u003cp\u003eFor caspase-3 staining, target cell Siha were washed after coculture with T cells to remove T cells, then trypsin (Gibco) digested to single cells. Fix and permeabilized as described previously using BD cytofix/Cytoperm kit, and stain anti-cleaved caspase-3 (BD Bioscience) before analysis with cytometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMonocyte activation analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTHP-1 cells were cultured in 12 well plate and treated with different doses of EDA-E7 and cultured in 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e for 10 minutes. Cells were collected and washed with PBS, then lysed with RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Roche). Purified and degenerated proteins were loaded onto 12% SDS-PAGE gels followed by electrophoretic transfer to nitrocellulose membranes. Primary antibodies for p65, P-p65 were purchased from abcam; anti-\u0026beta;-actin antibody were from CST company.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT-cell receptor (TCR) coupled single cell RNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollect T cells on day7 of T cells activation by EDA-E7 matured DCs for TCR coupled single cell RNA sequencing. Sequencing was completed by Beijing Genomics Institute (Beijing, China). To identify clonotypes, we used a 10\u0026times;Genomics Cell Ranger pipeline with alignment and annotation according to the manufacturer\u0026rsquo;s instruction. TCR were aligned to GRCh38 reference genome. In-frame TCR alpha-beta pairs were considered as dominant TCR of a single cell.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean\u0026plusmn;SEM. Statistical comparisons between groups were analyzed by a Student test. A p value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eRecombinant fusion protein EDA-E7 activates TLR4 signaling pathway and stimulate DCs maturation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEDA from fibronectin could activate TLR-4 signaling pathway of dendritic cells (DCs) and fusion protein EDA-OVA, EDA-E7 could stimulate specific CTL killing of OVA expression tumor cells and HPV-E7 infected tumor cell accordingly in mouse\u0026nbsp;[13]. Nevertheless, whether EDA-E7 could be used for activation of DCs and trigger antigen specific Cytotoxic T lymphocyte (CTL) killing in human setting is not known. To address this question, we amplified human EDA from human 293 cells\u0026nbsp;[1],\u0026nbsp;and constructed recombinant fusion protein of EDA-HPV16E7 (EDA-E7) expressing plasmid with his tag as shown in Fig1A. The protein sequence of EDA-E7 and control E7 was shown in FigS1A. EDA-E7 and control protein E7 were purified using anti-histidine antibodies. Protein purity was confirmed by SDS-PAGE stained with coomassie brilliant blue (FigS1B). As human monocyte cell line THP-1 cells express TLR4, we checked whether EDA-E7 could activate THP-1 TLR4 signaling pathway. Signaling through canonical TLR4 leads to phosphorylation of p65, one component of the NF-KB complex\u0026nbsp;[16]. Therefore, we applied western blotting to check the phosphorylation of p65, and found that p65 phosphorylation was upregulated upon EDA-E7 treatment in a dose dependent manner (Fig1A and FigS2).\u003c/p\u003e\n\u003cp\u003eTo study whether EDA-E7 could activate DCs. We firstly checked whether EDA-E7 recombinant protein could be captured by DCs. E7 and EDA-E7\u0026nbsp;were labeled with LinKine\u0026trade; FITC Labeling Kit (Abbkine), then co-cultured with DCs, which were purified from human PBMC. FITC positive DCs were observed in both E7 and EDA-E7 treated group as shown in Fig1B. Flow cytometry confirmed the binding of EDA-E7 to DCs with higher efficiency than E7 alone (Fig1B). Maturation of DCs upregulates the expression of cell surface MHC genes, co-stimulatory molecules as well as pro-inflammatory cytokines such as TNF\u0026alpha; and IL12[17]. Therefore, to functionally demonstrate the role of EDA-E7 on DCs activation, we evaluated the activation marker of DCs cells with flow cytometry for HLA-DR, HLA-ABC as well as co-stimulatory molecules\u0026nbsp;CD80, CD83 and CD86. The results showed that EDA-E7 upregulate both the MHC proteins and the costimulatory molecules with much better efficiency than E7 alone (Fig1C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e500uM EDA-E7 has the best effect to activate DCs in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEven though previous data in THP-1 cell showed a dose dependent manner for EDA-E7 on TLR4 pathway activation from 0uM to 0.1uM, we asked whether higher dose of EDA-E7 works more efficient for DCs activation in vitro. We tried 100uM, 200uM, 500uM, 800uM and 1000uM for DCs activation and used flow cytometry for MHC molecule and costimulatory molecule as activation marker. Mean flow index data showed that from 0uM to 500uM, activation effect of EDA-E7 on DCs increased with dosage. However, after 500uM, higher concentration of EDA-E7 does not achieve better activation (Fig2). We conclude that 500uM EDA-E7 is the best concentration for DCs activation \u003cem\u003ein vitro\u003c/em\u003e. Therefore, we will use 500uM EDA-E7 in this study unless otherwise mentioned.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCombined use of TLR activator upregulated EDA-E7 effect on DCs activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that TLR activators anti-CD40 agonist, poly (I:C), R848 and CpG2216 has the potential to stimulate the activation of DCs\u0026nbsp;[1]. We asked whether combined use of anti-CD40 agonist, poly (I:C), R848 and CpG2216 with EDA-E7 would work in synergy to stimulate DCs maturation. We treated DCs with EDA-E7, EDA-E7+anti-CD40, EDA-E7+poly (I:C), EDA-E7+R848, EDA-E7+CpG2216 and E7 as control. Flow cytometry was applied to evaluate the activation markers of DCs including antigen presenting molecule as well as costimulatory molecules. From the results we found that EDA-E7+anti-CD40 does not show better effect compared with EDA-E7, however, TLR4 activator poly (I:C), R848 and CpG2216 indeed upregulated the efficiency of EDA-E7 to stimulate DCs (Fig3A,3B). In addition to antigen presenting molecule as well as costimulatory molecules, we also checked the expression of pro-inflammatory cytokine IL-12 and TNF\u0026alpha; in the DCs with flow cytometry. In accordance with the expression of antigen presenting molecule and costimulatory pathway, IL12 and TNF\u0026alpha; expression by DCs were also elevated after stimulation with EDA-E7. Importantly, this effect was further upregulated by combination use of TLR4 activators poly (I:C), E7+R848, CpG2216. Interestingly, when combined use of EDA-E7 with anti-CD40 agonist, TNF\u0026alpha; also showed elevated expression even though no significant upregulation of IL12, MHC molecules or costimulatory molecules (Fig3C,3D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEDA-E7 matured DCs could activate T cells in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a specialized antigen-presenting cell, DCs are the key mediator of T cell immune response. They capture, ingest and process related antigens, then present the antigen to naive T cells and trigger a specific immune response. We thus asked whether EDA-E7 activated DCs could activate T cells\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e. We treat DCs with E7, EDA-E7 or EDA-E7 in combination with anti-CD40, poly (I:C), E7+R848, CpG2216, then co-culture the DCs with human T cells purified from PBMC. OX40 and 4-1BB were used as CD4+T cell activation markers while CD107 and 4-1BB were used as CD8+T cell activation markers as reported previously[18]. We firstly analyzed the proliferation of T cells, which could indicate the activation of T cells. From the data, we found that even we seed the same number of na\u0026iuml;ve T cells before activation, cell number increased after co-culture with EDA-E7stimulated DCs compared with E7 stimulated group on day 10 post activation. Moreover, combined use of EDA-E7 with TLR activators increased the proliferation of T cells compared to EDA-E7 used alone (Fig4A). On day10 post activation, we collected the T cells for the analysis of activation. Using flow cytometry we found that compared to E7 stimulated DCs, activation efficiency of EDA-E7 matured DCs on T cells was indeed upregulated.\u0026nbsp;Poly (I:C) treated group further increased\u0026nbsp;CD4+T cells activation percentage from 11.8% to 13.4%; while anti-CD40, poly(I:C) and R848 treated group increased CD8+T cells activation percentage from 2.78% to 3.91, 5.78 and 8.78 respectively. These data indicates that EDA-E7 treated DCs activate T cells more efficiently than E7, and combination of EDA-E7 with the TLR3 ligand poly (I:C), which promotes T cells proliferation and survival through the production of type I IFN[19, 20], has the best efficiency to further improve T cell activation (Fig4B, 4C ).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTCR coupled single cell RNA sequencing revealed TCR enrichment and cytotoxic property of T cells after co-culture with EDA-E7 activated DCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine that whether there is clonal selection and amplification of T cells after DCs stimulation, we analyzed the results from TCR coupled single cell sequencing for total T cells after stimulation with EDA-E7 matured DCs. From Fig5A we could see that each cluster was composed of different combinatorial subsets of the clonotypes. Clonal expansion was observed with clonal sizes ranging from\u0026nbsp;1 to 765 (Fig5B). CD8+T cells had more clonal cells than CD4+T cells and na\u0026iuml;ve CD4 T and CD8 T displayed very limited clonal expansion (data not shown). Cytotoxic clonetype1 which expressed high level of granzyme A, B, IFNG et, al showed higher expansion than other clonotypes (Fig5B). Pseudotime analysis indicate clonotype1 emerged as the earliest T cells clonotype activated by DCs (Fig5C). Top 10 frequency expanded clonotypes all showed high expression of GZMA, GZMB,IFNG,TNF,LAMP1 (Fig5D). To analyze the function of clonotypes, we used GO enrichment analysis to identify pathways that have been enriched in the T cells after stimulation. The results indicated biological process (BP) especially immune response related pathway were enriched in the T cells. For cellular components (CC) analysis we found extracellular components ranked most significantly upregulated, indicating immune related cytokines may be elevated in the activated T cells. Molecular function (MF) analysis found that cytokines activity was upregulated most significantly which is in accordance with BP and CC results (Fig5E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT cells activated by EDA-E7 matured DCs efficiently kills HPV16E7 infected SiHa\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince we have shown that EDA-E7 could stimulate DCs, and DCs would present E7 antigen to activate naive T cells. We asked whether the activated T cells could specifically target HPV16E7 infected cancer cells. EDA-E7 stimulated DCs were co-cultured with na\u0026iuml;ve T cells, then activated T cells were purified and co-cultured with HPV infected cervical cancer cell line SiHa. We tried effector T cells to target cell ratio (E:T) as 1:1, 5:1, 10:1 and analyzed the lysis efficiency at 12h and 24h post co-culture. As target cells SiHa were labeled with luciferase, we could use luciferase signal to determine the lysis percentage. From the data we can see that as early as 12h, T cells start to lysis the target cells, and EDA-E7 stimulated T cells has significantly better efficiency compared with E7 alone control group for all E:T ratio groups (Fig6A). We then used flow cytometry for caspase-3, which is the marker for apoptotic cells to further confirm the anti-tumor effect of the T cells and also found that EDA-E7 matured DCs activated T cells has better lytic efficiency compared to E7 stimulated alone (Fig6B).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCervical cancer is one of the main malignant tumors that endanger the health of women worldwide. The fact that 99% of cervical cancer was positive for HPV while type 16 HPV in Southwest of China is as high as 73.8% [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] makes the vaccination of cervical cancer via HPV possible. Compared to checkpoint inhibitor immunotherapy such as anti-PD-1 or anti-PD-L1 therapy, which relies largely on the expression of PD-L1 in cancer cells, vaccination with HPV antigen seems more promising. Indeed, preventive vaccine against HPV has already shown great potential to prevent 90% occurance of cervical cancer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, tumor immunosuppressive microenvironment including the recruitment of regulatory T cells and myeloid derived suppressor cells makes the immune response not efficient for therapeutic vaccination for established tumor. In addition, in vitro generation of HPV vaccine is expensive, time consuming as well as difficult to standardize each batch of product. Therefore, how to generate efficient therapeutic vaccine for HPV positive cervical cancer is of great interest.\u003c/p\u003e \u003cp\u003eThe spliced exon encoding the type III repeat extra domain A(EDA) from fibronectin could target antigens to DCs and induce maturation through TLR4 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, mouse derived EDA-E7 recombinant fused protein has been shown to induce maturation of DCs and was able to eradicate well-established tumors expressing HPVE7 protein in mouse system [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In this study, we generated human derived EDA-HPVE7 fused protein and confirmed that this recombinant protein maintains the pro-inflammatory property of the EDA domain as well as to induce the maturation of DCs through binding of HPV16E7. From Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB we can see that EDA-E7 could bind to DCs, upregulates antigen presenting molecules and costimulatory molecules. 500uM concentration of EDA-E7 was found to achieve the best activation of DCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). TLR agonist was reported to work in synergy with EDA-E7 to eradicate established tumor through induction of pro-inflammatory cytokines such as TNFα and IL12. In this study, we also found that the recombinant protein EDA-E7 and TLR agonist could work in synergy to promote the secretion of cytokines from DCs to achieve functional maturity. Since it is difficult to use humanized mouse model to mimic human immune system for eradicating established tumor \u003cem\u003ein vivo\u003c/em\u003e, we used \u003cem\u003ein vitro\u003c/em\u003e experiment to evaluate whether the DCs could induce antigen specific T cells. From T cell activation marker as well as target killing experiments, we can conclude that na\u0026iuml;ve T cells were indeed activated after incubation with EDA-E7 and TLR agonist matured DCs. TCR coupled single cell RNA sequencing indicated TCR clonal selection and amplification of T cells. \u003cem\u003eIn vitro\u003c/em\u003e T cell cytotoxic experiment indicated that T cell incubated with EDA-E7 matured DCs could successfully lyse HPV infective cervical cancer cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In future, we will continue to use humanized mouse models to verify the effect of EDA-E7 in inducing antigen specific T cells and eradication efficiency of HPV infected cervical cancer \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, we synthesized a human origin fusion protein EDA-E7, which could induce maturation of human DCs and activate anti-HPV infected cervical cancer immune responses \u003cem\u003ein vitro\u003c/em\u003e. Moreover, combined use with TLR agonist such as poly (I:C) will achieve better maturation of DCs and T cell activation. Our study would fill the gap between bench study and clinical application in human for the treatment of HPV infected cervical cancer using EDA-E7 vaccine.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we successfully synthesized fusion protein EDA-E7 from human fibronectin and human HPVE7, and found that EDA-E7 could be efficiently captured by human PBMC derived dendritic cells (DCs) \u003cem\u003ein vitro\u003c/em\u003e and induce DCs maturation. Importantly, this effect can work in synergy with the TLR ligand anti-CD40 agonist, polyinosinic-polycytidylic acid [poly (I:C)], R848 and CpG2216. EDA-E7 matured DCs could activate T cells and trigger anti-tumor response \u003cem\u003ein vitro\u003c/em\u003e. Single RNA sequencing and T cell target killing assay confirmed the activation of T cells by EDA-E7 matured DCs. These results demonstrated that therapeutic vaccination with EDA-E7 fusion protein is effective in human cervical carcinoma treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEuman papillomavirus infection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPVE7\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHPV oncoprotein E7\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtra domain A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDA-E7\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtra domain A- HPVE7 fusion protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMajor histocompatibility complex\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDendritic cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePoly (I\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eC):Polyinosinic-polycytidylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTLR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eToll-like receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eT-cell receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by Chongqing University Cancer Hospital ethics committee for the use of human PBMC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eThe data and related materials that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eThe authors indicate no potential competing of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (91959206 and 82120108019),and the funder of these grants is Prof. Cheng Qian, whose contribution to this study includes designing of the research, correction of the manuscript. General project of Chongqing Natural Science Foundation (cstc2020jcyj-msxmX0086), the funder of this grant if Ms. Juan Feng and her contribution to this study includes performing the experiments and data analysis. Postdoctoral Science Fund project of Chongqing Natural Science Foundation (cstc2020jcyj-bshX0009), funder of this grant is Dr. Na Zhuang, her contribution to this study is performing the experiments and data analysis. Fundamental Research Funds for the Central Universities (2021CDJYGRH-013), funder of this grant is Prof. Jiatao Li, whose contribution to this study includes data interpretation, research design and manuscript writing. Guidance program of Chongqing Research Institutes (cstc2019jxj1130015), funder of this grant if Prof.Limei Liu, and her contribution to this study is data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.F, Y.L, N.Z, L.L performed experiments and analyzed the data; Z.C analyzed the data; J.L interpreted the data and wrote the manuscript; C.Q, J.S designed the research, interpreted the data and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all members of the oncology laboratory for their technical help throughout this study. We thank Prof. Jesus Prieto and Juan Jose Lasarte from Centro de Investigacio´n Me´dica Aplicada CIMA, Pamplona, Spain, for providing EDA-E7 expressing plasmid pET20b.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMansilla C, et al. Eradication of large tumors expressing human papillomavirus E7 protein by therapeutic vaccination with E7 fused to the extra domain a from fibronectin. Int J Cancer. 2012;131(3):641\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaeda Y, et al. Effective renaturation of reduced lysozyme by gentle removal of urea. Protein Eng. 1995;8(2):201\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBray F, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394\u0026ndash;424.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoole DN, et al. A cross-sectional study to assess HPV knowledge and HPV vaccine acceptability in Mali. PLoS ONE. 2013;8(2):e56402.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTorre LA, et al. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArbyn M, et al., \u003cem\u003eProphylactic vaccination against human papillomaviruses to prevent cervical cancer and its precursors\u003c/em\u003e. Cochrane Database Syst Rev, 2018. 5(5): p. Cd009069.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, et al. Prevalence of HPV and variation of HPV 16/HPV 18 E6/E7 genes in cervical cancer in women in South West China. J Med Virol. 2014;86(11):1926\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoh WJ, et al. Cervical Cancer, Version 3.2019, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2019;17(1):64\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMezache L, et al. Enhanced expression of PD L1 in cervical intraepithelial neoplasia and cervical cancers. 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Cancer Lett. 2020;471:88\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cervical cancer, Human papillomavirus (HPV), extra domain A (EDA), Cancer immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-1782711/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1782711/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCervical cancer is a major cause of cancer death in women worldwide. Human papillomavirus (HPV) infection especially genotypes 16 and 18 is the main factor induces cervical lesions. Targeting HPV viral oncoproteins E6 and E7 is a new strategy for cervical cancer immunotherapy and has been associated with resolution of HPV-induced lesions. How to efficiently induce T cell target killing of HPV infected cervical cancer is of great potential benefit for cervical cancer treatment.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-E7) has been shown to efficiently induce dendritic cells maturation and trigger specific antitumor CD8\u0026thinsp;+\u0026thinsp;T cells response in mouse. In this study, we constructed EDA-E7 fusion protein of human origin and tested its function in dendritic cell maturation as well as specific antitumor T cell response.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that EDA-E7 could be efficiently captured by human PBMC derived dendritic cells (DCs) \u003cem\u003ein vitro\u003c/em\u003e and induce DCs maturation. Importantly, this effect can work in synergy with the TLR ligand anti-CD40 agonist, polyinosinic-polycytidylic acid [poly (I:C)], R848 and CpG2216. EDA-E7 matured DCs could activate T cells and trigger anti-tumor response \u003cem\u003ein vitro\u003c/em\u003e. Single RNA sequencing and T cell target killing assay confirmed the activation of T cells by EDA-E7 matured DCs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTherapeutic vaccination with EDA-E7 fusion protein is effective for human cervical carcinoma treatment.\u003c/p\u003e","manuscriptTitle":"EDA-E7 activated DCs induces specific cytotoxic T lymphocyte immune responses against HPV expressing cervical cancer in human setting","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-19 16:28:33","doi":"10.21203/rs.3.rs-1782711/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d140c1ff-13f6-4794-857b-cba3d71e844d","owner":[],"postedDate":"July 19th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-11-18T04:30:21+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-19 16:28:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1782711","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1782711","identity":"rs-1782711","version":["v1"]},"buildId":"-D5TCW68w8eVRRLyjaTIo","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}