Abstract：
BACKGROUND: Human Papillomavirus type 18 (HPV18) is a high-risk HPV that is commonly associated with cervical cancer. HPV18 oncogenes E6 and E7 are associated with the malignant transformation of cells, thus the identification of human leukocyte antigen (HLA)-restricted E6/E7 peptide-specific CD8 + T cell epitopes and the creation of a HPV18 E6/E7 expressing cervicovaginal tumor in HLA-A2 transgenic mice will be significant for vaccine development.
METHODS: In the below study, we characterized various human HLA class I-restricted HPV18 E6 and E7-specific CD8 + T cells mediated immune responses in HLA class I transgenic mice using DNA vaccines encoding HPV18E6 and HPV18E7. We then confirmed HLA-restricted E6/E7 specific CD8 + T cell epitopes using splenocytes from vaccinated mice stimulated with HPV18E6/E7 peptides. Furthermore, we used oncogenic DNA plasmids encoding HPV18E7E6(delD70), luciferase, cMyc, and AKT to create a spontaneous cervicovaginal carcinoma model in HLA-A2 transgenic mice.
RESULTS: Therapeutic HPV18 E7 DNA vaccination did not elicit any significant CD8 + T cell response in HLA-A1, HLA-24, HLA-B7, HLA-B44 transgenic or wild type C57BL/6 mice, but it did generate a strong HLA-A2 and HLA-A11 restricted HPV18E7-specific CD8 + T cell immune response. We found that a single deletion of aspartic acid (D) at location 70 in HPV18E6 DNA abolishes the presentation of HPV18 E6 peptide (aa67-75) by murine MHC class I. We found that the DNA vaccine with this mutant HPV18 E6 generated E6-specific CD8 + T cells in HLA-A2. HLA-A11, HLA-A24 and HLA-b40 transgenic mice. Of note, HLA-A2 restricted, HPV18 E7 peptide (aa7-15)- and HPV18 E6 peptide (aa97-105)-specific epitopes are endogenously processed by HPV18 positive Hela-AAD (HLA-A*0201/Dd) cells. Finally, we found that injection of DNA plasmids encoding HPV18E7E6(delD70), AKT, cMyc, and SB100 can result in the development of adenosquamous carcinoma in the cervicovaginal tract of HLA-A2 transgenic mice.
CONCLUSIONS: We characterized various human HLA class I-restricted HPV18 E6/E7 peptide specific CD8 + T cell epitopes in human HLA class I transgenic mice. We demonstrated that HPV18 positive Hela cells expressing chimeric HLA-A2 (AAD) do present both HLA-A2-restricted HPV18 E7 (aa7-15)- and HPV18 E6 (aa97-105)-specific CD8 + T cell epitopes. A mutant HPV18E6 that had a single deletion at location 70 obliterates the E6 presentation by murine MHC class I and remains oncogenic. The identification of these human MHC restricted HPV antigen specific epitopes as well as the HPV18E6/E7 expressing adenosquamous cell carcinoma model may have significant future translational potential.
METHODS: In the below study, we characterized various human HLA class I-restricted HPV18 E6 and E7-specific CD8 + T cells mediated immune responses in HLA class I transgenic mice using DNA vaccines encoding HPV18E6 and HPV18E7. We then confirmed HLA-restricted E6/E7 specific CD8 + T cell epitopes using splenocytes from vaccinated mice stimulated with HPV18E6/E7 peptides. Furthermore, we used oncogenic DNA plasmids encoding HPV18E7E6(delD70), luciferase, cMyc, and AKT to create a spontaneous cervicovaginal carcinoma model in HLA-A2 transgenic mice.
RESULTS: Therapeutic HPV18 E7 DNA vaccination did not elicit any significant CD8 + T cell response in HLA-A1, HLA-24, HLA-B7, HLA-B44 transgenic or wild type C57BL/6 mice, but it did generate a strong HLA-A2 and HLA-A11 restricted HPV18E7-specific CD8 + T cell immune response. We found that a single deletion of aspartic acid (D) at location 70 in HPV18E6 DNA abolishes the presentation of HPV18 E6 peptide (aa67-75) by murine MHC class I. We found that the DNA vaccine with this mutant HPV18 E6 generated E6-specific CD8 + T cells in HLA-A2. HLA-A11, HLA-A24 and HLA-b40 transgenic mice. Of note, HLA-A2 restricted, HPV18 E7 peptide (aa7-15)- and HPV18 E6 peptide (aa97-105)-specific epitopes are endogenously processed by HPV18 positive Hela-AAD (HLA-A*0201/Dd) cells. Finally, we found that injection of DNA plasmids encoding HPV18E7E6(delD70), AKT, cMyc, and SB100 can result in the development of adenosquamous carcinoma in the cervicovaginal tract of HLA-A2 transgenic mice.
CONCLUSIONS: We characterized various human HLA class I-restricted HPV18 E6/E7 peptide specific CD8 + T cell epitopes in human HLA class I transgenic mice. We demonstrated that HPV18 positive Hela cells expressing chimeric HLA-A2 (AAD) do present both HLA-A2-restricted HPV18 E7 (aa7-15)- and HPV18 E6 (aa97-105)-specific CD8 + T cell epitopes. A mutant HPV18E6 that had a single deletion at location 70 obliterates the E6 presentation by murine MHC class I and remains oncogenic. The identification of these human MHC restricted HPV antigen specific epitopes as well as the HPV18E6/E7 expressing adenosquamous cell carcinoma model may have significant future translational potential.
摘要：
背景：人乳头瘤病毒18型（HPV18）是一种高危HPV，通常与宫颈癌相关。HPV18癌基因E6和E7与细胞的恶性转化有关，因此,在HLA-A2转基因小鼠中鉴定人白细胞抗原(HLA)限制性E6/E7肽特异性CD8+T细胞表位和产生表达HPV18E6/E7的宫颈阴道肿瘤对于疫苗开发具有重要意义.
方法：在以下研究中，我们使用编码HPV18E6和HPV18E7的DNA疫苗在HLAI类转基因小鼠中表征了各种人HLAI类限制性HPV18E6和E7特异性CD8+T细胞介导的免疫应答。然后,我们使用来自用HPV18E6/E7肽刺激的接种小鼠的脾细胞证实了HLA限制性E6/E7特异性CD8+T细胞表位。此外，我们使用编码HPV18E7E6（delD70）的致癌DNA质粒，荧光素酶,cMyc,和AKT在HLA-A2转基因小鼠中创建自发性宫颈阴道癌模型。
结果：治疗性HPV18E7DNA疫苗接种在HLA-A1、HLA-24、HLA-B7、HLA-B44转基因或野生型C57BL/6小鼠中没有引起任何显著的CD8+T细胞反应，但它确实产生了强烈的HLA-A2和HLA-A11限制性HPV18E7特异性CD8+T细胞免疫应答。我们发现，在HPV18E6DNA中位置70处的天冬氨酸（D）的单个缺失消除了鼠I类MHC对HPV18E6肽（aa67-75）的呈递。我们发现具有该突变体HPV18E6的DNA疫苗在HLA-A2中产生了E6特异性CD8T细胞。HLA-A11、HLA-A24和HLA-b40转基因小鼠。值得注意的是，HLA-A2限制,HPV18E7肽(aa7-15)-和HPV18E6肽(aa97-105)-特异性表位由HPV18阳性Hela-AAD(HLA-A*0201/Dd)细胞内源性加工。最后,我们发现注射编码HPV18E7E6（delD70）的DNA质粒，AKT,cMyc,SB100可导致HLA-A2转基因小鼠宫颈阴道腺鳞癌的发展。
结论:我们在人I类HLA转基因小鼠中表征了各种人I类HLA限制性HPV18E6/E7肽特异性CD8+T细胞表位。我们证明了表达嵌合HLA-A2（AAD）的HPV18阳性Hela细胞确实同时存在HLA-A2限制性HPV18E7（aa7-15）和HPV18E6（aa97-105）特异性CD8T细胞表位。在位置70处具有单个缺失的突变体HPV18E6消除了鼠I类MHC的E6呈递并保持致癌。这些人MHC限制性HPV抗原特异性表位以及表达HPV18E6/E7的腺鳞状细胞癌模型的鉴定可能具有重要的未来翻译潜力。
方法：在以下研究中，我们使用编码HPV18E6和HPV18E7的DNA疫苗在HLAI类转基因小鼠中表征了各种人HLAI类限制性HPV18E6和E7特异性CD8+T细胞介导的免疫应答。然后,我们使用来自用HPV18E6/E7肽刺激的接种小鼠的脾细胞证实了HLA限制性E6/E7特异性CD8+T细胞表位。此外，我们使用编码HPV18E7E6（delD70）的致癌DNA质粒，荧光素酶,cMyc,和AKT在HLA-A2转基因小鼠中创建自发性宫颈阴道癌模型。
结果：治疗性HPV18E7DNA疫苗接种在HLA-A1、HLA-24、HLA-B7、HLA-B44转基因或野生型C57BL/6小鼠中没有引起任何显著的CD8+T细胞反应，但它确实产生了强烈的HLA-A2和HLA-A11限制性HPV18E7特异性CD8+T细胞免疫应答。我们发现，在HPV18E6DNA中位置70处的天冬氨酸（D）的单个缺失消除了鼠I类MHC对HPV18E6肽（aa67-75）的呈递。我们发现具有该突变体HPV18E6的DNA疫苗在HLA-A2中产生了E6特异性CD8T细胞。HLA-A11、HLA-A24和HLA-b40转基因小鼠。值得注意的是，HLA-A2限制,HPV18E7肽(aa7-15)-和HPV18E6肽(aa97-105)-特异性表位由HPV18阳性Hela-AAD(HLA-A*0201/Dd)细胞内源性加工。最后,我们发现注射编码HPV18E7E6（delD70）的DNA质粒，AKT,cMyc,SB100可导致HLA-A2转基因小鼠宫颈阴道腺鳞癌的发展。
结论:我们在人I类HLA转基因小鼠中表征了各种人I类HLA限制性HPV18E6/E7肽特异性CD8+T细胞表位。我们证明了表达嵌合HLA-A2（AAD）的HPV18阳性Hela细胞确实同时存在HLA-A2限制性HPV18E7（aa7-15）和HPV18E6（aa97-105）特异性CD8T细胞表位。在位置70处具有单个缺失的突变体HPV18E6消除了鼠I类MHC的E6呈递并保持致癌。这些人MHC限制性HPV抗原特异性表位以及表达HPV18E6/E7的腺鳞状细胞癌模型的鉴定可能具有重要的未来翻译潜力。
