Development of a Spontaneous HPV16 E6/E7-Expressing Head and Neck Squamous Cell Carcinoma in HLA-A2 Transgenic Mice

Peng, Shiwen; Xing, Deyin; Tsai, Ya Chea; Roden, Richard B.S.; Hung, Chien-Fu; Wu, T. C.

doi:10.1128/mbio.03252-21

Cited by 4 publications

(5 citation statements)

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“…HEK 293 cells were purchased from ATCC (Manassas, VA, USA). The generation of HEK 293 cells expressing HLA-A * 0201/D d (293-AAD) has been described previously [ 31 ]. The HPV18 positive human cervical cancer cell line, Hela cell, was purchased from ATCC.…”

Section: Methodsmentioning

confidence: 99%

“…These plasmids were purchased from Addgene. The generation of Pkt2-cMyc has been described previously [ 31 ]. To generate Pkt2-Luc-T2a-HPV18E7E6(del D70), 18E6 (delD70) was first amplified via PCR using the Pkt2-LucHPV18E7E6 [ 34 ] template and the following set of primers: 5′-CTGGCTCGAGGAGGGAAGGGGAAGCCTGCT-3′, 5′-GCTCCCGGATTCTGCTGTAGAAGATACACTTGTGGCAAGCGGCG-3′, 5′-CGCCGCTTGCCACAAGTGTATCTTCTACAGCAGAATCCGGGAGC-3′, AND 5′-AAACCAGCTAGCTGGTTATTACACCTGGGTCTC-3′.…”

Section: Methodsmentioning

confidence: 99%

“…Tumor growth was monitored using bioluminescence imaging (Xenogen IVIS spectrum bioluminescence imaging series 2000, Alameda, CA, USA) and gross inspection. Tumor-bearing mice were sacrificed when either the tumor diameter was greater than 15 mm or the mouse body weight was reduced by 10% compared with age-matched untreated control mice as described previously [ 31 ].…”

Section: Methodsmentioning

confidence: 99%

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Identification of human MHC-I HPV18 E6/E7-specific CD8 + T cell epitopes and generation of an HPV18 E6/E7-expressing adenosquamous carcinoma in HLA-A2 transgenic mice

et al. 2022

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Identification of human MHC-I HPV18 E6/E7-specific CD8 + T cell epitopes and generation of an HPV18 E6/E7-expressing adenosquamous carcinoma in HLA-A2 transgenic mice

et al. 2022

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“…In particular, we evaluated the binding of the immunodominant epitope corresponding to the region 49-57 of the E7 protein of the Human Papilloma Virus (HPV) to H2-D b , a class I MHC [14]. Notably, a mutation in this antigen (N53S) abolished the presentation of the murine H2-D b -restricted HPV16 E7 peptide in a head and neck squamous cell carcinoma mouse model [15]. Moreover, it has been shown that the same mutation eliminates the immunogenicity of E7 and is responsible for the evasion of the mutated TC-1 clones from the E7-specific immune responses induced by vaccination [16].…”

Section: Introductionmentioning

confidence: 99%

Structural and Dynamic-Based Characterization of the Recognition Patterns of E7 and TRP-2 Epitopes by MHC Class I Receptors through Computational Approaches

Balasco,

Tagliamonte,

Buonaguro

et al. 2024

IJMS

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A detailed comprehension of MHC-epitope recognition is essential for the design and development of new antigens that could be effectively used in immunotherapy. Yet, the high variability of the peptide together with the large abundance of MHC variants binding makes the process highly specific and large-scale characterizations extremely challenging by standard experimental techniques. Taking advantage of the striking predictive accuracy of AlphaFold, we report a structural and dynamic-based strategy to gain insights into the molecular basis that drives the recognition and interaction of MHC class I in the immune response triggered by pathogens and/or tumor-derived peptides. Here, we investigated at the atomic level the recognition of E7 and TRP-2 epitopes to their known receptors, thus offering a structural explanation for the different binding preferences of the studied receptors for specific residues in certain positions of the antigen sequences. Moreover, our analysis provides clues on the determinants that dictate the affinity of the same epitope with different receptors. Collectively, the data here presented indicate the reliability of the approach that can be straightforwardly extended to a large number of related systems.

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“…Previous studies demonstrated that the consistent overexpression of HPV E7 oncoproteins is required throughout the process of cervical epithelial cell carcinogenesis ( Domingos-Pereira et al, 2021 ). Hence, HPV E7 gene is an optimal target for the treatment and prevention of HPV-induced cancers ( Domingos-Pereira et al, 2021 ; Peng et al, 2022 ). However, since HPV life cycle is tightly linked to the host cell differentiation, the virus is extremely difficult to isolate and culture ( Wang et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

A replicative recombinant HPV16 E7 expression virus upregulates CD36 in C33A cells

Shao,

Wang,

Zheng

et al. 2023

Front. Microbiol.

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ObjectiveIn past decades, the role of high-risk HPV (HR-HPV) infection in cancer pathogenesis has been extensively studied. The viral E7 protein expressed in pre-malignant cells has been identified as an ideal target for immunological intervention. However, the cultivation of HPV in vitro remains a significant challenge, as well as the lack of methods for expressing the HPV E7 protein and generating replication-competent recombinant viral particles, which posed a major obstacle to further exploration of the function and carcinogenic mechanisms of the E7 oncoprotein. Therefore, it is imperative to investigate novel methodologies to construct replication-competent recombinant viral particles that express the HPV E7 protein to facilitate the study of its function.MethodsWe initiated the construction of recombinant viral particles by utilizing the ccdB-Kan forward/reverse screening system in conjunction with the Red/ExoCET recombinant system. We followed the infection of C33A cells with the obtained recombinant virus to enable the continuous expression of HPV16 E7. Afterwards, the total RNA was extracted and performed transcriptome sequencing using RNA-Seq technology to identify differentially expressed genes associated with HPV-induced oncogenicity.ResultsWe successfully established replicative recombinant viral particles expressing HPV16 E7 stably and continuously. The C33A cells were infected with recombinant viral particles to achieve overexpression of the E7 protein. Subsequently, RNA-Seq analysis was conducted to assess the changes in host cell gene expression. The results revealed an upregulation of the CD36 gene, which is associated with the HPV-induced oncogenic pathways, including PI3K-Akt and p53 signaling pathway. qRT-PCR analysis further identified that the upregulation of the CD36 gene due to the expression of HPV16 E7.ConclusionThe successful expression of HPV16 E7 in cells demonstrates that the replicated recombinant virus retains the replication and infection abilities of Ad4, while also upregulating the CD36 gene involved in the PI3K-Akt signaling and p53 pathways, thereby promoting cell proliferation. The outcome of this study provides a novel perspective and serves as a solid foundation for further exploration of HPV-related carcinogenesis and the development of replicative HPV recombinant vaccines capable of inducing protective immunity against HPV.

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Development of a Spontaneous HPV16 E6/E7-Expressing Head and Neck Squamous Cell Carcinoma in HLA-A2 Transgenic Mice

Abstract: Our data indicate that mutated HPV16 E6(R55K)(delK75) and mutated HPV16 E7(N53S) DNA abolishes the presentation of HPV16 E6 and E7 through murine MHC-I and results in their presentation through human HLA-A2 molecules. Additionally, the mutated HPV16 E6 and E7 remain oncogenic.

Cited by 4 publications

References 39 publications

Identification of human MHC-I HPV18 E6/E7-specific CD8 + T cell epitopes and generation of an HPV18 E6/E7-expressing adenosquamous carcinoma in HLA-A2 transgenic mice

Identification of human MHC-I HPV18 E6/E7-specific CD8 + T cell epitopes and generation of an HPV18 E6/E7-expressing adenosquamous carcinoma in HLA-A2 transgenic mice

Structural and Dynamic-Based Characterization of the Recognition Patterns of E7 and TRP-2 Epitopes by MHC Class I Receptors through Computational Approaches

A replicative recombinant HPV16 E7 expression virus upregulates CD36 in C33A cells

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