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

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.

Local radiotherapy and E7 RNA-LPX vaccination show enhanced therapeutic efficacy in preclinical models of HPV16+ cancer

Human papilloma virus (HPV) infection is a causative agent for several cancers types (genital, anal and head and neck region). The HPV E6 and E7 proteins are oncogenic drivers and thus are ideal candidates for therapeutic vaccination. We recently reported that a novel ribonucleic acid lipoplex (RNA-LPX)-based HPV16 vaccine, E7 RNA-LPX, mediates regression of mouse HPV16+ tumors and establishes protective T cell memory. An HPV16 E6/E7 RNA-LPX vaccine is currently being investigated in two phase I and II clinical trials in various HPV-driven cancer types; however, it remains a high unmet medical need for treatments for patients with radiosensitive HPV16+ tumors. Therefore, we set out to investigate the therapeutic efficacy of E7 RNA-LPX vaccine combined with standard-of-care local radiotherapy (LRT). We demonstrate that E7 RNA-LPX synergizes with LRT in HPV16+ mouse tumors, with potent therapeutic effects exceeding those of either monotherapy. Mode of action studies revealed that the E7 RNA-LPX vaccine induced high numbers of intratumoral-E7-specific CD8+T cells, rendering cold tumors immunologically hot, whereas LRT primarily acted as a cytotoxic therapy, reducing tumor mass and intratumor hypoxia by predisposing tumor cells to antigen-specific T cell-mediated killing. Overall, LRT enhanced the effector function of E7 RNA-LPX-primed T cell responses. The therapeutic synergy was dependent on total radiation dose, rather than radiation dose-fractionation. Together, these results show that LRT synergizes with E7 RNA-LPX and enhances its anti-tumor activity against HPV16+ cancer models. This work paves into a new translational therapy for HPV16+ cancer patients.

NSP4 as Adjuvant for Immunogenicity and Design of Effective Therapeutic HPV16 E6/E7/L1 DNA Vaccine in Tumoric and Healthy C57BL/6 Mice

Introduction: In humans, approximately 5% of all cancers are attributable to HPV infection. Prophylactic vaccines can inhibit viral migration and persistence. However, requirement to develop therapeutic treatments prevails. To achieve this goal, we designed a therapeutic HPV DNA vaccine encoding a construct of E6/E7/L1 and used NSP4 antigen as adjuvant to assess efficiency of this construct in generating antigen-specific antitumor immune responses.Material and Methods: Sixty female C57BL/6 mice (6–8 weeks old) were purchased from Institute Pasteur of Iran. 30 of them became cancerous, but 30 of them were healthy control. To amplify E6/E7/L1-pcDNA3, NSP4-pcDNA3, expression vector of DH5α and TC-1 cell line were used to generate a tumor. Mice were immunized with HPV DNA vaccine. Cell proliferation was assessed by MTT assay. Finally, we assessed cytokine responses (IL-2, IL-4, INF- γ), in the serum of mice spleen cells.Result: Mice receiving the NSP4/E6-E7-L1 vaccine had the highest stimulatory index compared to other groups but it was not significant. Interleukin 4/12 and INF-γ production were significantly higher in E6-E7-L1 / NSP4 group and E6-E7-L1 group compared to other groups (P <0.05). Among different groups, E6/E7/L1 + NSP4 group was able to slow down the tumor growth process, but it was not significant (p>0.05). Among the cytokines mentioned, IFN-γ and IL-12 are among the cytokines that stimulate the Th1 pathway and IL-4 cytokine stimulates the Th2 pathway and B lymphocytes.Conclusion: Our data suggest that present vaccine can stimulate innate and acquired immunity response, and can be a therapeutic vaccine in the tumoric mice.

HPV 16 E6 Promotes Growth and Metastasis of Esophageal Squamous Cell Carcinoma cells in vitro

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies. Increasing evidence has revealed that Human Papillomavirus (HPV) infection may be associated with the possible etiology of ESCC. Nevertheless, the precise role of HPV in ESCC remains unclear.MethodsProliferation and apoptosis capability of ESCC cells upon infection of HPV16 E6 were detected by CCK-8 assay and Western blotting analysis. Wound healing assay and Transwell experiment were conducted to determine the ability of migration and invasion, and the mRNA expression of E6AP, p53, miR-34a was determined by real-time PCR after transfected with HPV16 E6 plasmid. ResultsIn ESCC cells, the ability of proliferation, migration and invasion were increased, and the ability of apoptosis was decreased after transfected with HPV16 E6 plasmid. Furthermore, the mRNA level of E6AP, P53 and miR-34a were decreased in HPV16 E6-transfected cell lines.ConclusionsOur results not only provide evidence that in ESCC, HPV16 E6 promotes cell proliferation, migration and invasion, inhibits cell apoptosis, but also suggest that there may be a correlation between E6AP, P53 and miR-34a in HPV-transfected cell lines. These findings indicated that HPV16 E6 may play an important role in the development of ESCC.

HPV16 E6 Gene Polymorphisms and the Functions of the Mutation Site in Cervical Cancer Among Uygur and Han Women in Xinjiang

Background: To investigate the genotype distribution of human papillomavirus (HPV) in infected Uygur and Han women in Xinjiang; analyze the HPV16 E6 gene polymorphism site and relationship with the development of cervical cancer.Methods: The HPV16 E6 sequence was analyzed using the European standard prototype to perform an evolutionary tree. HPV16 E6-295T/350T, 295G/350G, and 295T/350G GV230 vectors were stably transfected into cervical cancer C33A cells to analyze the cell proliferation, migration and invasion, apoptosis by CCK8 and clonogenic assays, transwell and cell scratch assays, FACS experiments. Results: The total HPV infection rate was 26.390% (760/2879), whereas the Uygur 22.87% (196/857) and the Han was 27.89% (564/2022) (P < 0.05). Among 110 mutations, 65 cases of E6 genes were mutated at nucleotide 350 (T350G) with the leucine changing to valine (L83V). Moreover, there were 7 cases of E6 gene mutated at nucleotide 295 (T295G) with aspartic changing to glutamic (D64E). When E6 vector(s) of mutations sites were transfected into C33A cells, they were found to promote cellular proliferation, migration, invasion, and inhibit apoptosis. The 295T/350G had the strongest effect on C33A cells and 295G/350G was significantly stronger than 295T/350T (P < 0.05).Conclusions: The positive HPV infection rates differed between the Uygur and Han in Xinjiang, and the genotype distribution of infection was different. After transfecting C33A cells with different eukaryotic expression vectors, the 295T/350G mutation site promoted the proliferation，migration, and invasion of C33A cells to a greater extent than 295G/350G; however, 295G/350G had a stronger effect than 295T/350T.

Genetic Variability and Phylogeny of Human Papillomavirus Type 16 Based On E6, E7 and L1 Genes in Central China

In the current study, a total of 74 single-infected HPV16 samples from females attending the gynecological outpatient clinic in four cities of Henan province were collected and applied to the L1, E6 and E7 sequencing. Variations of the HPV16 L1, E6 and E7 genes were characterized by comparison with reference sequence and the secondary structure analysis were conducted. Phylogenetic trees based on the L1 and E6-E7 sequences were constructed separately. B-cell epitopes of the HPV16 E6 and E7 proteins were predicted further. A total of thirty-seven novel variations, including twenty L1 genes and seventeen E6-E7 genes were identified. Compared with the reference sequence, twenty-eight variations (1.8%, 28/1596) were identified in L1 gene sequences and 10/28 (35.7%) were non-synonymous mutations. For E6-E7 sequences, twenty-five novel gene changes (including 16 mutations (3.4%, 16/477) in E6 gene, 9 mutations (3.0%, 9/297) in E7 gene) were found, 18/25 (72.0%) were non-synonymous and 10/28 (35.7%) were non-synonymous mutations. Phylogenetic analysis showed that 56.8% (42/74) of the samples were A1 sublineages, 37.8% (28/74) were A4, 4.1% (3/74) were A3 and 1.4% (1/74) was A2 sublineages. On the prediction of B-cell epitopes, seven potent epitopes for E6 and four for E7 were identified. Amino mutations, including L90V, R62K, R142Q and F76L in E6, S63F and N29S/H in E7 changed the score. HPV16 variants prevalent in the central China belong to European A1 sublineages. Sequences of HPV16 L1, E6 and E7 in this study may provide assistant for the improvement of HPV vaccines.

Topical Application of Temperature-Sensitive Gel Containing Caerin 1.1 and 1.9 Peptides on TC-1 Tumour-Bearing Mice Induced High-Level Immune Response in the Tumour Microenvironment

The development of topical cream drugs that increase the immune activation of tumour-infiltrating lymphocytes against tumour and chronic viral infection-associated lesions is of great immunotherapeutic significance. This study demonstrates that the topical application of a temperature-sensitive gel containing caerin 1.1 and 1.9 peptides reduces nearly 50% of the tumour weight of HPV16 E6/E7-transformed TC-1 tumour-bearing mice via improving the tumour microenvironment. Confocal microscopy confirms the time-dependent penetration of caerin 1.9 through the epidermal layer of the ear skin structure of mice. Single-cell transcriptomic analysis shows that the caerin 1.1/1.9 gel expands the populations with high immune activation level and largely stimulates the pro-inflammatory activity of NK and dendritic cells. Closely associated with INFα response, Cebpb seems to play a key role in altering the function of all Arg1hi macrophages in the caerin group. In addition, the caerin gel treatment recruits almost two-fold more activated CD8+ T cells to the TME, relative to the untreated tumour, which shows a synergistic effect derived from the regulation of S1pr1, Ccr7, Ms4a4b and Gimap family expression. The TMT10plex-labelling proteomic quantification further demonstrates the activation of interferon-alpha/beta secretion and response to cytokine stimulus by the caerin gel, while the protein contents of several key regulators were elevated by more than 30%, such as Cd5l, Gzma, Ifit1, Irf9 and Stat1. Computational integration of the proteome with the single-cell transcriptome consistently suggested greater activation of NK and T cells with the topical application of caerin peptide gel.

Detection of Human Papillomavirus DNA in Paired Peripheral Blood and Cervix Samples in Patients with Cervical Lesions and Healthy Individuals

This study evaluated the presence of Human Papillomavirus (HPV) DNA in the cervix and peripheral blood of women with cervical intraepithelial neoplasia (CIN I, II, and III) and healthy individuals. Overall, 139 paired peripheral blood and cervix samples of healthy women and women with CIN I, II, and III (n = 68) were tested for HPV DNA by using standard procedures. Polymerase chain reaction (PCR) sequencing determined HPV types. Quantification of HPV16 E6 and E2 genes was performed to determine viral load and physical state. HPV DNA was detected in the cervix (21.1% in healthy individuals; 48.8–55.5% in CIN patients), blood (46.4% in healthy individuals; 44.1–77.7% in CIN patients) and paired peripheral blood and cervix samples (24% in healthy individuals; 32.5–44.4% in CIN patients). The most frequent types found in the cervix were HPV16, 18, 31, 33, 58, and 70, while HPV16, 18, 33, 58, and 66 were the most frequent types found in the blood. HPV DNA in the cervix was associated with previous sexually transmitted infections (STIs) (p = 0.023; OR: 2.978; CI:1.34–7.821), HPV DNA in the blood (p = 0.000; OR: 8.283; CI:3.700–18.540), and cervical lesions (CIN I/II or III) (p = 0.007). Binomial logistic regression showed that HPV DNA in the blood (p = 0.000; OR: 9.324; CI:3.612–24.072) and cervical lesions (p = 0.011; OR: 3.622; CI:1.338–9.806) were associated with HPV DNA in the cervix. However, we did not find an association between HPV DNA in the blood and cervical lesions (p = 0.385). Our results showed that only HPV DNA found in the cervix was associated with cervical lesions.