The polymorphism analysis and therapy vaccine target epitopes screening of HPV-35 E6 E7 among the threaten α-9 HPV in Sichuan area.

High-risk human papilloma virus (HR-HPV) persistent infection is closely associated with the development of cervical cancer and squamous intraepithelial lesion (SIL).The α-9 HPVs, which is predominantly composed of HR-HPV types, account for 75% of HR-HPV infection in Sichuan. The oncoproteins E6 and E7 of HPV play a crucial role in tumor initiation and progression. Notably, HPV-35 is the only HR-HPV type within the α-9 genus that is not included in the nine-valent HPV prophylactic vaccine. Cervical cell samples obtained from Sichuan were collected for HPV detection and genotyping. Among the 406 HPV-positive samples, 31 HPV-35 were detected, 24 HPV-35 E6 and 26 E7 were successfully amplified and sequenced, five nucleotide mutations in E6 and three in E7 were detected, T232C, T434G of E6 (W78R, I145R) and C67T, G84T of E7 (H23Y, L28F) were non-synonymy mutation. PAML 4.8 server was used to detect positive selection sites of HPV-35 E6, E7, and E6 is W78R. Phyre2 were used to predict and analyze protein structures, W78R made influences on protein structure. IEDB were used to screen epitopes vaccine target for HPV-35 affection therapy, and 5 HPV-35 E6 and 3 HPV-35 E7 most potential epitopes were obtained, the most potential peptides for therapy vaccine design were 79-91YRYSVYGETLEKQ, 45-60FACYDLCIVREGQPY, 124-135RFHNIGGRWTGR of E6; 3-19GEITTLQDYVLDLEPEA, 38-47TIDGPAGQAK, 70-88VQSTHIDIRKLEDLLMGTF of E7 and W78R mainly decreased the epitopes affinity.Conclusions Amino acid substitution in the positive selection sites of HPV-35 E6 and E7 genes have been found to influence protein structure and to decrease the overall affinity of antigen epitopes. This observation aligns with the evolutionary significance of positive selection site, which may confer advantages to the virus by making infected cells more challenging for the immune system to detect, thereby enhancing HPV's adaptability to the host environment. The polymorphism analysis of HPV-35 E6, E7 contributes to the enrichment of α-9 HPV data in Sichuan China, which is instrumental in improving the effectiveness of clinical detection. Furthermore, these findings provide a relevant theoretical foundation for the prevention and treatment of HPV-related diseases.

CER818: A Highly Specific and Sensitive HPV L1 High-Risk Serological Lateral Flow Rapid Test for Early Detection of Cervical Cancer and Its Precursor Lesions.

Objective: The objective of the study is to validate a new human papillomavirus (HPV) L1 high-risk specific serological assay in a case-control study. Methods: Serum samples of 138 patients (cervical intraepithelial neoplasia (CIN) 1, 2, and 3 and cervical cancer), 21 vaccinees, and 246 female controls were tested for the presence of HPV L1 high-risk specific antibodies. Results: HPV L1 high-risk antibodies were detected in 100% of the CIN1 and 2, 86.6% of the CIN3 and 82.4% of the cervical cancer cases, 100% of the vaccinees, and 3.9% of the female controls. Area under the curve (AUC) was calculated with 0.91 for controls versus CIN2+, 0.923 for controls versus CIN1+, and 0.968 for controls versus CIN1/2. Conclusion: The HPV L1 high-risk specific serological lateral flow rapid test shows promising data in the field of early detection of HPV high-risk induced cervical cancer and its precursor lesions. This easy-to-use, robust, and affordable approach could offer a chance to reach women in low- or middle-income countries (LMICs) that could not be reached by HPV molecular testing-based cervical cancer screening programs.

Manipulating host secreted protein gene expression: an indirect approach by HPV11/16 E6/E7 to suppress PBMC cytokine secretion.

Human papillomavirus (HPV) 11/16 E6/E7 proteins have been recognized to be pivotal in viral pathogenesis. This study sought to uncover the potential mechanisms of how HPV11/16 E6/E7-transfected keratinocytes inhibit cytokine secretion in peripheral blood mononuclear cells (PBMC). Upon co-culturing HPV11/16 E6/E7-transfected keratinocytes with PBMC in a non-contact manner, we observed a marked decrease in various cytokines secreted by PBMC. To determine if this suppression was mediated by specific common secreted factors, we conducted transcriptomic sequencing on these transfected cells. This analysis identified 53 common differentially secreted genes in all four HPV-transfected cells. Bioinformatics analysis demonstrated these genes were predominantly involved in immune regulation. Results from quantitative PCR (qPCR) and an extensive literature review suggested the downregulation of 12 genes (ACE2, BMP3, BPIFB1, CLU, CST6, CTF1, HMGB2, MMP12, PDGFA, RNASE7, SULF2, TGM2), and upregulation of 7 genes (CCL17, CCL22, FBLN1, PLAU, S100A7, S100A8, S100A9), may be crucial in modulating tumor immunity and combating pathogenic infections, with genes S100A8 and S100A9, and IL-17 signaling pathway being particularly noteworthy. Thus, HPV11/16 E6/E7 proteins may inhibit cytokine secretion of immune cells by altering the expression of host-secreted genes. Further exploration of these genes may yield new insights into the complex dynamics of HPV infection.

Molecular dynamics simulation and purification of chimeric L1/L2 protein from human papillomavirus type 52 expressed in Escherichia coli BL21 (DE3).

The available prophylactic vaccines for human papillomavirus (HPV) in the market are only effective against specific types of HPV, rendering them ineffective for other types of HPV infections. The objective of this research is to investigate the stability of the recombinant protein constructed, namely chimeric L1/L2 protein from HPV type 52, with improved cross-neutralization ability. The 3D model, predicted using Alphafold, Robetta, I-Tasser, and refined with Galaxy Refinement, is validated using Ramachandran plot analysis. The stability is verified through molecular dynamics simulations, considering parameters such as RMSD, RMSF, Rg, and SASA, where stable conditions are observed. The chimeric L1/L2 protein from HPV type 52 is purified using affinity chromatography, and the His-tag is cleaved using SUMO protease to obtain pure chimeric protein with the size of ~ 55 kDa. Western blot analysis confirms binding to anti-L1 HPV type 52 polyclonal antibody. The obtained vaccine candidate can be utilized as an effective prophylactic vaccine against HPV.

Differential tumor immune microenvironment coupled with tumor progression or tumor eradication in HPV-antigen expressing squamous cell carcinoma (SCC) models.

Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C-100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C-225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack.

Enhancement of Tumor Antigen-specific T-cell Responses by Immune Checkpoint Blockade in Human Papillomavirus-related Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is clinically and immunologically distinct from HPV-negative HNSCC. Herein, we investigated the presence of tumor antigens HPV E6/E7 and wild-type p53-specific T-cell responses, and the impact of immune checkpoint blockade in patients with HPV-positive HNSCC. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) from patients with HPV-positive HNSCC were stimulated with HPV E6/E7 or wild-type p53-derived peptide mixture and evaluated using the interferon-γ enzyme-linked immunosorbent spot assay. Flow cytometry was performed to analyze the proportion of T-cell subsets and T cells expressing immune checkpoint molecules. RESULTS: HPV E6/E7-specific T cells were detected in 22 (95.7%) of 23 patients, whereas wild-type p53-specific T cells were detected in 3 (15.0%) of 20 patients. Seven (43.8%) of 16 patients exhibited wild-type p53-specific T-cell responses, as determined using whole proteins instead of peptides. Immune checkpoint blockade enhanced wild-type p53-specific T-cell responses in 9 (45.0%) of 20 patients. Flow cytometric analysis of PBMCs revealed that responders exhibiting enhanced wild-type p53-specific T-cell responses following immune checkpoint blockade had a significantly higher proportion of Ki-67+CD4+ T cells, Ki-67+CD8+ T cells, regulatory T cells, PD-1+CD4+ T cells, and TIM-3+CD4+ T cells than non-responders. CONCLUSION: Our findings indicate that tumor antigen-specific T cells are present in the peripheral blood of patients with HPV-positive HNSCC. Blockade of checkpoint pathways can enhance T-cell responses in certain patients, probably via activated T cells, Tregs, and/or exhausted CD4+ T cells.

Final outcomes analysis of the cell product SQZ-PBMC-HPV Phase 1 trial in incurable HPV16+ solid tumors shows improved overall survival in patients with increased CD8+ T cell tumor infiltration.

Cancer vaccines strive to induce robust, antigen-targeted, T-cell-mediated immune responses but have struggled to produce meaningful regression in solid tumors. An autologous cell vaccine, SQZ-PBMC-HPV, was developed by SQZ Biotechnologies using microfluidic squeezing technology to load PBMCs with HPV16 E6 and E7 antigens in HLA-A*02+ patients. The SQZ-PBMC-HPV-101 Phase 1 trial (NCT04084951) enrolled patients with incurable HPV16+ cancers. Here, we present a post hoc analysis of the relationship between Posttreatment CD8+ T cell infiltration and patient outcomes. SQZ-PBMC-HPV was administered as monotherapy every 3 weeks. Tumor samples were collected pre-dose and post-dose 4 weeks after treatment start. Biomarkers including CD8, MHC-I, E6, E7, GZMB, and Ki67 were evaluated by immunohistochemistry, immunofluorescence, and RNA in situ hybridization, and were correlated with clinical response, survival, and drug product composition. Eighteen patients had paired pre- and post-dose biopsies. Six (33%) had an increase in CD8+ T cell density in tumor parenchyma between screening and C2D8. Patients with increased CD8+ T cell density had improved disease control rate (66.7% vs 16.7%) and median overall survival (606.5 days vs 170.0 days, p = 0.0078). Drug product was significantly enriched for higher T cells and lower monocytes in the increased CD8+ T cell density group. In patients with incurable HPV16+ solid tumors treated with SQZ-PBMC-HPV, an increase in CD8+ T cell density within the tumor parenchyma was associated with superior disease control rate and overall survival. The product composition for patients with increased CD8+ T cell density was enriched for T cells.

Development of an mRNA-based therapeutic vaccine mHTV-03E2 for high-risk HPV-related malignancies.

Human papillomavirus (HPV) 16 and 18 infections are related to many human cancers. Despite several preventive vaccines for high-risk (hr) HPVs, there is still an urgent need to develop therapeutic HPV vaccines for targeting pre-existing hrHPV infections and lesions. In this study, we developed a lipid nanoparticle (LNP)-formulated mRNA-based HPV therapeutic vaccine (mHTV)-03E2, simultaneously targeting the E2/E6/E7 of both HPV16 and HPV18. mHTV-03E2 dramatically induced antigen-specific cellular immune responses, leading to significant CD8+ T cell infiltration and cytotoxicity in TC-1 tumors derived from primary lung epithelial cells of C57BL/6 mice expressing HPV E6/E7 antigens, mediated significant tumor regression, and prolonged animal survival, in a dose-dependent manner. We further demonstrated significant T cell immunity against HPV16/18 E6/E7 antigens for up to 4 months post-vaccination in immunological and distant tumor rechallenging experiments, suggesting robust memory T cell immunity against relapse. Finally, mHTV-03E2 synergized with immune checkpoint blockade to inhibit tumor growth and extend animal survival, indicating the potential in combination therapy. We conclude that mHTV-03E2 is an excellent candidate therapeutic mRNA vaccine for treating malignancies caused by HPV16 or HPV18 infections.

The potential use of therapeutics and prophylactic mRNA vaccines in human papillomavirus (HPV).

Cervical cancer (CC) and other malignant malignancies are acknowledged to be primarily caused by persistent human papillomavirus (HPV) infection. Historically, vaccinations against viruses that produce neutralizing antibodies unique to the virus have been an affordable way to manage viral diseases. CC risk is decreased, but not eliminated, by HPV vaccinations. Since vaccinations have been made available globally, almost 90% of HPV infections have been successfully avoided. On the lesions and diseases that are already present, however, no discernible treatment benefit has been shown. As a result, therapeutic vaccines that elicit immune responses mediated by cells are necessary for the treatment of established infections and cancers. mRNA vaccines possess remarkable potential in combating viral diseases and malignancy as a result of their superior industrial production, safety, and efficacy. Furthermore, considering the expeditiousness of production, the mRNA vaccine exhibits promise as a therapeutic approach targeting HPV. Given that the HPV-encoded early proteins, including oncoproteins E6 and E7, are consistently present in HPV-related cancers and pre-cancerous lesions and have crucial functions in the progression and persistence of HPV-related diseases, they serve as ideal targets for therapeutic HPV vaccines. The action mechanism of HPV and HPV-related cancer mRNA vaccines, their recent advancements in clinical trials, and the potential for their therapeutic applications are highlighted in this study, which also offers a quick summary of the present state of mRNA vaccines. Lastly, we highlight a few difficulties with mRNA HPV vaccination clinical practice and provide our thoughts on further advancements in this quickly changing sector. It is expected that mRNA vaccines will soon be produced quickly for clinical HPV prevention and treatment.

High-risk HPV oncoproteins E6 and E7 and their interplay with the innate immune response: Uncovering mechanisms of immune evasion and therapeutic prospects.

Human papillomaviruses (HPVs) are double-stranded DNA (dsDNA) tumor viruses causally associated with 5% of human cancers, comprising both anogenital and upper aerodigestive tract carcinomas. Despite the availability of prophylactic vaccines, HPVs continue to pose a significant global health challenge, primarily due to inadequate vaccine access and coverage. These viruses can establish persistent infections by evading both the intrinsic defenses of infected tissues and the extrinsic defenses provided by professional innate immune cells. Crucial for their evasion strategies is their unique intraepithelial life cycle, which effectively shields them from host detection. Thus, strategies aimed at reactivating the innate immune response within infected or transformed epithelial cells, particularly through the production of type I interferons (IFNs) and lymphocyte-recruiting chemokines, are considered viable solutions to counteract the adverse effects of persistent infections by these oncogenic viruses. This review focuses on the complex interplay between the high-risk HPV oncoproteins E6 and E7 and the innate immune response in epithelial cells and HPV-associated cancers. In particular, it details the molecular mechanisms by which E6 and E7 modulate the innate immune response, highlighting significant progress in our comprehension of these processes. It also examines forward-looking strategies that exploit the innate immune system to ameliorate existing anticancer therapies, thereby providing crucial insights into future therapeutic developments.

Design and evaluation of a multi-epitope DNA vaccine against HPV16.

Cervical cancer, among the deadliest cancers affecting women globally, primarily arises from persistent infection with high-risk human papillomavirus (HPV). To effectively combat persistent infection and prevent the progression of precancerous lesions into malignancy, a therapeutic HPV vaccine is under development. This study utilized an immunoinformatics approach to predict epitopes of cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) using the E6 and E7 oncoproteins of the HPV16 strain as target antigens. Subsequently, through meticulous selection of T-cell epitopes and other necessary elements, a multi-epitope vaccine was constructed, exhibiting good immunogenic, physicochemical, and structural characteristics. Furthermore, in silico simulations showed that the vaccine not only interacted well with toll-like receptors (TLR2/TLR3/TLR4), but also induced a strong innate and adaptive immune response characterized by elevated Th1-type cytokines, such as interferon-gamma (IFN-γ) and interleukin-2 (IL2). Additionally, our study investigated the effects of different immunization intervals on immune responses, aiming to optimize a time-efficient immunization program. In animal model experiments, the vaccine exhibited robust immunogenic, therapeutic, and prophylactic effects. Administered thrice, it consistently induced the expansion of specific CD4 and CD8 T cells, resulting in substantial cytokines release and increased proliferation of memory T cell subsets in splenic cells. Overall, our findings support the potential of this multi-epitope vaccine in combating HPV16 infection and signify its candidacy for future HPV vaccine development.

Through the stringent selection of T-cell epitopes and other necessary elements, a novel multi-epitope vaccine targeting HPV 16 E6 and E7 oncoproteins was constructed using an immunoinformatics approach.The vaccine designed can induce both cellular and humoral immune responses, encompassing all the required immunogenic, physicochemical, and structural characteristics for an ideal vaccine design. Moreover, it offers decent worldwide coverage.In animal studies, the vaccine demonstrated strong immune responses, including expansion of CD4 and CD8 T cells, cytokine release, and enhanced memory T cell proliferation, resulting in long-term anti-tumor effects, inhibition of tumor growth, and prolonged survival in tumor-bearing mice.The immunological evaluation of the designed vaccine suggests its potential as a novel vaccine candidate against HPV 16.

Immunological Response against Breast Lineage Cells Transfected with Human Papillomavirus (HPV).

Breast cancer is the most common neoplasm worldwide. Viral infections are involved with carcinogenesis, especially those caused by oncogenic Human Papillomavirus (HPV) genotypes. Despite the detection of HPV in breast carcinomas, the virus's activity against this type of cancer remains controversial. HPV infection promotes remodeling of the host's immune response, resulting in an immunosuppressive profile. This study assessed the individual role of HPV oncogenes in the cell line MDA-MB-231 transfected with the E5, E6, and E7 oncogenes and co-cultured with peripheral blood mononuclear cells. Immunophenotyping was conducted to evaluate immune system modulation. There was an increase in CD4+ T cell numbers when compared with non-transfected and transfected MDA-MB-231, especially in the Treg profile. Pro-inflammatory intracellular cytokines, such as IFN-γ, TNF-α, and IL-17, were impaired by transfected cells, and a decrease in the cytolytic activity of the CD8+ and CD56+ lymphocytes was observed in the presence of HPV oncogenes, mainly with E6 and E7. The E6 and E7 oncogenes decrease monocyte expression, activating the expected M1 profile. In the monocytes found, a pro-inflammatory role was observed according to the cytokines released in the supernatant. In conclusion, the MDA-MB-231 cell lineage transfected with HPV oncogenes can downregulate the number and function of lymphocytes and monocytes.

Phylogenetic analysis and antigenic epitope prediction for E6 and E7 of Alpha-papillomavirus 9 in Taizhou, China.

BACKGROUND: Alpha-papillomavirus 9 (α-9) is a member of the human papillomavirus (HPV) α genus, causing 75% invasive cervical cancers worldwide. The purpose of this study was to provide data for effective treatment of HPV-induced cervical lesions in Taizhou by analysing the genetic variation and antigenic epitopes of α-9 HPV E6 and E7. METHODS: Cervical exfoliated cells were collected for HPV genotyping. Positive samples of the α-9 HPV single type were selected for E6 and E7 gene sequencing. The obtained nucleotide sequences were translated into amino acid sequences (protein primary structure) using MEGA X, and positive selection sites of the amino acid sequences were evaluated using PAML. The secondary and tertiary structures of the E6 and E7 proteins were predicted using PSIPred, SWISS-MODEL, and PyMol. Potential T/B-cell epitopes were predicted by Industrial Engineering Database (IEDB). RESULTS: From 2012 to 2023, α-9 HPV accounted for 75.0% (7815/10423) of high-risk HPV-positive samples in Taizhou, both alone and in combination with other types. Among these, single-type-positive samples of α-9 HPV were selected, and the entire E6 and E7 genes were sequenced, including 298 HPV16, 149 HPV31, 185 HPV33, 123 HPV35, 325 HPV52, and 199 HPV58 samples. Compared with reference sequences, 34, 12, 10, 2, 17, and 17 nonsynonymous nucleotide mutations were detected in HPV16, 31, 33, 35, 52, and 58, respectively. Among all nonsynonymous nucleotide mutations, 19 positive selection sites were selected, which may have evolutionary significance in rendering α-9 HPV adaptive to its environment. Immunoinformatics predicted 57 potential linear and 59 conformational B-cell epitopes, many of which are also predicted as CTL epitopes. CONCLUSION: The present study provides almost comprehensive data on the genetic variations, phylogenetics, positive selection sites, and antigenic epitopes of α-9 HPV E6 and E7 in Taizhou, China, which will be helpful for local HPV therapeutic vaccine development.

Antibodies against high-risk human papillomavirus proteins as markers for noncervical HPV-related cancers in a Black South African population, according to HIV status.

Human papillomavirus (HPV) proteins may elicit antibody responses in the process toward HPV-related malignancy. However, HPV seroepidemiology in noncervical HPV-related cancers remains poorly understood, particularly in populations with a high prevalence of human immunodeficiency virus (HIV). Using a glutathione S-transferase-based multiplex serology assay, antibodies against E6, E7 and L1 proteins of HPV16 and HPV18 were measured in sera of 535 cases of noncervical HPV-related cancers (anal (n = 104), vulval (n = 211), vaginal (n = 49), penile (n = 37) and oropharyngeal (n = 134)) and 6651 non-infection-related cancer controls, from the Johannesburg Cancer Study that recruited Black South African with newly diagnosed cancer between 1995 and 2016. Logistic and Poisson regression models were used to calculate adjusted odds ratios (aOR) and prevalence ratios (aPR) and 95% confidence intervals (CI) in cases versus controls. HPV16 E6 was more strongly associated with noncervical HPV-related cancers than HPV16 L1 or E7, or HPV18 proteins: anal (females (HPV16 E6 aOR = 11.50;95%CI:6.0-22.2), males (aOR = 10.12;95%CI:4.9-20.8), vulval (aOR = 11.69;95%CI:7.9-17.2), vaginal (aOR = 10.26;95%CI:5.0-21), penile (aOR = 18.95;95%CI:8.9-40), and oropharyngeal (females (aOR = 8.95;95%CI:2.9-27.5), males (aOR = 3.49;95%CI:1.8-7.0)) cancers. HPV16-E6 seropositivity ranged from 24.0% to 35.1% in anal, vulval, vaginal and penile cancer but was significantly lower (11.2%) in oropharyngeal cancer. After adjustment for HIV, prevalence of which increased from 22.2% in 1995-2005 to 54.1% in 2010-2016, HPV16 E6 seropositivity increased by period of diagnosis (aPR for 2010-2016 vs. 1995-2006 = 1.84;95%CI:1.1-3.0). Assuming HPV16 E6 seroprevalence reflects HPV attributable fraction, the proportion of certain noncervical-HPV-related cancers caused by HPV is increasing over time in South Africa. This is expected to be driven by the increasing influence of HIV.

Dual T cell receptor/chimeric antigen receptor engineered NK-92 cells targeting the HPV16 E6 oncoprotein and the tumor-associated antigen L1CAM exhibit enhanced cytotoxicity and specificity against tumor cells.

The human papillomavirus type 16 (HPV16) causes a large fraction of genital and oropharyngeal carcinomas. To maintain the transformed state, the tumor cells must continuously synthesize the E6 and E7 viral oncoproteins, which makes them tumor-specific antigens. Indeed, specific T cell responses against them have been well documented and CD8+ T cells engineered to express T cell receptors (TCRs) that recognize epitopes of E6 or E7 have been tested in clinical studies with promising results, yet with limited clinical success. Using CD8+ T cells from peripheral blood of healthy donors, we have identified two novel TCRs reactive to an unexplored E618-26 epitope. These TCRs showed limited standalone cytotoxicity against E618-26-HLA-A*02:01-presenting tumor cells. However, a single-signaling domain chimeric antigen receptor (ssdCAR) targeting L1CAM, a cell adhesion protein frequently overexpressed in HPV16-induced cancer, prompted a synergistic effect that significantly enhanced the cytotoxic capacity of NK-92/CD3/CD8 cells armored with both TCR and ssdCAR when both receptors simultaneously engaged their respective targets, as shown by live microscopy of 2-D and 3-D co-cultures. Thus, virus-specific TCRs from the CD8+ T cell repertoire of healthy donors can be combined with a suitable ssdCAR to enhance the cytotoxic capacity of the effector cells and, indirectly, their specificity.

Prevention and treatment of HPV-related cancer through a mRNA vaccine expressing APC-targeting antigen.

Persistent human papillomavirus (HPV) infection is associated with multiple malignancies. Developing therapeutic vaccines to eliminate HPV-infected and malignant cells holds significant value. In this study, we introduced a lipid nanoparticle encapsulated mRNA vaccine expressing tHA-mE7-mE6. Mutations were introduced into E6 and E7 of HPV to eliminate their tumourigenicity. A truncated influenza haemagglutinin protein (tHA), which binds to the CD209 receptor on the surface of dendritic cells (DCs), was fused with mE7-mE6 in order to allow efficient uptake of antigen by antigen presenting cells. The tHA-mE7-mE6 (mRNA) showed higher therapeutic efficacy than mE7-mE6 (mRNA) in an E6 and E7+ tumour model. The treatment resulted in complete tumour regression and prevented tumour formation. Strong CD8+ T-cell immune response was induced, contributing to preventing and curing of E6 and E7+ tumour. Antigen-specific CD8+ T were found in spleens, peripheral blood and in tumours. In addition, the tumour infiltration of DC and NK cells were increased post therapy. In conclusion, this study described a therapeutic mRNA vaccine inducing strong anti-tumour immunity in peripheral and in tumour microenvironment, holding promising potential to treat HPV-induced cancer and to prevent cancer recurrence.

Human papillomavirus 68 prevalence and genetic variability based on E6/E7 genes in Sichuan.

HPV68 is a common HR-HPV, its persistent infection is closely related with the occurrence of cervical cancer. In this study, 2939 (27.60%, 2939/10650) positive samples were detected, and 174 (5.92%, 174/2939) were HPV68. 150 HPV68 E6-E7 were successful sequenced, 4 non-synonymous mutations were detected in E6, and E7 were 12. N133S non-synonymous mutations of HPV 68 E6 and C67G, T68 A/M of HPV68 E7 are E6, E7 positive selection sites, they all located in the key domains and major motifs of E6/E7 protein, the above amino-acid substitutions changed the protein structure, disturbed the interaction with other protein or cellular factors and make a difference in epitopes affinity, may affect the pathogenicity and adaptability of HPV68 to the environment. The enrichment of HPV68 data is of great significance for understanding the inherent geographical and biological differences of HPV68 in China.

Depletion of Human Papilloma Virus E6- and E7-Oncoprotein-Specific T-Cell Responses in Women Living With HIV.

Background: Cervical cancer - caused by persistent High Risk Human Papilloma Virus (HR HPV) infections - is the second most common cancer affecting women globally. HIV infection increases the risk for HPV persistence, associated disease progression and malignant cell transformation. We therefore hypothesized that this risk increase is directly linked to HIV infection associated dysfunction or depletion of HPV-oncoprotein-specific T-cell responses. Methods: The 2H study specifically included HIV+ and HIV- women with and without cervical lesions and cancer to analyze HPV oncogene-specific T cell responses in relation to HPV infection, cervical lesion status and HIV status. Oncoprotein E6 and E7 specific T-cell responses were quantified for the most relevant types HPV16, 18 and 45 and control antigens (CMV-pp65) and M.tb-PPD in 373 women, using fresh peripheral blood mononuclear cells in an IFN-γ release ELISpot assay. Results: Overall, systemic E6- and E7-oncoprotein-specific T-cell responses were infrequent and of low magnitude, when compared to CMV-pp65 and M.tb-PPD (p < 0.001 for all HR HPV types). Within HIV negative women infected with either HPV16, 18 or 45, HPV16 infected women had lowest frequency of autologous-type-E6/E7-specific T-cell responses (33%, 16/49), as compared to HPV18 (46% (6/13), p = 0.516) and HPV45 (69% (9/13), p = 0.026) infected women. Prevalent HPV18 and 45, but not HPV16 infections were linked to detectable oncoprotein-specific T-cell responses, and for these infections, HIV infection significantly diminished T-cell responses targeting the autologous infecting genotype. Within women living with HIV, low CD4 T-cell counts, detectable HIV viremia as well as cancerous and precancerous lesions were significantly associated with depletion of HPV oncoprotein-specific T-cell responses. Discussion: Depletion of HPV-oncoprotein-specific T-cell responses likely contributes to the increased risk for HR HPV persistence and associated cancerogenesis in women living with HIV. The low inherent immunogenicity of HPV16 oncoproteins may contribute to the exceptional potential for cancerogenesis associated with HPV16 infections.

Germline determinants of humoral immune response to HPV-16 protect against oropharyngeal cancer.

Although several oropharyngeal cancer (OPC) susceptibility loci have been identified, most previous studies lacked detailed information on human papillomavirus (HPV) status. We conduct a genome-wide analysis by HPV16 serology status in 4,002 oral cancer cases (OPC and oral cavity cancer (OCC)) and 5,256 controls. We detect four susceptibility loci pointing to a distinct genetic predisposition by HPV status. Our most notable finding in the HLA region, that is now confirmed to be specific of HPV(+)OPC risk, reveal two independent loci with strong protective effects, one refining the previously reported HLA class II haplotype association. Antibody levels against HPV16 viral proteins strongly implicate the protective HLA variants as major determinants of humoral response against L1 capsid protein or E6 oncoprotein suggesting a natural immune response against HPV(+)OPC promoted by HLA variants. This indicates that therapeutic vaccines that target E6 and attenuate viral response after established HPV infections might protect against HPV(+)OPC.

Functional HPV-specific PD-1+ stem-like CD8 T cells in head and neck cancer.

T cells are important in tumour immunity but a better understanding is needed of the differentiation of antigen-specific T cells in human cancer1,2. Here we studied CD8 T cells in patients with human papillomavirus (HPV)-positive head and neck cancer and identified several epitopes derived from HPV E2, E5 and E6 proteins that allowed us to analyse virus-specific CD8 T cells using major histocompatibility complex (MHC) class I tetramers. HPV-specific CD8 T cells expressed PD-1 and were detectable in the tumour at levels that ranged from 0.1% to 10% of tumour-infiltrating CD8 T lymphocytes (TILs) for a given epitope. Single-cell RNA-sequencing analyses of tetramer-sorted HPV-specific PD-1+ CD8 TILs revealed three transcriptionally distinct subsets. One subset expressed TCF7 and other genes associated with PD-1+ stem-like CD8 T cells that are critical for maintaining T cell responses in conditions of antigen persistence. The second subset expressed more effector molecules, representing a transitory cell population, and the third subset was characterized by a terminally differentiated gene signature. T cell receptor clonotypes were shared between the three subsets and pseudotime analysis suggested a hypothetical differentiation trajectory from stem-like to transitory to terminally differentiated cells. More notably, HPV-specific PD-1+TCF-1+ stem-like TILs proliferated and differentiated into more effector-like cells after in vitro stimulation with the cognate HPV peptide, whereas the more terminally differentiated cells did not proliferate. The presence of functional HPV-specific PD-1+TCF-1+CD45RO+ stem-like CD8 T cells with proliferative capacity shows that the cellular machinery to respond to PD-1 blockade exists in HPV-positive head and neck cancer, supporting the further investigation of PD-1 targeted therapies in this malignancy. Furthermore, HPV therapeutic vaccination efforts have focused on E6 and E7 proteins; our results suggest that E2 and E5 should also be considered for inclusion as vaccine antigens to elicit tumour-reactive CD8 T cell responses of maximal breadth.