Human Papillomavirus Antigens Research Articles

Manganese-Modified Aluminum Adjuvant Enhances both Humoral and Cellular Immune Responses.

Aluminum adjuvants remain the most commonly used vaccine adjuvants. Being rather effective in triggering humoral immunity, however, aluminum adjuvants usually show limited abilities in activating cellular immunities. Herein, by adding manganese ions during the preparation of aluminum adjuvant, a manganese-modified aluminum (Mn-Al) adjuvant is obtained, which can effectively stimulate both humoral and cellular immune responses. Such Mn-Al adjuvant can enhance antigen adsorption and promote antigen internalization by dendritic cells (DCs). Subsequently, the released Mn2+ can activate the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes pathway to further promote DC activation. When combines with the model antigen ovalbumin (OVA), the Mn-Al-adjuvantes vaccine can induce high levels of antigen-specific antibody titers and high proportions of antigen-specific cytotoxic T cells in vivo. Moreover, the Mn-Al-adjuvanted vaccine elicited stronger antigen-specific humoral and cellular immune responses than high-dose of the aluminum-based adjuvant. Additionally, immunization of mice with OVA in the presence of the Mn-Al adjuvant significantly inhibited the growth of B16-OVA tumors. Furthermore, when formulated with human papillomavirus antigens, Mn-Al-adjuvanted vaccines show better in vivo vaccination performance than aluminum-adjuvanted vaccines. Therefore, the manganese-modified aluminum adjuvant may thus become a new vaccine adjuvant with the potential to replace conventional aluminum adjuvants.

Preclinical Immunogenicity and Efficacy Studies for Therapeutic Vaccines for Human Papillomavirus-Type-16-Associated Cancer.

The objective of this study was to conduct preclinical immunogenicity and efficacy studies with several therapeutic vaccines for human papillomavirus (HPV)-16-associated cancers expressing the early antigens E5, E6, and E7 with or without E2. The viral oncoproteins were either expressed by themselves as fusion proteins or the fusion proteins were inserted genetically into herpes simplex virus (HSV)-1 glycoprotein D (gD) which, upon binding to the herpes virus entry mediator (HVEM), inhibits an early T cell checkpoint mediated by the B and T cell mediator (BTLA). This, in turn, lowers the threshold for T cell activation and augments and broadens CD8+ T cell responses to the antigens. The fusion antigens were expressed by chimpanzee adenovirus (AdC) vectors. Expression of the HPV antigens within gD was essential for vaccine immunogenicity and efficacy against challenge with TC-1 cells, which express E7 and E6 of HPV-16 but neither E5 nor E2. Unexpectedly, inclusion of E2 increased both CD8+ T cell responses to the other oncoproteins of HPV-16 and the effectiveness of the vaccines to cause the regression of sizable TC-1 tumors.

A phase 2 study to evaluate efficacy and safety of PRGN-2009, a novel gorilla adenovirus-based immunotherapy, in combination with pembrolizumab versus pembrolizumab monotherapy in patients with recurrent or metastatic cervical cancer.

TPS5623 Background: The association between cervical cancer and human papillomavirus (HPV) is well understood, with nearly all cervical cancer known to be caused by high risk HPV infection. HPV16 and HPV18 are the most common high-risk HPV types and are responsible for about 70% of cervical cancer cases. Recurrent or metastatic cervical cancer is currently treated with pembrolizumab alone or in combination with chemotherapy, however t there remains a significant need for treatments for patients that recur or do not respond at all to pembrolizumab treatment. PRGN-2009 is a gorilla adenovirus-based immunotherapy targeting cancers caused by HPV16 or HPV18. PRGN-2009 includes a novel HPV antigen design to induce HPV16/18-specific T cell immune responses against cancer cells. The rationale for combining the PRGN-2009 with immune checkpoint inhibitor is based on preclinical and clinical data demonstrating that combination of cancer vaccines and immune checkpoint inhibitors can trigger intensified immunogenicity leading to increased antitumor efficacy compared to either treatment alone. PRGN-2009 has been characterized in both in vitroand in vivostudies to support the scientific rationale and the safety of PRGN-2009 has been demonstrated in evaluated in initial Phase 1/2 studies in patients with HPV+ malignancies. Methods: This is a randomized, Phase 2 study to evaluate efficacy and safety of PRGN-2009 in combination with pembrolizumab compared to pembrolizumab alone in patients with recurrent or metastatic cervical cancer. Key inclusion criteria include recurrent or metastatic cervical cancer (histologically or cytologically confirmed) resistant to pembrolizumab. Subjects will be randomized 1:1 to Cohort 1: Combination of PRGN-2009, 5 x 1011 PU (Day 0, Day 21 and Day 42, then q6w), plus pembrolizumab, 400mg q6w, or to Cohort 2: pembrolizumab alone (400 mg q6w). Subjects randomized to Cohort 2 (pembrolizumab monotherapy) and who have confirmed disease progression (per RECIST 1.1) after at least one cycle of pembrolizumab and have had no Grade ≥ 3 adverse events related to treatment will be offered the option to crossover to Cohort 1 (PRGN-2009 plus pembrolizumab). All subjects will be evaluated for safety and efficacy of PRGN-2009 in combination with pembrolizumab, and for biological correlative assays. The primary endpoint is objective response rate (ORR) following treatment with PRGN-2009 in combination with pembrolizumab or pembrolizumab monotherapy, and will be tested using Fleming’s two-stage design and the stopping boundaries for futility and efficacy applied. The study is currently recruiting patients with recurrent or metastatic cervical cancer (NCT06157151). Clinical trial information: NCT06157151 .

Safety and immunogenicity of Innovax bivalent human papillomavirus vaccine in girls 9–14 years of age: Interim analysis from a phase 3 clinical trial

BackgroundWorld Health Organization human papillomavirus (HPV) vaccination recommendations include a single- or two-dose schedule in individuals 9–20 years old and advice for generating data on single-dose efficacy or immunobridging. The ongoing Phase 3 trial of Innovax’s bivalent (types 16 and 18) HPV vaccine (Cecolin®) assesses in low- and middle-income countries alternative dosing schedules and generates data following one dose in girls 9–14 years old. Interim data for the 6-month dosing groups are presented. MethodsIn Bangladesh and Ghana, 1,025 girls were randomized to receive either two doses of Cecolin at 6-, 12-, or 24-month intervals; one dose of Gardasil® followed by one dose of Cecolin at month 24; or two doses of Gardasil 6 months apart (referent). Serology was measured by enzyme-linked immunosorbent assay (ELISA) and, in a subset, by neutralization assays. Primary objectives include immunological non-inferiority of the Cecolin schedules to referent one month after the second dose. Safety endpoints include reactogenicity and unsolicited adverse events for 7 and 30 days post-vaccination, respectively, as well as serious adverse events throughout the study. ResultsInterim analyses included data from the two groups on a 0, 6-month schedule with 205 participants per group. One month after Dose 2, 100% of participants were seropositive by ELISA and had seroconverted for both antigens. Non-inferiority of Cecolin to Gardasil was demonstrated. Six months following one dose, over 96% of participants were seropositive by ELISA for both HPV antigens, with a trend for higher geometric mean concentration following Cecolin administration. Reactogenicity and safety were comparable between both vaccines. ConclusionsCecolin in a 0, 6-month schedule elicits robust immunogenicity. Non-inferiority to Gardasil was demonstrated one month after a 0, 6-month schedule. Immunogenicity following one dose was comparable to Gardasil up to six months. Both vaccines were safe and well tolerated (ClinicalTrials.gov No. 04508309).

Inter-epitope spacer variation within polytopic L2-based human papillomavirus antigens affects immunogenicity

The human papillomavirus minor capsid protein L2 is being extensively explored in pre-clinical studies as an attractive vaccine antigen capable of inducing broad-spectrum prophylactic antibody responses. Recently, we have developed two HPV vaccine antigens – PANHPVAX and CUT-PANHPVAX- both based on heptameric nanoparticle antigens displaying polytopes of the L2 major cross-neutralizing epitopes of eight mucosal and twelve cutaneous HPV types, respectively. Prompted by the variable neutralizing antibody responses against some of the HPV types targeted by the antigens observed in previous studies, here we investigated the influence on immunogenicity of six distinct glycine-proline spacers inserted upstream to a specific L2 epitope. We show that spacer variants differentially influence antigen immunogenicity in a mouse model, with the antigen constructs M8merV6 and C12merV6 displaying a superior ability in the induction of neutralizing antibodies as determined by pseudovirus-based neutralization assays (PBNAs). L2-peptide enzyme-linked immunosorbent assay (ELISA) assessments determined the total anti-L2 antibody level for each antigen variant, showing for the majority of sera a correlation with their repective neutralizing antibody level. Surface Plasmon Resonance revealed that L2 epitope-specific, neutralizing monoclonal antibodies (mAbs) display distinct avidities to different antigen spacer variants. Furthermore, mAb affinity toward individual spacer variants was well correlated with their neutralizing antibody induction capacity, indicating that the mAb affinity assay predicts L2-based antigen immunogenicity. These observations provide insights on the development and optimization of L2-based HPV vaccines.

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.

Skin-Grafting and Dendritic Cell "Boosted" Humanized Mouse Models Allow the Pre-Clinical Evaluation of Therapeutic Cancer Vaccines.

Vaccines have been hailed as one of the most remarkable medical advancements in human history, and their potential for treating cancer by generating or expanding anti-tumor T cells has garnered significant interest in recent years. However, the limited efficacy of therapeutic cancer vaccines in clinical trials can be partially attributed to the inadequacy of current preclinical mouse models in recapitulating the complexities of the human immune system. In this study, we developed two innovative humanized mouse models to assess the immunogenicity and therapeutic effectiveness of vaccines targeting human papillomavirus (HPV16) antigens and delivering tumor antigens to human CD141+ dendritic cells (DCs). Both models were based on the transference of human peripheral blood mononuclear cells (PBMCs) into immunocompromised HLA-A*02-NSG mice (NSG-A2), where the use of fresh PBMCs boosted the engraftment of human cells up to 80%. The dynamics of immune cells in the PBMC-hu-NSG-A2 mice demonstrated that T cells constituted the vast majority of engrafted cells, which progressively expanded over time and retained their responsiveness to ex vivo stimulation. Using the PBMC-hu-NSG-A2 system, we generated a hyperplastic skin graft model expressing the HPV16-E7 oncogene. Remarkably, human cells populated the skin grafts, and upon vaccination with a DNA vaccine encoding an HPV16-E6/E7 protein, rapid rejection targeted to the E7-expressing skin was detected, underscoring the capacity of the model to mount a vaccine-specific response. To overcome the decline in DC numbers observed over time in PBMC-hu-NSG-A2 animals, we augmented the abundance of CD141+ DCs, the specific targets of our tailored nanoemulsions (TNEs), by transferring additional autologous PBMCs pre-treated in vitro with the growth factor Flt3-L. The Flt3-L treatment bolstered CD141+ DC numbers, leading to potent antigen-specific CD4+ and CD8+ T cell responses in vivo, which caused the regression of pre-established triple-negative breast cancer and melanoma tumors following CD141+ DC-targeting TNE vaccination. Notably, using HLA-A*02-matching PBMCs for humanizing NSG-A2 mice resulted in a delayed onset of graft-versus-host disease and enhanced the efficacy of the TNE vaccination compared with the parental NSG strain. In conclusion, we successfully established two humanized mouse models that exhibited strong antigen-specific responses and demonstrated tumor regression following vaccination. These models serve as valuable platforms for assessing the efficacy of therapeutic cancer vaccines targeting HPV16-dysplastic skin and diverse tumor antigens specifically delivered to CD141+ DCs.

Therapeutic Efficacy of a VSV-GP-based Human Papilloma Virus Vaccine in a Murine Cancer Model

Human papilloma virus (HPV) infections are associated with almost all cervical cancers and to a lower extend also with anogenital or oropharyngeal cancers. HPV proteins expressed in HPV-associated tumors are attractive antigens for cancer vaccination strategies as self-tolerance, which is associated with most endogenous tumor-associated antigens, does not need to be overcome. In this study, we generated a live attenuated cancer vaccine based on the chimeric vesicular stomatitis virus VSV-GP, which has previously proven to be a potent vaccine vector and oncolytic virus. Genes at an earlier position in the genome more to the 3′ end are expressed stronger compared to genes located further downstream. By inserting an HPV16-derived antigen cassette consisting of E2, E6 and E7 into VSV-GP either at first (HPVp1) or fifth (HPVp5) position in VSV-GP’s genome we aimed to analyze the effect of vaccine antigen position and consequently expression level on viral fitness, immunogenicity, and anti-tumoral efficacy in a syngeneic mouse tumor model. HPVp1 expressed higher amounts of HPV antigens compared to HPVp5 in vitro but had a slightly delayed replication kinetic which overall translated into increased HPV-specific T cell responses upon vaccination of mice. Immunization with both vectors protected mice in prophylactic and in therapeutic TC-1 tumor models with HPVp1 being more effective in the prophylactic setting. Taken together, VSV-GP is a promising candidate as therapeutic HPV vaccine and first position of the vaccine antigen in a VSV-derived vector seems to be superior to fifth position.

Development and application of HPV31-specific monoclonal antibodies

Human papillomavirus (HPV) is the infectious etiological agent for diseases such as cervical cancer. Developing HPV vaccines is of great significance for cancer prevention. During the development of polyvalent HPV vaccines, such as 9-valent and 15-valent HPV vaccines, specifical identification of different HPV antigen is very important. The homemade HPV31 L1 VLPs was used as an immunogen to generate HPV 31 monoclonal antibodies (mAbs) through hybridoma cell culture. We generated several mAbs through hybridoma cell culture and selected two specific neutralizing antibodies, 19B2 and 19C4, which were further analyzed and characterized for binding activity, neutralizing activity, and specificity by enzyme-linked immunosorbent assays (ELISA). It is shown that both 19B2 and 19C4 could specifically recognize HPV31 L1 virus-like particles (VLPs). These two specific mAbs were further used to assemble a HPV31 antigen ELISA test kit. The assembled kit with high detection performance could be widely applied for the quantitative detection of HPV31 antigen during vaccine production as well as in the preliminary analysis of HPV31 infection.

Abstract C029: The pattern of mutant p53 protein expression in cervical cancer-associated single and co-infection with human papillomavirus and Epstein-Barr virus

Abstract Introduction: There is mounting evidence indicating that the co-existence of Human papillomavirus (HPV), and Epstein-Barr virus (EBV) is associated with aggressive and poorly differentiated squamous cell carcinomas (SCC) phenotype. However, there is a paucity of data on the prevalence of EBV in cases of cervical cancer in Southern Nigeria or the effect of both viruses on p53 gene expression among patients diagnosed with cervical cancer. Therefore, this study investigated the prevalence of mutant p53 (mtp53) in single and co-presence of HPV and EBV-LMP1 in invasive and non-invasive cases of cervical cancer in order to determine the role of EBV in cervical carcinogenesis. Methods: This retrospective cohort study included 105 viable archived formalin-fixed paraffin-embedded tissues of cervical cancer, diagnosed between 2016 and 2019 in two tertiary health care facilities in Southern Nigeria. Tissue sections were stained for mtp53, EBV-LMP1, and HPV using the immunohistochemical technique and scored based on staining intensity and documented as 0, +1, +2, and +3. Tissues with scores of 0 and +1 were considered negative whereas those with scores of +2 and +3 were considered positive. Descriptive, Chi-square test and Pearson’s correlation were carried out and statistical significance was set at ≤0.05. Result: The mean age of participants with cervical cancer was 53.32 ± 12.54 years. The prevalence of invasive and non-invasive cases of cervical cancer were 62.9% and 37.1%, respectively. The prevalence of well, moderately, and poorly differentiated cervical cancer were 54.3%, 34.3%, and 11.4%, respectively. The prevalence of single EBV-LMP1, and HPV antigen were 21.0% (n= 21) and 14.2% (n= 15), respectively whereas the prevalence of EBV-LMP1/HPV co-infection was 42.9% (n= 45). The prevalence of HPV/EBV-LMP1 co=presence was high in invasive cervical cancer compared with non-invasive cervical cancer cases (45.5% versus 30.8%) at p= 0.154. The prevalence of mtp53 was higher in EBV-LMP1/HPV co-presence compared with HPV, and EBV-LMP1 mono-presence (80.0%, 60.0%, and 14.3%, respectively) at p= 0.001. Conclusion: This study suggests that EBV increases the risk of cervical carcinogenesis in HPV-infected individuals in Southern Nigeria. Thus, EBV DNA should equally be investigated during HPV testing of suspected cervical cancer cases to identify individuals with poorer prognoses. Citation Format: Jude Okoye, Anthony Ajuluchukwu Ngokere, Charles Chinedum Onyenekwe, Ukpai Eze, Okechi Obioma. The pattern of mutant p53 protein expression in cervical cancer-associated single and co-infection with human papillomavirus and Epstein-Barr virus [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C029.

The association of cervicovaginal Langerhans cells with clearance of human papillomavirus.

Human papillomavirus (HPV) clearance is important in eliminating cervical cancer which contributes to high morbidity and mortality in women. Nevertheless, it remains largely unknown about key players in clearing pre-existing HPV infections. HPV antigens can be detected by the most important cervical antigen-presenting cells (Langerhans cells, LCs), of which the activities can be affected by cervicovaginal microbiota. In this review, we first introduce persistent HPV infections and then describe HPV-suppressed LCs activities, including but not limited to antigen uptake and presentation. Given specific transcriptional profiling of LCs in cervical epithelium, we also discuss the impact of cervicovaginal microbiota on LCs activation as well as the promise of exploring key microbial players in activating LCs and HPV-specific cellular immunity.

Albumin and interferon-β fusion protein serves as an effective vaccine adjuvant to enhance antigen-specific CD8+ T cell-mediated antitumor immunity

BackgroundType I interferons (IFN) promote dendritic cells maturation and subsequently enhance generation of antigen-specific CD8 +T cell for the control of tumor. Using type I interferons as an adjuvant to...

Binding antibody levels to vaccine (HPV6/11/16/18) and non-vaccine (HPV31/33/45/52/58) HPV antigens up to 7 years following immunization with either Cervarix® or Gardasil® vaccine

Human Papillomavirus (HPV) bivalent (Cervarix®) and quadrivalent (Gardasil®) vaccines demonstrate robust efficacy against vaccine types and cross-protection against related non-vaccine types. Here we evaluate the breadth, magnitude and durability of the vaccine-induced antibody response against vaccine (HPV6/11/16/18) and non-vaccine (HPV31/33/45/52/58) type antigens up to 7 years following vaccination of 12‐15 year old girls in a three dose schedule and contrast these data with the levels of antibody typically seen in natural infection. Vaccine-type antibody levels waned over the 7-year follow up period but remained at least an order of magnitude above the typical antibody levels elicited by natural infection. Seropositivity to non-vaccine types remained high 7 years after initial vaccination, but antibody levels approached those typically generated following natural infection. Empirical data on the breadth, magnitude, specificity and durability of the immune response elicited by the HPV vaccines contribute to improving the evidence base supporting this important public health intervention.

Therapeutic DNA Vaccines against HPV-Related Malignancies: Promising Leads from Clinical Trials.

In 2014 and 2021, two nucleic-acid vaccine candidates named MAV E2 and VGX-3100 completed phase III clinical trials in Mexico and U.S., respectively, for patients with human papillomavirus (HPV)-related, high-grade squamous intraepithelial lesions (HSIL). These well-tolerated but still unlicensed vaccines encode distinct HPV antigens (E2 versus E6+E7) to elicit cell-mediated immune responses; their clinical efficacy, as measured by HSIL regression or cure, was modest when compared with placebo or surgery (conization), but both proved highly effective in clearing HPV infection, which should help further optimize strategies for enhancing vaccine immunogenicity, toward an ultimate goal of preventing malignancies in millions of patients who are living with persistent, oncogenic HPV infection but are not expected to benefit from current, prophylactic vaccines. The major roadblocks to a highly efficacious and practical product remain challenging and can be classified into five categories: (i) getting the vaccines into the right cells for efficient expression and presentation of HPV antigens (fusion proteins or epitopes); (ii) having adequate coverage of oncogenic HPV types, beyond the current focus on HPV-16 and -18; (iii) directing immune protection to various epithelial niches, especially anogenital mucosa and upper aerodigestive tract where HPV-transformed cells wreak havoc; (iv) establishing the time window and vaccination regimen, including dosage, interval and even combination therapy, for achieving maximum efficacy; and (v) validating therapeutic efficacy in patients with poor prognosis because of advanced, recurrent or non-resectable malignancies. Overall, the room for improvements is still large enough that continuing efforts for research and development will very likely extend into the next decade.

Novel Antigenic Targets of HPV Therapeutic Vaccines.

Human papillomavirus (HPV) infection is the cause of the majority of cervical cancers and head and neck cancers worldwide. Although prophylactic vaccines and cervical cancer screening programs have shown efficacy in preventing HPV-associated cervical cancer, cervical cancer is still a major cause of morbidity and mortality, especially in third world countries. Furthermore, head and neck cancer cases caused by HPV infection and associated mortality are increasing. The need for better therapy is clear, and therapeutic vaccination generating cytotoxic T cells against HPV proteins is a promising strategy. This review covers the current scene of HPV therapeutic vaccines in clinical development and discusses relevant considerations for the design of future HPV therapeutic vaccines and clinical trials, such as HPV protein expression patterns, immunogenicity, and exhaustion in relation to the different stages and types of HPV-associated lesions and cancers. Ultimately, while the majority of the HPV therapeutic vaccines currently in clinical testing target the two HPV oncoproteins E6 and E7, we suggest that there is a need to include more HPV antigens in future HPV therapeutic vaccines to increase efficacy and find that especially E1 and E2 could be promising novel targets.

Tumor-infiltrating FoxP3+ T cells are associated with poor prognosis in oral squamous cell carcinoma.

ObjectivesSquamous cell carcinoma is the most common malignancy in the oral cavity. Moreover, human papillomavirus (HPV) infection has been recently implicated in the onset of oral squamous cell carcinoma (OSCC). Regulatory T cells (Tregs) are Forkhead box P3 (FoxP3) positive and are normally involved in the mechanism by which organisms escape attacks from their own immune system; however, in tumors, these cells are known to suppress antitumor immunity and block the attack against tumors. The present study evaluated the associations of the number of Tregs and HPV infection with prognoses in patients with OSCC.Material and methodsSamples from 106 patients diagnosed with OSCC were evaluated by immunohistochemical staining for the identification of FoxP3+ Tregs and HPV. The relationship between the observed number of Foxp3‐positive cells, the presence/absence of HPV infection and associations with clinicopathological indicators were analyzed.ResultsTissues were classified into high (High) and low (Low) Treg count groups, with 69 patients classified as High and 37 classified as Low. The prognoses were significantly better in the Low group compared with the High group (p = 0.04). FoxP3 expression may have had some effect on nodal metastases (p = 0.09). HPV antigens were detected in 65 patients, but there were no significant associations with prognosis (p = 0.34). HPV‐infected tumors were more common in the gums and tongues than in the lips, cheeks, and floor of the mouth (p = 0.05).ConclusionsThese results indicate that Tregs in tumor sites are associated with worsened prognoses of patients with OSCC and suggest potential therapies targeting Tregs in OSCC.

Human Papillomavirus (HPV)–Specific T-Cells Can be Generated from Unimmunized Donors for Third Party Cell Therapy of HPV-Associated Neoplasms

▪HPV is a known cause of oropharyngeal cancer, cervical cancer, and other genital neoplasms. Although treatments exist for HPV-associated malignancies, patients unresponsive to these therapies have a poor prognosis. Recent findings from vaccine studies suggest that T cell immunity is essential for disease control. As “off the shelf” third party EBV-specific T-cells have been successful in treating patients with EBV-associated tumors, we hypothesized that the development of a manufacturing platform for HPV-specific T cells from healthy donors could be used in a third party setting to treat patients with HPV-associated cancers. Most protocols for generating virus-specific T-cells require prior exposure of the donor to the targeted virus. Since the seroprevalence of high-risk HPV types varies greatly by age and ethnicity, manufacturing of donor-derived HPV-specific T cells is challenging. Hence, we implemented systematic changes to GMP-compliant protocols to improve antigen presentation, priming, and expansion for the manufacture of HPV-specific T cells.By optimizing dendritic cell maturation and function using lipopolysaccharide (LPS) and IFNg, and adding IL-21 for priming, we achieved reliable expansion of naïve T-cells specific for oncoproteins E6 and E7 of the HPV16 serotype to clinically relevant numbers (mean 578-fold expansion, n=10). Ex vivo expanded, HPV-specific T cells secreted IFNγ (137±148 IFNγ spot forming cells, SFC/1x105 cells and 68±145 IFNγ SFC/1x105cells against HPV E6 and E7 respectively, compared to 7±8 IFNγ SFC/1x105 cells against an irrelevant antigen, n=10) and other cytokines (in five evaluated products,T cells secreted high levels of IL6 (3792±966 pg/mL), IL7 (18000±0 pg/mL), IL8 (4766 ±2271 pg/mL), IP10 (234 ±220pg/mL), MIP1b (4915.848±2050.065 pg/mL) and TNFa (1945±823pg/mL). In contrast to T cells directed against other viral antigens, most T cells expanded against HPV antigens were CD4+ with a mean of 68.6±23.3% vs 18.2±20.9% CD8+ T cells (n=10). Expanded populations primarily recognized antigens through MHC class II confirmed by a decrease in responses to HPV antigen in the presence of HLA class II blocking antibodiesIn summary, we successfully generated HPV16 specific T cells from healthy donors, and established the conditions for a robust and reproducible. GMP-compliant protocol using this product as an off the shelf T cell immunotherapeutic for patients with HPV-positive malignancies. Other groups have previously developed T cell therapies for HPV targets, but most efforts have focused on generating autologous products for patients with HPV malignancies. Other studies using TCR gene modified T cells have been developed, but the strategy limits the HLA types that can be targeted. Since immune dysfunction frequently accompanies patients with HPV malignancies, the development of an “off the shelf” third-party HPV-specific T-cell product potentially represents a readily available effective cell therapeutic for patients with HPV-associated cancers. DisclosuresBollard:Torque: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Neximmune: Consultancy, Membership on an entity's Board of Directors or advisory committees.

ADXS11-001 LM-LLO as specific immunotherapy in cervical cancer

ABSTRACT Human papillomavirus (HPV) infection is a well-known cause of cervical cancer. Therapeutic cancer vaccines are part of the current therapeutic options for HPV-associated cancers. Axalimogen filolisbac (ADXS11-001) is an immunotherapy based on live attenuated Listeria monocytogenes-listeriolysin O (Lm-LLO), designed by biological engineering to secrete an antigen-adjuvant fusion protein, composed of a truncated fragment of LLO fused to HPV. The proposed mechanism of action is that Lm-based vectors infect antigen-presenting cells (APC) and secrete HPV-LLO fusion proteins within the APC cytoplasm, these proteins are processed and presented to cytotoxic T lymphocytes (CTL), thus generating a new population of CTLs specific to HPV antigens. These HPV-specific CTLs destroy HPV infected cells. ADXS11-001 has demonstrated safety results in phase I–II studies in women with cervical cancer and is being assessed in clinical trials in patients with HPV-positive anal canal and head and neck cancers.

Association of Human Papilloma Virus in Oral Squamous Cell Carcinoma: An Alarming Need for Human Papillomavirus 16 Screening in Cancer Patients.

Background:The percentage of cancers of the tongue and palatine tonsils has continued to increase by 2%–4% among younger men. This increased prevalence of a subsection of oropharyngeal carcinoma can be associated with human papillomavirus (HPV). Among the head-and-neck cancers, a strong association with HPV infection is evident with oropharyngeal cancers, particularly tonsillar and basal tongue cancers.Objectives:Oral carcinoma, with an overall incidence of 16.1 adults per 100,000, is one of the leading malignancies worldwide, presenting a noticeable geographic variation in its distribution. Squamous cell carcinoma (SCC) being the most common of all oral malignancies, the objective of the study is to detect the HPV antigen p16 over-expression in patients with oral SCC using immunohistochemistry (IHC).Materials and Methods:Oral SCC (OSCC) diagnosed formalin-fixed-paraffin embedded blocks were processed for IHC.Results:Out of 50 cases, 3 were deferred due to insufficient tumor sample and 2/47 cases were p16 positive and the site was the lateral border of the tongue.Conclusion:The HPV antigen overexpression in patients with OSCC was investigated to detect the incidence of HPV in SCC of oral cavity. P16 was used as a marker for the detection of OSCC using IHC in HPV-induced OSCC. Positives were detected thus concluding the significance of studying HPV expression during diagnosis.

Characterization of human papillomavirus (HPV) 16 E6 seropositive individuals without HPV-associated malignancies after 10 years of follow-up in the UK Biobank.

BackgroundAntibodies against the HPV16 oncoprotein E6 are promising biomarkers for HPV16-driven oropharyngeal cancer (HPV16-OPC) due to their high sensitivity and specificity, and prospective manifestation. In previous studies, 0•7% of controls without HPV-associated malignancies were HPV16 E6 seropositive of which only a minority is expected to develop HPV16-driven cancer. We aimed to characterise HPV16 E6 antibodies in individuals without HPV-associated malignancies.MethodsWe analysed serum antibodies against HPV16 E6, E7, L1 and HPV18 L1 in a random sample (n = 9,695) of the prospective UK Biobank cohort (UKB). Excluding individuals with potentially HPV-associated malignancies (n = 192), we assessed risk factors for seropositivity by logistic regression.FindingsIn individuals without potentially HPV-associated malignancies (n = 9,503), the HPV16 E6 seroprevalence was 0•8%. Seropositivity against HPV16 E6 and all other HPV antigens was strongly associated with sexual behaviour. The seroprevalence of HPV16 E6, L1 and HPV18 L1 increased with the number of lifetime sex partners (ptrend<0•005), and all HPV antibodies were associated with same-sex intercourse (ORE6 3•1, 95%CI 1•4–6•9; reference category: no same-sex intercourse). HPV16 E6 and L1 seropositivity were associated with young age (≤17 years) at sexual debut (ORE6 2•0, 95%CI 1•1–3•7) compared with individuals reporting sexual debut at age ≥20 years.InterpretationThis is the first study characterising HPV16 E6 antibodies in the general UK population. Their strong association with sexual behaviour, and overlapping risk factor profiles with other HPV antibodies support their relevance for HPV16-OPC disease prediction. However, additional risk stratification will be required to identify individuals at highest risk to develop HPV16-OPC.