It is time to improve the diagnostic workup of oropharyngeal cancer with circulating tumor HPV DNA: Systematic review and meta-analysis.

The possibility of detecting circulating tumor HPV DNA (ctHPVDNA) in plasma in patients with oropharyngeal cancer has been demonstrated in several reports. However, these data are from small cohorts and available tests for detection of ctHPVDNA are not fully validated. The aim is to evaluate sensitivity, specificity, and accuracy of ctHPVDNA by ddPCR to define its efficacy in the clinical setting for the diagnosis of HPV + OPSCC. A comprehensive search of three different databases: MEDLINE, Embase, and Cochrane Library databases. A total of 998 patients were evaluated from the 13 studies. OPSSC p16+ were 729, while controls p16- were 269. The meta-analytic study estimated the diagnostic performance of ctHPVDNA as follows: pooled sensitivity and specificity of 0.90 (95% CI: 0.82-0.94) and 0.94 (95% CI: 0.85-0.98), respectively; positive and negative likelihood ratios of 12.6 (95% CI: 4.9-32.1) and 0.05 (95% CI: 0.02-0.13), respectively. ddPCR for ctHPVDNA has good accuracy, sensitivity, and specificity for diagnosis of HPV + OPSCC. ctHPVDNA kinetic represents a great reliable opportunity to improve diagnostic and therapeutic management of cancer patients and could open new perspectives for understanding tumor biology.

hMENA isoforms regulate cancer intrinsic type I IFN signaling and extrinsic mechanisms of resistance to immune checkpoint blockade in NSCLC.

BACKGROUND: Understanding how cancer signaling pathways promote an immunosuppressive program which sustains acquired or primary resistance to immune checkpoint blockade (ICB) is a crucial step in improving immunotherapy efficacy. Among the pathways that can affect ICB response is the interferon (IFN) pathway that may be both detrimental and beneficial. The immune sensor retinoic acid-inducible gene I (RIG-I) induces IFN activation and secretion and is activated by actin cytoskeleton disturbance. The actin cytoskeleton regulatory protein hMENA, along with its isoforms, is a key signaling hub in different solid tumors, and recently its role as a regulator of transcription of genes encoding immunomodulatory secretory proteins has been proposed. When hMENA is expressed in tumor cells with low levels of the epithelial specific hMENA11a isoform, identifies non-small cell lung cancer (NSCLC) patients with poor prognosis. Aim was to identify cancer intrinsic and extrinsic pathways regulated by hMENA11a downregulation as determinants of ICB response in NSCLC. Here, we present a potential novel mechanism of ICB resistance driven by hMENA11a downregulation. METHODS: Effects of hMENA11a downregulation were tested by RNA-Seq, ATAC-Seq, flow cytometry and biochemical assays. ICB-treated patient tumor tissues were profiled by Nanostring IO 360 Panel enriched with hMENA custom probes. OAK and POPLAR datasets were used to validate our discovery cohort. RESULTS: Transcriptomic and biochemical analyses demonstrated that the depletion of hMENA11a induces IFN pathway activation, the production of different inflammatory mediators including IFNß via RIG-I, sustains the increase of tumor PD-L1 levels and activates a paracrine loop between tumor cells and a unique macrophage subset favoring an epithelial-mesenchymal transition (EMT). Notably, when we translated our results in a clinical setting of NSCLC ICB-treated patients, transcriptomic analysis revealed that low expression of hMENA11a, high expression of IFN target genes and high macrophage score identify patients resistant to ICB therapy. CONCLUSIONS: Collectively, these data establish a new function for the actin cytoskeleton regulator hMENA11a in modulating cancer cell intrinsic type I IFN signaling and extrinsic mechanisms that promote protumoral macrophages and favor EMT. These data highlight the role of actin cytoskeleton disturbance in activating immune suppressive pathways that may be involved in resistance to ICB in NSCLC.

Targeting tumor-stroma communication by blocking endothelin-1 receptors sensitizes high-grade serous ovarian cancer to PARP inhibition.

PARP inhibitors (PARPi) have changed the treatment paradigm of high-grade serous ovarian cancer (HG-SOC). However, the impact of this class of inhibitors in HG-SOC patients with a high rate of TP53 mutations is limited, highlighting the need to develop combinatorial therapeutic strategies to improve responses to PARPi. Here, we unveil how the endothelin-1/ET-1 receptor (ET-1/ET-1R) axis, which is overexpressed in human HG-SOC and associated with poor prognosis, instructs HG-SOC/tumor microenvironment (TME) communication via key pro-malignant factors and restricts the DNA damage response induced by the PARPi olaparib. Mechanistically, the ET-1 axis promotes the p53/YAP/hypoxia inducible factor-1α (HIF-1α) transcription hub connecting HG-SOC cells, endothelial cells and activated fibroblasts, hence fueling persistent DNA damage signal escape. The ET-1R antagonist macitentan, which dismantles the ET-1R-mediated p53/YAP/HIF-1α network, interferes with HG-SOC/stroma interactions that blunt PARPi efficacy. Pharmacological ET-1R inhibition by macitentan in orthotopic HG-SOC patient-derived xenografts synergizes with olaparib to suppress metastatic progression, enhancing PARPi survival benefit. These findings reveal ET-1R as a mechanistic determinant in the regulation of HG-SOC/TME crosstalk and DNA damage response, indicating the use of macitentan in combinatorial treatments with PARPi as a promising and emerging therapy.

The Effectiveness of Therapeutic Vaccines for the Treatment of Cervical Intraepithelial Neoplasia 3: A Systematic Review and Meta-Analysis.

Cervical cancer (CC) is a disease that affects many women worldwide, especially in low-income countries. The human papilloma virus (HPV) is the main causative agent of this disease, with the E6 and E7 oncoproteins being responsible for the development and maintenance of transformed status. In addition, HPV is also responsible for the appearance of cervical intraepithelial neoplasia (CIN), a pre-neoplastic condition burdened by very high costs for its screening and therapy. So far, only prophylactic vaccines have been approved by regulatory agencies as a means of CC prevention. However, these vaccines cannot treat HPV-positive women. A search was conducted in several databases (PubMed, Scopus, Web of Science, and ClinicalTrials.gov) to systematically identify clinical trials involving therapeutic vaccines against CIN 3. Histopathological regression data, immunological parameters, safety, DNA clearance, and vaccine efficacy were considered from each selected study, and from the 102 articles found, 8 were selected based on the defined inclusion criteria. Histopathological regression from CIN 3 to CIN < 1 was 22.1% (95% CI: 0.627−0.967; p-value = 0.024), showing a vaccine efficacy of 23.6% (95% CI; 0.666−0.876; p-value < 0.001). DNA clearance was assessed, and the risk of persistent HPV DNA was 23.2% (95% CI: 0.667−0.885; p-value < 0.001). Regarding immunological parameters, immune responses by specific T-HPV cells were more likely in vaccinated women (95% CI: 1.245−9.162; p-value = 0.017). In short, these studies favored the vaccine group over the placebo group. This work indicated that therapeutic vaccines are efficient in the treatment of CIN 3, even after accounting for publication bias.

Deconvolution of malignant pleural effusions immune landscape unravels a novel macrophage signature associated with worse clinical outcome in lung adenocarcinoma patients.

BACKGROUND: Immune checkpoint inhibitors are still unable to provide clinical benefit to the large majority of non-small cell lung cancer (NSCLC) patients. A deeper characterization of the tumor immune microenvironment (TIME) is expected to shed light on the mechanisms of cancer immune evasion and resistance to immunotherapy. Here, we exploited malignant pleural effusions (MPEs) from lung adenocarcinoma (LUAD) patients as a model system to decipher TIME in metastatic NSCLC. METHODS: Mononuclear cells from MPEs (PEMC) and peripheral blood (PBMC), cell free pleural fluid and/or plasma were collected from a total of 24 LUAD patients and 12 healthy donors. Bulk-RNA sequencing was performed on total RNA extracted from PEMC and matched PBMC. The DEseq2 Bioconductor package was used to perform differential expression analysis and CIBERSORTx for the regression-based immune deconvolution of bulk gene expression data. Cytokinome analysis of cell-free pleural fluid and plasma samples was performed using a 48-Plex Assay panel. THP-1 monocytic cells were used to assess macrophage polarization. Survival analyses on NSCLC patients were performed using KM Plotter (LUAD, N=672; lung squamous cell carcinoma, N=271). RESULTS: Transcriptomic analysis of immune cells and cytokinome analysis of soluble factors in the pleural fluid depicted MPEs as a metastatic niche in which all the components required for an effective antitumor response are present, but conscripted in a wound-healing, proinflammatory and tumor-supportive mode. The bioinformatic deconvolution analysis revealed an immune landscape dominated by myeloid subsets with the prevalence of monocytes, protumoral macrophages and activated mast cells. Focusing on macrophages we identified an MPEs-distinctive signature associated with worse clinical outcome in LUAD patients. CONCLUSIONS: Our study reports for the first time a wide characterization of MPEs LUAD microenvironment, highlighting the importance of specific components of the myeloid compartment and opens new perspectives for the rational design of new therapies for metastatic NSCLC.

Natural Bioactives: Back to the Future in the Fight against Human Papillomavirus? A Narrative Review.

Human papillomavirus (HPV) still represents an important threat to health worldwide. Better therapy in terms of further improvement of outcomes and attenuation of related side-effects is desirable. The pharmaceutical industry has always targeted natural substances-phytochemicals in particular-to identify lead compounds to be clinically validated and industrially produced as antiviral and anticancer drugs. In the field of HPV, numerous naturally occurring bioactives and dietary phytochemicals have been investigated as potentially valuable in vitro and in vivo. Interference with several pathways and improvement of the efficacy of chemotherapeutic agents have been demonstrated. Notably, some clinical trials have been conducted. Despite being endowed with general safety, these natural substances are in urgent need of further assessment to foresee their clinical exploitation. This review summarizes the basic research efforts conducted so far in the study of anti-HPV properties of bio-actives with insights into their mechanisms of action and highlights the variety of their natural origin in order to provide comprehensive mapping throughout the different sources. The clinical studies available are reported, as well, to highlight the need of uniformity and consistency of studies in the future to select those natural compounds that may be suited to clinical application.

Modifying Anthocyanins Biosynthesis in Tomato Hairy Roots: A Test Bed for Plant Resistance to Ionizing Radiation and Antioxidant Properties in Space.

Gene expression manipulation of specific metabolic pathways can be used to obtain bioaccumulation of valuable molecules and desired quality traits in plants. A single-gene approach to impact different traits would be greatly desirable in agrospace applications, where several aspects of plant physiology can be affected, influencing growth. In this work, MicroTom hairy root cultures expressing a MYB-like transcription factor that regulates the biosynthesis of anthocyanins in Petunia hybrida (PhAN4), were considered as a testbed for bio-fortified tomato whole plants aimed at agrospace applications. Ectopic expression of PhAN4 promoted biosynthesis of anthocyanins, allowing to profile 5 major derivatives of delphinidin and petunidin together with pelargonidin and malvidin-based anthocyanins, unusual in tomato. Consistent with PhAN4 features, transcriptomic profiling indicated upregulation of genes correlated to anthocyanin biosynthesis. Interestingly, a transcriptome reprogramming oriented to positive regulation of cell response to biotic, abiotic, and redox stimuli was evidenced. PhAN4 hairy root cultures showed the significant capability to counteract reactive oxygen species (ROS) accumulation and protein misfolding upon high-dose gamma irradiation, which is among the most potent pro-oxidant stress that can be encountered in space. These results may have significance in the engineering of whole tomato plants that can benefit space agriculture.

Reduction of Cell Proliferation by Acute C2H6O Exposure.

Endogenous acetaldehyde production from the metabolism of ingested alcohol exposes hematopoietic progenitor cells to increased genotoxic risk. To develop possible therapeutic strategies to prevent or reverse alcohol abuse effects, it would be critical to determine the temporal progression of acute ethanol toxicity on progenitor cell numbers and proliferative status. We followed the variation of the cell proliferation rate in bone marrow and spleen in response to acute ethanol intoxication in the MITO-Luc mouse, in which NF-Y-dependent cell proliferation can be assessed in vivo by non-invasive bioluminescent imaging. One week after ethanol administration, bioluminescent signals in bone marrow and spleen decreased below the level corresponding to physiological proliferation, and they progressively resumed to pre-treatment values in approximately 4 weeks. Boosting acetaldehyde catabolism by administration of an aldehyde dehydrogenase activity activator or administration of polyphenols with antioxidant activity partially restored bone marrow cells' physiological proliferation. These results indicate that in this mouse model, bioluminescent alteration reflects the reduction of the physiological proliferation rate of bone marrow progenitor cells due to the toxic effect of aldehydes generated by alcohol oxidation. In summary, this study presents a novel view of the impact of acute alcohol intake on bone marrow cell proliferation in vivo.

Multi-omic approach identifies a transcriptional network coupling innate immune response to proliferation in the blood of COVID-19 cancer patients.

Clinical outcomes of COVID-19 patients are worsened by the presence of co-morbidities, especially cancer leading to elevated mortality rates. SARS-CoV-2 infection is known to alter immune system homeostasis. Whether cancer patients developing COVID-19 present alterations of immune functions which might contribute to worse outcomes have so far been poorly investigated. We conducted a multi-omic analysis of immunological parameters in peripheral blood mononuclear cells (PBMCs) of COVID-19 patients with and without cancer. Healthy donors and SARS-CoV-2-negative cancer patients were also included as controls. At the infection peak, cytokine multiplex analysis of blood samples, cytometry by time of flight (CyTOF) cell population analyses, and Nanostring gene expression using Pancancer array on PBMCs were performed. We found that eight pro-inflammatory factors (IL-6, IL-8, IL-13, IL-1ra, MIP-1a, IP-10) out of 27 analyzed serum cytokines were modulated in COVID-19 patients irrespective of cancer status. Diverse subpopulations of T lymphocytes such as CD8+T, CD4+T central memory, Mucosal-associated invariant T (MAIT), natural killer (NK), and γδ T cells were reduced, while B plasmablasts were expanded in COVID-19 cancer patients. Our findings illustrate a repertoire of aberrant alterations of gene expression in circulating immune cells of COVID-19 cancer patients. A 19-gene expression signature of PBMCs is able to discriminate COVID-19 patients with and without solid cancers. Gene set enrichment analysis highlights an increased gene expression linked to Interferon α, γ, α/ß response and signaling which paired with aberrant cell cycle regulation in cancer patients. Ten out of the 19 genes, validated in a real-world consecutive cohort, were specific of COVID-19 cancer patients independently from different cancer types and stages of the diseases, and useful to stratify patients in a COVID-19 disease severity-manner. We also unveil a transcriptional network involving gene regulators of both inflammation response and proliferation in PBMCs of COVID-19 cancer patients.

ePCL Electrospun Microfibrous Layers for Immune Assays: Sensitive ELISA for the Detection of Serum Antibodies Against HPV16 E7 Oncoprotein.

Human papillomavirus (HPV) type 16 is the etiologic agent of more than 50% anal/cervical cancers and about 20% oropharyngeal cancers. HPV16 E6 and E7 oncogenes favor the transformation and are essential for maintaining the transformed status. Serum anti-E6 and anti-E7 antibodies appear to have prognostic significance for HPV-associated cancers. However, most of the previous attempts to establish diagnostic tools based on serum detection of E6 and/or E7 antibodies have been unsuccessful, mainly due to the low accuracy of applied tests. This paper reports on a feasibility study to prove the possibility to easily immobilize HPV16 E7 onto electrospun substrates for application in diagnostic tools. In this study, poly(ε-caprolactone) electrospun scaffolds (called ePCL) are used to provide a microstructured substrate with a high surface-to-volume ratio, capable of binding E7 proteins when used for enzyme-linked immunosorbent assay (ELISA) tests. ePCL functionalized with E7 exhibited superior properties compared to standard polystyrene plates, increasing the detection signal from serum antibodies by 5-6 times. Analysis of the serum samples from mice immunized with HPV16 E7 DNA vaccine showed higher efficiency of this new anti-E7 ePCL-ELISA test vs control in E7-specific antibody detection. In addition, ePCL-E7-ELISA is prepared with a relatively low amount of antigen, decreasing the manufacturing costs.

HPV Infection in Middle Ear Squamous Cell Carcinoma: Prevalence, Genotyping and Prognostic Impact.

Middle ear squamous cell carcinoma (MESCC) is rare. Human Papilloma Virus (HPV) infection has been found in a significant number of cases of MESCC. Despite the emerging role of HPV in oncogenesis, its role in the pathogenesis and prognosis of MESCC is not known. This study aims to identify the prognostic impact of alpha and beta HPV in MESCC and its correlation with p16 protein. We retrospectively investigated 33 patients with MESCC surgically treated between 2004 and 2016. HPV DNA was ascertained by polymerase chain reaction (PCR) and P16INK4a detection was performed. Disease-specific survival (DSS) and cumulative incidence of recurrence were calculated in relation to HPV presence and genotype. p16 sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in predicting HPV infection were calculated. HPV was detected in 66.7% of patients (36.4% alpha HPV, 63.6% beta HPV). Five-year DSS was 55.0% and was not statistically related to HPV presence (p = 0.55) or genotype (p = 0.87). Five-year cumulative incidence of recurrence was 46 %, and was not statistically related to HPV presence (p = 0.22) or genotype (p = 0.44). p16 sensitivity, specificity, PPV, and NPV in predicting HPV infection were 27.3%, 36.4%, 46.2%, and 20.0%, respectively. In our experience, beta HPV was more frequent than alpha HPV in MESCC. Neither HPV presence nor HPV genotypes relate to DSS or cumulative incidence of recurrence. p16 expression was not predictive for HPV infection in MESCC. The role of HPV infection in oncogenesis, maintenance, and prognosis of MESCC seems to be different from that in oropharynx and skin cancer.

Construction of a recombinant avipoxvirus expressing the env gene of Zika virus as a novel putative preventive vaccine.

BACKGROUND: Zika virus (ZIKV) has been declared a public health emergency that requires development of an effective vaccine, as it might represent an international threat. METHODS: Here, two novel DNA-based (pVAXzenv) and fowlpox-based (FPzenv) recombinant putative vaccine candidates were constructed that contained the cPrME genes of ZIKV. The env gene inserted into the fowlpox vector was verified for correct transgene expression by Western blotting and by immunofluorescence in different cell lines. The production of virus-like particles as a result of env gene expression was also demonstrated by electron microscopy. BALB/c mice were immunosuppressed with dexamethasone and immunized following a prime-boost strategy in a heterologous protocol where pVAXzenv was followed by FPzenv, to evaluate the immunogenicity of the Env protein. The mice underwent a challenge with an epidemic ZIKV after the last boost. RESULTS: These data show that the ZIKV Env protein was correctly expressed in both normal human lung fibroblasts (MRC-5 cells) and green monkey kidney (Vero) cells infected with FPzenv, and that the transgene expression lasted for more than 2 weeks. After mucosal administration of FPzenv, the immunized mice showed specific and significantly higher humoral responses compared to the control mice. However, virus neutralizing antibodies were not detected using plaque reduction assays. CONCLUSIONS: Although BALB/c mice appear to be an adequate model for ZIKV infection, as it mimics the natural mild infection in human beings, inadequate immune suppression seemed to occur by dexamethasone and different immune suppression strategies should be applied before challenge to reveal any protection of the mice.

Intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for therapy of established HPV-associated tumors.

BACKGROUND: The oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells. METHODS: Envisioning clinical application of the best characterized anti-HPV16 E6 and -HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed. RESULTS: We showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway. CONCLUSION: Based on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.

Plant-Derived Natural Compounds in Genetic Vaccination and Therapy for HPV-Associated Cancers.

Antigen-specific immunotherapy and, in particular, DNA vaccination provides an established approach for tackling human papillomavirus (HPV) cancers at different stages. DNA vaccines are stable and have a cost-effective production. Their intrinsic low immunogenicity has been improved by several strategies with some success, including fusion of HPV antigens with plant gene sequences. Another approach for the control of HPV cancers is the use of natural immunomodulatory agents like those derived from plants, that are able to interfere in carcinogenesis by modulating many different cellular pathways and, in some instances, to reduce chemo- and radiotherapy resistance of tumors. Indeed, plant-derived compounds represent, in many cases, an abundantly available, cost-effective source of molecules that can be either harvested directly in nature or obtained from plant cell cultures. In this review, an overview of the most relevant data reported in literature on the use of plant natural compounds and genetic vaccines that include plant-derived sequences against HPV tumors is provided. The purpose is also to highlight the still under-explored potential of multimodal treatments implying DNA vaccination along with plant-derived agents.

Beta HPV Type 15 Can Interfere With NF-κB Activity and Apoptosis in Human Keratinocytes.

E7 protein from cutaneous as well as mucosal HPV types can alter NF-κB activity. Conflicting literature data show a HPV-induced up- or down-regulation of the NF-κB pathway in different cell lines. In a previous study we detected the expression of E7 gene of HPV15 in a subungual tumor of a patient affected by incontinentia pigmenti (IP). IP is a rare X-linked genodermatosis in which the IKKγ gene is altered. From observations in transgenic IKKγ defective mice, it was suggested that IKK-deficient cells may undergo rapid hyper-proliferation and apoptosis/necrosis, leading to increased pro-inflammatory cytokine production in the neighboring IKK-positive cells. The objective of this study was to ascertain if beta HPV 15 can alter apoptosis and NF-κB pathway in normal and IKKγ-deficient keratinocytes. The human immortalized keratinocyte cell line (HaCaT), and human primary keratinocyte (HPK) cells were transduced with a retrovirus expressing E6-E7 proteins of HPV 15 and IKKγ was successful silenced mimicking the HPV15 infection and IP. HPV15 E6-E7 gene expression improved NF-κB activity in human keratinocytes even when IKKγ was silenced by siRNA. In IKKγ silenced keratinocyte cells, TNF-α-induced apoptosis was strongly reduced by the expression of HPV15 E6-E7 genes. Beta HPV15 exerted this anti-apoptotic activity by decreasing pro-apoptotic BAK and cleaved Caspase 3 proteins. In conclusion, we can speculate that presence of persistent infection by beta papillomavirus might influence the biological fate of IP by altering NF-κB activation and apoptosis in IKKγ mutated cells, favoring their survival and possibly the development of tumors in the late stage of disease. Taken together, our data reinforce the importance of host genetic background in the pathogenesis of HPV-associated skin lesions.

HPV16 E5 Mediates Resistance to PD-L1 Blockade and Can Be Targeted with Rimantadine in Head and Neck Cancer.

There is a critical need to understand mechanisms of resistance and to develop combinatorial strategies to improve responses to checkpoint blockade immunotherapy (CBI). Here, we uncover a novel mechanism by which the human papillomavirus (HPV) inhibits the activity of CBI in head and neck squamous cell carcinoma (HNSCC). Using orthotopic HNSCC models, we show that radiation combined with anti-PD-L1 immunotherapy significantly enhanced local control, CD8+ memory T cells, and induced preferential T-cell homing via modulation of vascular endothelial cells. However, the HPV E5 oncoprotein suppressed immune responses by downregulating expression of major histocompatibility complex and interfering with antigen presentation in murine models and patient tumors. Furthermore, tumors expressing HPV E5 were rendered entirely resistant to anti-PD-L1 immunotherapy, and patients with high expression of HPV16 E5 had worse survival. The antiviral E5 inhibitor rimantadine demonstrated remarkable single-agent antitumor activity. This is the first report that describes HPV E5 as a mediator of resistance to anti-PD-1/PD-L1 immunotherapy and demonstrates the antitumor activity of rimantadine. These results have broad clinical relevance beyond HNSCC to other HPV-associated malignancies and reveal a powerful mechanism of HPV-mediated immunosuppression, which can be exploited to improve response rates to checkpoint blockade. SIGNIFICANCE: This study identifies a novel mechanism of resistance to anti-PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/4/732/F1.large.jpg.

Bioproduction of a Therapeutic Vaccine Against Human Papillomavirus in Tomato Hairy Root Cultures.

Human papillomavirus (HPV) tumor disease is a critical public health problem worldwide, especially in the developing countries. The recognized pathogenic function of E5, E6, and E7 oncoproteins offers the opportunity to devise therapeutic vaccines based on engineered recombinant proteins. The potential of plants to manufacture engineered compounds for pharmaceutical purposes, from small to complex protein molecules, allows the expression of HPV antigens and, possibly, the regulation of immune functions to develop very specific therapies as a reinforcement to available nonspecific therapies and preventive vaccination also in developed countries. Among plant-based expression formats, hairy root cultures are a robust platform combining the benefits of eukaryotic plant-based bioreactors, with those typical of cell cultures. In this work, to devise an experimental therapeutic vaccine against HPV, hairy root cultures were used to express a harmless form of the HPV type 16 E7 protein (E7*) fused to SAPKQ, a noncytotoxic form of the saporin protein from Saponaria officinalis, that we had shown to improve E7-specific cell-mediated responses as a fusion E7*-SAPKQ DNA vaccine. Hairy root clones expressing the E7*-SAPKQ candidate vaccine were obtained upon infection of leaf explants of Solanum lycopersicum using a recombinant plant expression vector. Yield was approximately 35.5 µg/g of fresh weight. Mouse immunization with vaccine-containing crude extracts was performed together with immunological and biological tests to investigate immune responses and anticancer activity, respectively. Animals were primed with either E7*-SAPKQ DNA-based vaccine or E7*-SAPKQ root extract-based vaccine and boosted with the same (homologous schedule) or with the other vaccine preparation (heterologous schedule) in the context of TC-1 experimental mouse model of HPV-associated tumor. All the formulations exhibited an immunological response associated to anticancer activity. In particular, DNA as prime and hairy root extract as boost demonstrated the highest efficacy. This work, based on the development of low-cost technologies, highlights the suitability of hairy root cultures as possible biofactories of therapeutic HPV vaccines and underlines the importance of the synergic combination of treatment modalities for future developments in this field.

Immunotherapeutic Potential of Mollusk Hemocyanins in Combination with Human Vaccine Adjuvants in Murine Models of Oral Cancer.

Mollusk hemocyanins have been used for decades in immunological and clinical applications as natural, nontoxic, nonpathogenic, and nonspecific immunostimulants for the treatment of superficial bladder cancer, as carriers/adjuvants of tumor-associated antigens in cancer vaccine development and as adjuvants to dendritic cell-based immunotherapy, because these glycoproteins induce a bias towards Th1 immunity. Here, we analyzed the preclinical therapeutic potential of the traditional keyhole limpet hemocyanin (KLH) and two new hemocyanins from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH) in mouse models of oral squamous cell carcinoma. Due to the aggressiveness and deadly malignant potential of this cancer, the hemocyanins were applied in combination with adjuvants, such as alum, AddaVax, and QS-21, which have been shown to be safe and effective in human vaccines, to potentiate their antitumor activity. The immunogenic performance of the hemocyanins in combination with the adjuvants was compared, and the best formulation was evaluated for its antitumor effects in two murine models of oral cancer: MOC7 cells implanted in the flank (heterotopic) and bioluminescent AT-84 E7 Luc cells implanted in the floor of the mouth (orthotopic). The results demonstrated that the hemocyanins in combination with QS-21 showed the greatest immunogenicity, as reflected by a robust, specific humoral response predominantly characterized by IgG2a antibodies and a sustained cellular response manifesting as a delayed hypersensitivity reaction. The KLH- and FLH-QS-21 formulations showed reduced tumor development and greater overall survival. Hemocyanins, as opposed to QS-21, had no cytotoxic effect on either oral cancer cell line cultured in vitro, supporting the idea that the antitumor effects of hemocyanins are associated with their modulation of the immune response. Therefore, hemocyanin utilization would allow a lower QS-21 dosage to achieve therapeutic results. Overall, our study opens a new door to further investigation of the use of hemocyanins plus adjuvants for the development of immunotherapies against oral carcinoma.