Discovery and characterization of novel DNA viruses in Apis mellifera: expanding the honey bee virome through metagenomic analysis.

To date, many viruses have been discovered to infect honey bees. In this study, we used high-throughput sequencing to expand the known virome of the honey bee, Apis mellifera, by identifying several novel DNA viruses. While the majority of previously identified bee viruses are RNA, our study reveals nine new genomes from the Parvoviridae family, tentatively named Bee densoviruses 1 to 9. In addition, we characterized a large DNA virus, Apis mellifera filamentous-like virus (AmFLV), which shares limited protein identities with the known Apis mellifera filamentous virus. The complete sequence of AmFLV, obtained by a combination of laboratory techniques and bioinformatics, spans 152,678 bp. Linear dsDNA genome encodes for 112 proteins, of which 49 are annotated. Another large virus we discovered is Apis mellifera nudivirus, which belongs to a group of Alphanudivirus. The virus has a length of 129,467 bp and a circular dsDNA genome, and has 106 protein encoding genes. The virus contains most of the core genes of the family Nudiviridae. This research demonstrates the effectiveness of viral binning in identifying viruses in honey bee virology, showcasing its initial application in this field.IMPORTANCEHoney bees contribute significantly to food security by providing pollination services. Understanding the virome of honey bees is crucial for the health and conservation of bee populations and also for the stability of the ecosystems and economies for which they are indispensable. This study unveils previously unknown DNA viruses in the honey bee virome, expanding our knowledge of potential threats to bee health. The use of the viral binning approach we employed in this study offers a promising method to uncovering and understanding the vast viral diversity in these essential pollinators.

Varroa destructor parasitism and Deformed wing virus infection in honey bees are linked to peroxisome-induced pathways.

The ectoparasitic mite Varroa destructor transmits and triggers viral infections that have deleterious effects on honey bee colonies worldwide. We performed a manipulative experiment in which worker bees collected at emergence were exposed to Varroa for 72 h, and their proteomes were compared with those of untreated control bees. Label-free quantitative proteomics identified 77 differentially expressed A. mellifera proteins (DEPs). In addition, viral proteins were identified by orthogonal analysis, and most importantly, Deformed wing virus (DWV) was found at high levels/intensity in Varroa-exposed bees. Pathway enrichment analysis suggested that the main pathways affected included peroxisomal metabolism, cyto-/exoskeleton reorganization, and cuticular proteins. Detailed examination of individual DEPs revealed that additional changes in DEPs were associated with peroxisomal function. In addition, the proteome data support the importance of TGF-ß signaling in Varroa-DWV interaction and the involvement of the mTORC1 and Hippo pathways. These results suggest that the effect of DWV on bees associated with Varroa feeding results in aberrant autophagy. In particular, autophagy is selectively modulated by peroxisomes, to which the observed proteome changes strongly corresponded. This study complements previous research with different study designs and suggests the importance of the peroxisome, which plays a key role in viral infections.

Genome sequence of parvovirus from budgerigar (Melopsittacus undulatus).

Here, we report a parvovirus genome identified in Melopsittacus undulatus. The genome is 4,547 bp long and codes for two major open reading frames (ORFs): the non-structural replicase protein 1 (NS1) and the structural capsid gene (VP1). Phylogenetic analysis shows that this virus belongs to the genus Chaphamaparvovirus.

Can variants, reinfection, symptoms and test types affect COVID-19 diagnostic performance? A large-scale retrospective study of AG-RDTs during circulation of Delta and Omicron variants, Czechia, December 2021 to February 2022.

BackgroundThe sensitivity and specificity of selected antigen detection rapid diagnostic tests (AG-RDTs) for SARS-CoV-2 were determined in the unvaccinated population when the Delta variant was circulating. Viral loads, dynamics, symptoms and tissue tropism differ between Omicron and Delta.AimWe aimed to compare AG-RDT sensitivity and specificity in selected subgroups during Omicron vs Delta circulation.MethodsWe retrospectively paired AG-RDT results with PCRs registered in Czechia's Information System for Infectious Diseases from 1 to 25 December 2021 (Delta, n = 20,121) and 20 January to 24 February 2022 (Omicron, n = 47,104).ResultsWhen confirmatory PCR was conducted on the same day as AG-RDT as a proxy for antigen testing close to peak viral load, the average sensitivity for Delta was 80.4% and for Omicron 81.4% (p < 0.05). Sensitivity in vaccinated individuals was lower for Omicron (OR = 0.94; 95% confidence interval (CI): 0.87-1.03), particularly in reinfections (OR = 0.83; 95% CI: 0.75-0.92). Saliva AG-RDT sensitivity was below average for both Delta (74.4%) and Omicron (78.4%). Tests on the European Union Category A list had higher sensitivity than tests in Category B. The highest sensitivity for Omicron (88.5%) was recorded for patients with loss of smell or taste, however, these symptoms were almost 10-fold less common than for Delta. The sensitivity of AG-RDTs performed on initially asymptomatic individuals done 1, 2 or 3 days before a positive PCR test was consistently lower for Omicron compared with Delta.ConclusionSensitivity for Omicron was lower in subgroups that may become more common if SARS-CoV-2 becomes an endemic virus.

Urinary shedding of common DNA viruses and their possible association with bladder cancer: a qPCR-based study.

After a decade of human urinary microbiota research, little is known about the composition of the urinary virome and its association with health and disease. This study aimed to investigate the presence of 10 common DNA viruses in human urine and their putative association with bladder cancer (BC). Catheterized urine samples were collected from patients undergoing endoscopic urological procedures under anesthesia. After DNA extraction from the samples, viral DNA sequences were detected using real-time PCR. Viruria rates were compared between BC patients and controls. A total of 106 patients (89 males and 17 females) were included in the study. Fifty-seven (53.8%) were BC patients and 49 (46.2%) had upper urinary tract stones or bladder outlet obstruction. The viruses detected in the urine were human cytomegalovirus (2.0%), Epstein-Barr virus (6.0%), human herpesvirus-6 (12.5%), human papillomavirus (15.2%), BK polyomavirus (15.5%), torque teno virus (44.2%), and JC polyomavirus (47.6%), while no adenoviruses, herpes simplex virus 1 and 2, or parvoviruses were found. There were statistically significant differences in HPV viruria rates between cancer patients and controls (24.5% vs. 4.3%, p=0.032 after adjustment for age and gender). Viruria rates increased from benign to non-muscle-invasive and muscle-invasive tumors. Patients with a history of BC have higher HPV viruria rates than controls. Whether this relationship is a causal one remains to be established by further research.

PD1+CD8+ Cells Are an Independent Prognostic Marker in Patients with Head and Neck Cancer.

Head and neck squamous cell carcinomas (HNSCCs) belong to a group of diverse tumors, which can be induced by infection with human papillomavirus (HPV) or tobacco and alcohol consumption. The viral etiology of HNSCC relates to better clinical outcomes reflecting a different immune system response. Here, we retrospectively analyzed 97 tissue samples from oral and oropharyngeal carcinomas associated and non-associated with HPV infection using multispectral fluorescent immunohistochemistry. To evaluate the immune cell infiltration in tumor and stroma compartments, we designed four panels of four to five antibodies. We detected more T lymphocytes in the stroma, compared to the tumor parenchyma. In HPV positive (HPV+) in comparison to HPV negative (HPV-) tumors, higher counts of CD3+CD4+, CD3+CD8+, PD1+CD4+, PD1+CD8+ T cells, and ICOS- Treg cells were detected while more ICOS+ Treg cells and CTLA4+CD4+ T cells were observed in HPV- than in HPV+ tumors. The results of the univariate and multivariate analyses confirmed the predominant impact of HPV status on prognosis. More importantly, the number of CD8+PD-1+ T cells was identified as an independent factor, influencing the overall and/or disease-specific survival of patients with oral cavity or oropharyngeal carcinomas.

Role of population and test characteristics in antigen-based SARS-CoV-2 diagnosis, Czechia, August to November 2021.

BackgroundAnalyses of diagnostic performance of SARS-CoV-2 antigen rapid diagnostic tests (AG-RDTs) based on long-term data, population subgroups and many AG-RDT types are scarce.AimWe aimed to analyse sensitivity and specificity of AG-RDTs for subgroups based on age, incidence, sample type, reason for test, symptoms, vaccination status and the AG-RDT's presence on approved lists.MethodsWe included AG-RDT results registered in Czechia's Information System for Infectious Diseases between August and November 2021. Subpopulations were analysed based on 346,000 test results for which a confirmatory PCR test was recorded ≤ 3 days after the AG-RDT; 38 AG-RDTs with more than 100 PCR-positive and 300 PCR-negative samples were individually evaluated.ResultsAverage sensitivity and specificity were 72.4% and 96.7%, respectively. We recorded lower sensitivity for age groups 0-12 (65.5%) and 13-18 years (65.3%). The sensitivity level rose with increasing SARS-CoV-2 incidence from 66.0% to 76.7%. Nasopharyngeal samples had the highest sensitivity and saliva the lowest. Sensitivity for preventive reasons was 63.6% vs 86.1% when testing for suspected infection. Sensitivity was 84.8% when one or more symptoms were reported compared with 57.1% for no symptoms. Vaccination was associated with a 4.2% higher sensitivity. Significantly higher sensitivity levels pertained to AG-RDTs on the World Health Organization Emergency Use List (WHO EUL), European Union Common List and the list of the United Kingdom's Department of Health and Social Care.ConclusionAG-RDTs from approved lists should be considered, especially in situations associated with lower viral load. Results are limited to SARS-CoV-2 delta variant.

Prevalence of genital and oral human papillomavirus infection among psoriasis patients on biologic therapy.

Current knowledge about human papillomavirus (HPV) infection in psoriasis patients treated with biologics is limited. In this study we evaluated the prevalence of oral and genital HPV infection in psoriasis patients treated with biologics or topical therapy for at least 6 months. The presence of HPV DNA in oral rinse and genital smears was evaluated. In total, 267 patients who met the inclusion criteria and agreed to participate were enrolled: 110 (41.2%) on topical therapy, 84 (31.5%) on anti-TNF-alpha therapy, 31 (11.6%) on anti-IL-12/23 therapy and 42 (15.7%) on anti-IL-17 therapy. The presence of genital HPV infection was detected in 34.6% of men receiving anti-TNF-α treatment, in 25.0% of patients on anti-IL-12/23 and 18.8% of patients on anti-IL-17 therapy. The difference in prevalence was not statistically different from men on topical treatment (26.3%). Prevalence of oral HPV infection was higher across all of the biologic groups (11.9% for anti-TNF-α, 12.9% for anti-IL-12/23 and 19.0% for anti-IL-17) compared to patients on topical therapy (7.3%), but statistically significant only for anti-IL-17 (p < 0.05). The presence of oral HPV infection in patients treated with biologics was significantly higher (44.0%) in patients on long-term biologic treatment (>8 years) compared to patients taking biologics for a shorter period (9.1%; p < 0.01). Our results suggest that patients on biologics for psoriasis have a higher prevalence of oral HPV infection compared to patients on topical treatment. Long-term treatment with biologics seems to be associated with a higher prevalence of oral HPV infection, independent of previous conventional immunosuppressive therapy.

Outcomes After Human Papillomavirus Vaccination in Patients With Recurrent Respiratory Papillomatosis: A Nonrandomized Clinical Trial.

Importance: Recurrent respiratory papillomatosis (RRP) is a rare benign chronic disease of the larynx etiologically linked with the infection of low-risk human papillomavirus (HPV). Combination of surgical and immunomodulatory therapy has limited success. Possible use of prophylactic HPV vaccine that includes HPV-6 and HPV-11 antigens has been studied. Objective: To evaluate if the HPV vaccination is associated with a lower number of recurrences requiring surgical intervention in patients with new and recurrent RRP. Design, Setting, and Participants: This was a non-placebo-controlled intervention study. Enrollment data were collected from October 2011 to August 2013. The patients were followed up at 1 month, 12 months, and 5 years after the third dose of the vaccine and clinically monitored until December 31, 2018. Data were analyzed from 2019 to 2021. Altogether, 50 adults with active RRP were enrolled and followed up in referral centers. For the final outcome, follow-up data for 42 patients were available. Eight patients who did not fulfill the protocol were excluded. Interventions: All patients received HPV vaccine as an adjuvant treatment and were clinically followed up. When RRP progression or a significant recurrent lesion was detected, surgical removal via direct laryngoscopy was indicated. No adjuvant therapy with antiviral or biological agents was used. Main Outcomes and Measures: This study compared the prevaccination and postvaccination positivity for HPV-specific antibodies. The main outcome was the difference in the frequency of RRP recurrences in the prevaccination and postvaccination period. Results: A total of 50 patients with RRP were enrolled (median [SD] age, 41.5 [12.3] years [range, 21-73 years]; 39 [78%] men and 11 [22%] women). After HPV vaccination, patients with previously no HPV-specific antibodies showed seroconversion, and all patients developed 100-fold higher levels of HPV vaccine type-specific antibodies compared with the prevaccination period. In patients with recurrent RRP, decreased frequency of recurrences requiring surgical treatment was present after vaccination (from 0.85 to 0.36 recurrences/y). No difference in postvaccination recurrences was found between patients with newly diagnosed and recurrent RRP. Conclusions and Relevance: In this nonrandomized clinical trial, the frequency of RRP recurrences was significantly lower after HPV vaccination, and patients with RRP thus had a reduced burden of disease. Because no difference was detected in the frequency of recurrent postvaccination lesions in patients with new and recurrent disease, it appears that both groups showed equal benefit following HPV vaccination. These findings suggest that the earlier that patients with RRP receive HPV vaccine, the sooner they may show reduced burden of disease. Trial Registration: EudraCT Identifier: 2011-002667-14; ClinicalTrials.gov Identifier: NCT01375868.

The Virome of Healthy Honey Bee Colonies: Ubiquitous Occurrence of Known and New Viruses in Bee Populations.

Honey bees are globally important pollinators threatened by many different pathogens, including viruses. We investigated the virome of honey bees collected at the end of the beekeeping season (August/September) in Czechia, a Central European country. Samples were examined in biological replicates to assess the homogeneity, stability, and composition of the virome inside a single hive. By choice of healthy workers from colonies, where Varroa destructor was under control, we could identify ubiquitous bee viruses. Deformed wing virus (DWV) was highly prevalent, even though the bees were healthy, without any noticeable disease signs. The overall virome composition (consisting of honey bee-, plant-, and bacterium-infecting viruses) was driven primarily by the hive and its location. However, honey bee-specific viruses showed an uneven distribution within the same hive. In addition, our results point to an unusual cooccurrence between two rhabdoviruses and reveal the presence of five distinct lineages of Lake Sinai viruses (LSVs) clustering with other LSV strains described globally. Comparison of our results with the virome of Australian honey bees, the last truly Varroa- and DWV-free population, showed a strong difference with respect to DWV and a set of diverse members of the Picornavirales, of which the latter were absent in our samples. We hypothesize that the occurrence of DWV introduced by Varroa strongly affects the virome structure despite the mite being under control. IMPORTANCE The Western honey bee, Apis mellifera, is a vital part of our ecosystem as well as cultural heritage. Annual colony losses endanger beekeeping. In this study, we examined healthy bees from the heart of Central Europe, where honey bee colonies have been commonly affected by varroosis over 5 decades. Our virome analysis showed the presence of ubiquitous viruses in colonies where the mite Varroa destructor was under control and no honey bee disease signs were observed. Compared to previous studies, an important part of our study was the analysis of multiple replicates from individual hives. Our overall results indicate that the virome structure (including bee-infecting viruses, plant-infecting viruses, and bacteriophages) is stable within hives; however, the bee-infecting viruses varied largely within interhive replicates, suggesting variation of honey bee viruses within individual bees. Of interest was the striking difference between the viromes of our 39 pools and 9 pools of honey bee viromes previously analyzed in Australia. It could be suggested that Varroa not only affects DWV spread in bee colonies but also affects diverse members of the Picornavirales, which were strongly decreased in Czech bees compared to the Varroa- and DWV-naive Australian bees.

Lack of Conserved miRNA Deregulation in HPV-Induced Squamous Cell Carcinomas.

Squamous cell carcinomas (SCCs) in the anogenital and head and neck regions are associated with high-risk types of human papillomaviruses (HR-HPV). Deregulation of miRNA expression is an important contributor to carcinogenesis. This study aimed to pinpoint commonly and uniquely deregulated miRNAs in cervical, anal, vulvar, and tonsillar tumors of viral or non-viral etiology, searching for a common set of deregulated miRNAs linked to HPV-induced carcinogenesis. RNA was extracted from tumors and nonmalignant tissues from the same locations. The miRNA expression level was determined by next-generation sequencing. Differential expression of miRNAs was calculated, and the patterns of miRNA deregulation were compared between tumors. The total of deregulated miRNAs varied between tumors of different locations by two orders of magnitude, ranging from 1 to 282. The deregulated miRNA pool was largely tumor-specific. In tumors of the same location, a low proportion of miRNAs were exclusively deregulated and no deregulated miRNA was shared by all four types of HPV-positive tumors. The most significant overlap of deregulated miRNAs was found between tumors which differed in location and HPV status (HPV-positive cervical tumors vs. HPV-negative vulvar tumors). Our results imply that HPV infection does not elicit a conserved miRNA deregulation in SCCs.

ARG1 mRNA Level Is a Promising Prognostic Marker in Head and Neck Squamous Cell Carcinomas.

Head and neck squamous cell carcinomas (HNSCC) can be induced by smoking or alcohol consumption, but a growing part of cases relate to a persistent high-risk papillomavirus (HPV) infection. Viral etiology has a beneficial impact on the prognosis, which may be explained by a specific immune response. Tumor associated macrophages (TAMs) represent the main immune population of the tumor microenvironment with a controversial influence on the prognosis. In this study, the level, phenotype, and spatial distribution of TAMs were evaluated, and the expression of TAM-associated markers was compared in HPV positive (HPV+) and HPV negative (HPV-) tumors. Seventy-three formalin and embedded in paraffin (FFPE) tumor specimens were examined using multispectral immunohistochemistry for the detection of TAM subpopulations in the tumor parenchyma and stroma. Moreover, the mRNA expression of TAM markers was evaluated using RT-qPCR. Results were compared with respect to tumor etiology, and the prognostic significance was evaluated. In HPV- tumors, we observed more pro-tumorigenic M2 in the stroma and a non-macrophage arginase 1 (ARG1)-expressing population in both compartments. Moreover, higher mRNA expression of M2 markers-cluster of differentiation 163 (CD163), ARG1, and prostaglandin-endoperoxide synthase 2 (PTGS2)-was detected in HPV- patients, and of M1 marker nitric oxide synthase 2 (NOS2) in HPV+ group. The expression of ARG1 mRNA was revealed as a negative prognostic factor for overall survival of HNSCC patients.

Aspartate ß-hydroxylase as a target for cancer therapy.

As metastasis is a major cause of death in cancer patients, new anti-metastatic strategies are needed to improve cancer therapy outcomes. Numerous pathways have been shown to contribute to migration and invasion of malignant tumors. Aspartate ß-hydroxylase (ASPH) is a key player in the malignant transformation of solid tumors by enhancing cell proliferation, migration, and invasion. ASPH also promotes tumor growth by stimulation of angiogenesis and immunosuppression. These effects are mainly achieved via the activation of Notch and SRC signaling pathways. ASPH expression is upregulated by growth factors and hypoxia in different human tumors and its inactivation may have broad clinical impact. Therefore, small molecule inhibitors of ASPH enzymatic activity have been developed and their anti-metastatic effect confirmed in preclinical mouse models. ASPH can also be targeted by monoclonal antibodies and has also been used as a tumor-associated antigen to induce both cluster of differentiation (CD) 8+ and CD4+ T cells in mice. The PAN-301-1 vaccine against ASPH has already been tested in a phase 1 clinical trial in patients with prostate cancer. In summary, ASPH is a promising target for anti-tumor and anti-metastatic therapy based on inactivation of catalytic activity and/or immunotherapy.

Prevalence and Risk Factors for Oral HPV in Healthy Population, in Central Europe.

Background/Aim: The incidence of oropharyngeal tumours induced by human papillomaviruses (HPV) is ever increasing. Information about oral HPV prevalence and its risk factors are very important for future screening and early diagnosis of the disease. The present study aimed to assess oral HPV prevalence in healthy population and risk factors for HPV infection, since this data is scarce or even missing in Central Europe. Patients and Methods: HPV prevalence in oral rinse and HPV-specific antibodies in peripheral blood were investigated in two groups of healthy participants. Group I consisted of 294 students who reached sexual maturity after the HPV vaccine had been licensed with mean age 23.2 years, and Group II of 215 unvaccinated participants with the mean age 55.7 years. Additionally, the risk factors were evaluated. Results: In Group I, 2% of participants were positive for oral HPV DNA. A statistically significantly higher rate (8.8%) was found in Group II. The seropositivity rates for anamnestic HPV antibodies were comparable in both groups. None of the analysed risk factors was significantly associated with oral HPV positivity. Conclusion: The lower prevalence of oral HPV DNA in younger participants suggests the positive influence of vaccination against oral HPV.

Prognostic value of posttreatment HPV-specific antibodies in patients with oropharyngeal tumors.

BACKGROUND: The presence of human papillomavirus (HPV)-specific antibodies in patients with head and neck cancer at enrollment has prognostic significance. In cervical carcinoma patients, the decrease of HPV E6/E7-specific antibodies appears to be associated with a better prognosis. METHODS: This prospective study with follow-up focused on the persistence and prognostic value of antibodies specific for HR HPV-derived VLPs and HPV16 E6/E7 oncoproteins in patients with oropharyngeal cancers. In this study, we analyzed sera of 93 patients taken a year after the end of treatment and sera from 58 of these patients taken up to 14 years after treatment. RESULTS: The level of HPV-specific antibodies decreased on the 1-year follow-up and the decrease during the long follow-up was statistically significant. For HPV16 E7 antibodies the decrease was steeper in nonrecurrent patients. While the level of antibodies at enrollment was not predictive of recurrences, the decrease of HPV16 E6 antibodies at 1-year follow up was associated with better overall as well as disease-specific survival of patients. CONCLUSIONS: The data suggest that the pretreatment level of HPV-specific antibodies is not predictive of the occurrence of recurrences but the decrease HPV16 E6 antibodies on the 1-year follow-up is predictive of better survival of HN patients.

Implementation of Mass Cytometry for Immunoprofiling of Patients with Solid Tumors.

Monitoring immune responses to solid cancers may be a better prognostic tool than conventional staging criteria, and it can also serve as an important criterion for the selection of individualized therapy. Multiparametric phenotyping by mass cytometry extended possibilities for immunoprofiling. However, careful optimization of each step of such method is necessary for obtaining reliable results. Also, with respect to procedure length and costs, sample preparation, staining, and storage should be optimized. Here, we designed a panel of 31 antibodies which allows for identification of several subpopulations of lymphoid and myeloid cells in a solid tumor and peripheral blood simultaneously. For sample preparation, disaggregation of tumor tissue with two different collagenases combined with DNase I was compared, and removal of dead or tumor cells by magnetic separation was evaluated. Two possible procedures of barcoding for single-tube staining of several samples were examined. While the palladium-based barcoding affected the stability of several antigens, the staining with two differently labeled CD45 antibodies was suitable for cells isolated from a patient's blood and tumor. The storage of samples in the intercalation solution for up to two weeks did not influence results of the analysis, which allowed the measurement of samples collected within this interval on the same day. This procedure optimized on samples from patients with head and neck squamous cell carcinoma enabled identification of various immune cells including rare subpopulations.

Genome-wide miRNA profiling reinforces the importance of miR-9 in human papillomavirus associated oral and oropharyngeal head and neck cancer.

Head and neck cancer is the sixth most common malignancy worldwide, predominantly developing from squamous cell epithelia (HNSCC). The main HNSCC risk factors are tobacco, excessive alcohol use, and the presence of human papillomavirus (HPV). HPV positive (+) cancers are etiologically different from other HNSCC and often show better prognosis. The current knowledge regarding HNSCC miRNA profiles is still incomplete especially in the context of HPV+ cancer. Thus, we analyzed 61 freshly collected primary oral (OSCC) and oropharyngeal (OPSCC) SCC samples. HPV DNA and RNA was found in 21% cases. The Illumina whole-genome small-RNA profiling by next-generation sequencing was done on 22 samples and revealed 7 specific miRNAs to HPV+ OSCC, 77 to HPV+ OPSCC, and additional 3 shared with both; 51 miRNAs were specific to HPV- OPSCC, 62 to HPV- OSCC, and 31 shared with both. The results for 9 miRNAs (miR-9, -21, -29a, -100, -106b, -143 and -145) were assessed by reverse transcription-quantitative polymerase chain reaction on the whole study population. The data was additionally confirmed by reanalyzing publicly available miRNA sequencing Cancer Genome Atlas consortium (TCGA) HNSCC data. Cell signaling pathway analysis revealed differences between HPV+ and HPV- HNSCC. Our findings compared with literature data revealed extensive heterogeneity of miRNA deregulation with only several miRNAs consistently affected, and miR-9 being the most likely HPV related miRNA.

Detailed Characteristics of Tonsillar Tumors with Extrachromosomal or Integrated Form of Human Papillomavirus.

The human papillomavirus (HPV) integration, the critical step in viral carcinogenesis, most frequently occurs in the E2 gene, which results in its inactivation and in an increase of E6/E7 transcription. However, in a substantial number of tumors, the virus is present in an extrachromosomal form. For those tumors, the transformation mechanisms are not fully elucidated. Here we evaluated the possible mechanism of inactivating the E2 without interruption of the gene, methylation or mutation of the E2 binding sites (E2BSs) in HPV16-positive tonsillar tumors by next-generation and Sanger sequencing. Viral genome status was analyzed by the amplification of papillomavirus oncogene transcripts assay (APOT) and mRNA mapping, and expression of viral oncogenes was performed by qPCR. The methylation of E2BSs was significantly higher in tumors with an integrated, in comparison to extrachromosomal, form of the viral genome. No mutations were detected in the E2BSs. The viral oncogenes were equally expressed in samples with an integrated and extrachromosomal form of the virus. Only the nucleotide variants were identified in the E2 gene. No proposed mechanism of E2 inactivation was confirmed in tonsillar tumors with an extrachromosomal form of the HPV genome. The expression of E6/E7 genes seems to be sufficient to initiate and maintain the carcinogenic process.

Experimental Combined Immunotherapy of Tumours with Major Histocompatibility Complex Class I Downregulation.

Combined immunotherapy constitutes a novel, advanced strategy in cancer treatment. In this study, we investigated immunotherapy in the mouse TC-1/A9 model of human papillomavirus type 16 (HPV16)-associated tumors characterized by major histocompatibility complex class I (MHC-I) downregulation. We found that the induction of a significant anti-tumor response required a combination of DNA vaccination with the administration of an adjuvant, either the synthetic oligodeoxynucleotide ODN1826, carrying immunostimulatory CpG motifs, or α-galactosylceramide (α-GalCer). The most profound anti-tumor effect was achieved when these adjuvants were applied in a mix with a one-week delay relative to DNA immunization. Combined immunotherapy induced tumor infiltration with various subsets of immune cells contributing to tumor regression, of which cluster of differentiation (CD) 8⁺ T cells were the predominant subpopulation. In contrast, the numbers of tumor-associated macrophages (TAMs) were not markedly increased after immunotherapy but in vivo and in vitro results showed that they could be repolarized to an anti-tumor M1 phenotype. A blockade of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) immune checkpoint had a negligible effect on anti-tumor immunity and TAMs repolarization. Our results demonstrate a benefit of combined immunotherapy comprising the activation of both adaptive and innate immunity in the treatment of tumors with reduced MHC-I expression.

Dysfunction of HPV16-specific CD8+ T cells derived from oropharyngeal tumors is related to the expression of Tim-3 but not PD-1.

BACKGROUND: Human papillomavirus (HPV) type 16 infection is one of the most important etiological agents of oropharyngeal squamous cell carcinoma. Patients with HPV-associated carcinomas of the head and neck were reported to have a better clinical outcome than patients with HPV-negative tumors. Because HPV16 E6 and E7 oncoproteins are highly immunogenic and constitutively expressed, HPV-specific T cell immunity may play the key role in improving the prognosis of these patients. METHODS: Tumor-derived T cells were expanded in high levels of IL-2 and stimulated with HPV16 E6/E7 peptides in the presence or absence of anti-PD-1 monoclonal antibody nivolumab and soluble Tim-3. RESULTS: HPV16-specific tumor-infiltrating T cells were present in 73.1% of HPV-associated oropharyngeal tumors. HPV16 specific CD8+ TILs were able to produce IFNγ upon specific stimulation and predominantly expressed PD-1 but not Tim-3. Specific IFNγ production was further enhanced after a blockade of both PD-1 and Tim-3 pathways but not after a PD-1 blockade alone. Additionally, the specific stimulation of anti-HPV16 CD8+ T cells suppressed Tim-3 upregulation after the PD-1 blockade. CONCLUSION: Our data provide the rationale for combination cancer immunotherapy approaches, including the dual blockade of PD-1 and Tim-3 and, potentially, the use of HPV16-directed therapeutic vaccines.