Restriction of viral gene expression and replication prevents immortalization of human keratinocytes by a beta-human papillomavirus.

SignificanceHigh-risk (HR) human papillomaviruses (HPV) from the genus alpha cause anogenital and oropharyngeal cancers, whereas the contribution of HPV from the genus beta to the development of cutaneous squamous cell cancer is still under debate. HR-HPV genomes display potent immortalizing activity in human keratinocytes, the natural target cell for HPV. This paper shows that immortalization of keratinocytes by the beta-HPV49 genome requires the inactivation of the viral E8^E2 repressor protein and the presence of the E6 and E7 oncoproteins but also of the E1 and E2 replication proteins. This reveals important differences in the carcinogenic properties of HR-HPV and beta-HPV but also warrants further investigations on the distribution and mutation frequencies of beta-HPV in human cancers.

Prevalence of Human Papillomavirus (HPV) and HPV Type Distribution in Penile Samples in Young Men in Denmark: Results 10 Years After Implementation of a Girls-Only HPV Vaccination Program.

BACKGROUND: In Denmark, a girls-only human papillomavirus (HPV) vaccination program was initiated in 2008-2009. The study aim was to assess the HPV prevalence and type distribution in younger men prior to HPV vaccination in men. METHODS: The study population was younger men who attended information days regarding military service. At random days (2019-2020), 280 men were included. We collected questionnaire data regarding risk factors for HPV infection and a penile swab for HPV testing. We compared results in this study with those from a previous study of young men (2006-2007). RESULTS: The majority of participants (94%) were 18-20 years old. The median number of lifetime sexual partners was 4. Altogether, 130 men (46.4%) were HPV positive. No infections with HPV types 6, 11, 16, 18, 31, and 45 were detected. The most frequent type was HPV-51 (detected in 11.1%). Comparison showed that the odds of high-risk HPV type infection were higher in 2019-2020 (prevalence odds ratio [POR], 1.7 [95% confidence interval {CI}, 1.1-2.7]) compared with 2006-2007. In contrast, the odds were lower (POR, 0.3 [95% CI, .1-.6]) for HPV types targeted by the 9-valent HPV vaccine. CONCLUSIONS: The multicohort girls-only vaccination program has to a large degree protected young men against the HPV types included in the licensed vaccines. This does not speak against gender-neutral vaccination as the HPV prevalence is still high, although consisting largely of less carcinogenic HPV types.

Evidence for direct interaction between the oncogenic proteins E6 and E7 of high-risk human papillomavirus (HPV).

Human papillomaviruses (HPVs) are DNA tumor viruses that infect mucosal and cutaneous epithelial cells of more than 20 vertebrates. High-risk HPV causes about 5% of human cancers worldwide, and the viral proteins E6 and E7 promote carcinogenesis by interacting with tumor suppressors and interfering with many cellular pathways. As a consequence, they immortalize cells more efficiently in concert than individually. So far, the networks of E6 and E7 with their respective cellular targets have been studied extensively but independently. However, we hypothesized that E6 and E7 might also interact directly with each other in a novel interaction affecting HPV-related carcinogenesis. Here, we report a direct interaction between E6 and E7 proteins from carcinogenic HPV types 16 and 31. We demonstrated this interaction via cellular assays using two orthogonal methods: coimmunoprecipitation and flow cytometry-based FRET assays. Analytical ultracentrifugation of the recombinant proteins revealed that the stoichiometry of the E6/E7 complex involves two E7 molecules and two E6 molecules. In addition, fluorescence polarization showed that (I) E6 binds to E7 with a similar affinity for HPV16 and HPV31 (in the same micromolar range) and (II) that the binding interface involves the unstructured N-terminal region of E7. The direct interaction of these highly conserved papillomaviral oncoproteins may provide a new perspective for studying HPV-associated carcinogenesis and the overall viral life cycle.

Molecular characterization of human adenoviruses associated with pediatric respiratory infections in Karachi, Pakistan.

Human adenoviruses (HAdVs) are a diverse group of viruses associated with respiratory infections in humans worldwide. However, there is a lack of research on the genetic diversity and epidemiology of HAdVs in Pakistan. This study characterized HAdVs in pediatric patients with respiratory tract infections in Karachi, Pakistan, between 2022 and 2023. We analyzed 762 nasopharyngeal samples of children ≤ 5 years. DNA extraction, followed by PCR targeting E2B and hexon genes, was carried out. Data analysis was performed on SPSS 25.0, and phylogenetic analysis of hexon gene was performed on MEGA 11. HAdV was detected in 7.34% (56/762) of patients round the year, but at a significantly higher rate during the winter season. Age was insignificantly associated with HAdV incidence (p = 0.662), but more than 62.5% (35/56) of positive cases were younger than 10 months. The circulating HAdVs were identified as six different types from species B (78.57%) and C (21.42%), with the majority of isolates found to be like B3. HAdV was found to be co-infected with bocavirus (5.4%) and measles (7.14%). These findings revealed a high frequency and genetic diversity of respiratory HAdVs in Karachi, Pakistan. We conclude that periodic and continuous surveillance of adenoviruses and other respiratory pathogens is necessary to improve the prognosis and management of respiratory diseases, thereby reducing the child mortality rate in Pakistan.

Comparison of human papillomavirus-based cervical cancer screening strategies in Tanzania among women with and without HIV.

Cervical cancer is the most common female cancer in Eastern Africa, and the World Health Organization (WHO) recommends human papillomavirus (HPV)-based screening as a key element to eliminate the disease. In this cross-sectional study from Tanzania, we compared nine HPV-based cervical cancer screening strategies, including HPV testing at standard cut-off; HPV testing at increased viral load cut-offs; HPV testing with partial/extended genotyping, and HPV testing with visual inspection with acetic acid (VIA). We pooled data collected during 2008 to 2009 and 2015 to 2017 from 6851 women aged 25 to 65. Cervical cytology samples were HPV tested with Hybrid Capture 2, and HPV positive samples were genotyped with INNO-LiPA Extra II. Human immunodeficiency virus (HIV) testing and VIA were done according to local standards. We calculated sensitivity, specificity, positive and negative predictive value of screening strategies, with high-grade cytological lesions as reference, separately for women with and without HIV. HPV testing at standard cut-off (1.0 relative light units [RLU]) had highest sensitivity (HIV+: 97.8%; HIV-: 91.5%), but moderate specificity (HIV+: 68.1%; HIV-: 85.7%). Increasing the cut-off for HPV positivity to higher viral loads (5.0/10.0 RLU) increased specificity (HIV+: 74.2%-76.5%; HIV-: 89.5%-91.2%), with modest sensitivity reductions (HIV+: 91.3%-95.7%; HIV-: 83.5%-87.8%). Limiting test positivity to HPV types 16/18/31/33/35/45/52/58 improved specificity while maintaining high sensitivity (HIV+: 90.2%; HIV-: 81.1%). Triage with VIA and/or partial genotyping for HPV16/18 or HPV16/18/45 had low sensitivities (≤65%). In conclusion, HPV testing alone, or HPV testing with extended genotyping or increased viral load cut-offs, may improve cervical cancer screening in Sub-Saharan Africa.

Detection of circulating cell-free HPV DNA of 13 HPV types for patients with cervical cancer as potential biomarker to monitor therapy response and to detect relapse.

BACKGROUND: HPV-related cervical cancer (CC) is the fourth most frequent cancer in women worldwide. Cell-free tumour DNA is a potent biomarker to detect treatment response, residual disease, and relapse. We investigated the potential use of cell-free circulating HPV-DNA (cfHPV-DNA) in plasma of patients with CC. METHODS: cfHPV-DNA levels were measured using a highly sensitive next-generation sequencing-based approach targeting a panel of 13 high-risk HPV types. RESULTS: Sequencing was performed in 69 blood samples collected from 35 patients, of which 26 were treatment-naive when the first liquid biopsy sample was retrieved. cfHPV-DNA was successfully detected in 22/26 (85%) cases. A significant correlation between tumour burden and cfHPV-DNA levels was observed: cfHPV-DNA was detectable in all treatment-naive patients with advanced-stage disease (17/17, FIGO IB3-IVB) and in 5/9 patients with early-stage disease (FIGO IA-IB2). Sequential samples revealed a decrease of cfHPV-DNA levels in 7 patients corresponding treatment response and an increase in a patient with relapse. CONCLUSIONS: In this proof-of-concept study we demonstrated the potential of cfHPV-DNA as a biomarker for therapy monitoring in patients with primary and recurrent CC. Our findings facilitate the development of a sensitive and precise, non-invasive, inexpensive, and easily accessible tool in CC diagnosis, therapy monitoring and follow-up.

Transcription Properties of Beta-HPV8 and HPV38 Genomes in Human Keratinocytes.

Persistent infections with high-risk human papillomaviruses (HR-HPV) from the genus alpha are established risk factors for the development of anogenital and oropharyngeal cancers. In contrast, HPV from the genus beta have been implicated in the development of cutaneous squamous cell cancer (cSCC) in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. Keratinocytes are the in vivo target cells for HPV, but keratinocyte models to investigate the replication and oncogenic activities of beta-HPV genomes have not been established. A recent study revealed, that beta-HPV49 immortalizes normal human keratinocytes (NHK) only, when the viral E8^E2 repressor (E8-) is inactivated (T. M. Rehm, E. Straub, T. Iftner, and F. Stubenrauch, Proc Natl Acad Sci U S A 119:e2118930119, 2022, https://doi.org/10.1073/pnas.2118930119). We now demonstrate that beta-HPV8 and HPV38 wild-type or E8- genomes are unable to immortalize NHK. Nevertheless, HPV8 and HPV38 express E6 and E7 oncogenes and other transcripts in transfected NHK. Inactivation of the conserved E1 and E2 replication genes reduces viral transcription, whereas E8- genomes display enhanced viral transcription, suggesting that beta-HPV genomes replicate in NHK. Furthermore, growth of HPV8- or HPV38-transfected NHK in organotypic cultures, which are routinely used to analyze the productive replication cycle of HR-HPV, induces transcripts encoding the L1 capsid gene, suggesting that the productive cycle is initiated. In addition, transcription patterns in HPV8 organotypic cultures and in an HPV8-positive lesion from an EV patient show similarities. Taken together, these data indicate that NHK are a suitable system to analyze beta-HPV8 and HPV38 replication. IMPORTANCE High-risk HPV, from the genus alpha, can cause anogenital or oropharyngeal malignancies. The oncogenic properties of high-risk HPV are important for their differentiation-dependent replication in human keratinocytes, the natural target cell for HPV. HPV from the genus beta have been implicated in the development of cutaneous squamous cell cancer in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. Currently, the replication cycle of beta-HPV has not been studied in human keratinocytes. We now provide evidence that beta-HPV8 and 38 are transcriptionally active in human keratinocytes. Inactivation of the viral E8^E2 repressor protein greatly increases genome replication and transcription of the E6 and E7 oncogenes, but surprisingly, this does not result in immortalization of keratinocytes. Differentiation of HPV8- or HPV38-transfected keratinocytes in organotypic cultures induces transcripts encoding the L1 capsid gene, suggesting that productive replication is initiated. This indicates that human keratinocytes are suited as a model to investigate beta-HPV replication.

EXPRESSION OF E8

Human papillomavirus (HPV) E1 and E2 proteins activate genome replication. E2 also modulates viral gene expression and is involved in the segregation of viral genomes. In addition to full length E2, almost all PV share the ability to encode an E8^E2 protein, that is a fusion of E8 with the C-terminal half of E2 which mediates specific DNA-binding and dimerization. HPV E8^E2 acts as a repressor of viral gene expression and genome replication. To analyze the function of E8^E2 in vivo, we used the Mus musculus PV1 (MmuPV1)-mouse model system. Characterization of the MmuPV1 E8^E2 protein revealed that it inhibits transcription from viral promoters in the absence and presence of E1 and E2 proteins and that this is partially dependent upon the E8 domain. MmuPV1 genomes, in which the E8 ATG start codon was disrupted (E8-), displayed a 10- to 25-fold increase in viral gene expression compared to wt genomes in cultured normal mouse tail keratinocytes in short-term experiments. This suggests that the function and mechanism of E8^E2 is conserved between MmuPV1 and HPVs. Surprisingly, challenge of athymic nude Foxn1nu/nu mice with MmuPV1 E8- genomes did not induce warts on the tail in contrast to wt MmuPV1. Furthermore, viral gene expression was completely absent at E8- MmuPV1 sites 20 - 22 weeks after DNA challenge on the tail or quasivirus challenge in the vaginal vault. This reveals that expression of E8^E2 is necessary to form tumors in vivo and that this is independent from the presence of T-cells.IMPORTANCE HPV encode an E8^E2 protein which acts as repressors of viral gene expression and genome replication. In cultured normal keratinocytes, E8^E2 is essential for long-term episomal maintenance of HPV31 genomes, but not for HPV16. To understand E8^E2's role in vivo, the Mus musculus PV1 (MmuPV1)-mouse model system was used. This revealed that E8^E2's function as a repressor of viral gene expression is conserved. Surprisingly, MmuPV1 E8^E2 knock out genomes did not induce warts in T-cell deficient mice. This shows for the first time that expression of E8^E2 is necessary for tumor formation in vivo independently of T cell immunity. This indicates that E8^E2 could be an interesting target for anti-viral therapy in vivo.

Comprehensive clinical and virological characterization of three cases of fulminant liver failure owing to HSV1 primary infection.

Herpes simplex virus 1 (HSV-1) is a frequently unrecognized, yet deadly cause of acute liver failure (ALF). We, therefore, analysed three cases of fatal HSV-1-induced ALF. All patients shared clinical (extremely elevated transaminases, LDH and AST/LDH ratio < 1) and virological characteristics (ratio of viral load in plasma versus throat swabs: 60-700-fold, lack of anti-HSV-1-IgG antibodies or low IgG-avidity during primary infection), which may help to identify patients at risk. Additionally, in vitro chemosusceptibility assays revealed high efficacy of the helicase-primase inhibitors (HPI), pritelivir and drug-candidate IM-250 compared to acyclovir (ACV) using HSV-1-isolates from two patients; hence, ACV/HPI-combinations might offer new therapeutic options for HSV-induced ALF.

A TGF-ß- and p63-Responsive Enhancer Regulates IFN-κ Expression in Human Keratinocytes.

Type I IFNs have antiviral and immune-modulating activities. IFN-α/-ß have very low basal expression levels but are strongly induced upon activation of pattern recognition receptors. In contrast, IFN-κ is constitutively expressed in uninfected keratinocytes and responds only weakly to pattern recognition receptor activation. IFN-κ expression has been implicated in the pathogenesis of inflammatory skin diseases and in limiting human papillomavirus replication in human keratinocytes. We have identified an enhancer ∼5 kb upstream of the IFNK gene driving its expression in keratinocytes. The enhancer consists of binding sites for the transcription factors jun-B, SMAD3/4, AP-2α/γ, and p63, of which the latter two are key regulators of keratinocyte biology. The jun-B and SMAD3/4 elements confer activation by the TGF-ß pathway. Furthermore, inhibition of ERK1/2 kinases activates IFN-κ expression. Our study provides a framework for the cell type-specific, constitutive expression of IFN-κ and its modulation by signal transduction pathways in human keratinocytes.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 in Households with Children, Southwest Germany, May-August 2020.

Resolving the role of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in households with members from different generations is crucial for containing the current pandemic. We conducted a large-scale, multicenter, cross-sectional seroepidemiologic household transmission study in southwest Germany during May 11-August 1, 2020. We included 1,625 study participants from 405 households that each had ≥1 child and 1 reverse transcription PCR-confirmed SARS-CoV-2-infected index case-patient. The overall secondary attack rate was 31.6% and was significantly higher in exposed adults (37.5%) than in children (24.6%-29.2%; p = <0.015); the rate was also significantly higher when the index case-patient was >60 years of age (72.9%; p = 0.039). Other risk factors for infectiousness of the index case-patient were SARS-CoV-2-seropositivity (odds ratio [OR] 27.8, 95% CI 8.26-93.5), fever (OR 1.93, 95% CI 1.14-3.31), and cough (OR 2.07, 95% CI 1.21-3.53). Secondary infections in household contacts generate a substantial disease burden.

Structure of High-Risk Papillomavirus 31 E6 Oncogenic Protein and Characterization of E6/E6AP/p53 Complex Formation.

The degradation of p53 is a hallmark of high-risk human papillomaviruses (HPVs) of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely related HPV16 and HPV31. HPV16 E6 and HPV31 E6 display high sequence identity (∼67%). We report here, for the first time, the structure of HPV31 E6 bound to the LxxLL motif of E6AP. HPV16 E6 and HPV31 E6 are structurally very similar, in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31 E6 to the LxxLL motif of E6AP and p53, respectively, are reduced 2-fold and 5.4-fold compared to 16 E6. The affinity of E6-E6AP-p53 ternary complex formation parallels the efficacy of the subsequent reaction, namely, degradation of p53. Therefore, closely related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathological impacts.IMPORTANCE Variations of carcinogenicity of human papillomaviruses are related to variations of the E6 and E7 interactome. While different HPV species and genera are known to target distinct host proteins, the fine differences between E6 and E7 of closely related HPVs, supposed to target the same cellular protein pools, remain to be addressed. We compare the oncogenic E6 proteins of the closely related high-risk HPV31 and HPV16 with regard to their structure and their efficiency of ternary complex formation with their cellular targets p53 and E6AP, which results in p53 degradation. We solved the crystal structure of 31 E6 bound to the E6AP LxxLL motif. HPV16 E6 and 31 E6 structures are highly similar, but a few sequence variations lead to different protein contacts within the ternary complex and, as quantified here, an overall lower binding affinity of 31 E6 than 16 E6. These results align with the observed lower p53 degradation potential of 31 E6.

HPV types, cervical high-grade lesions and risk factors for oncogenic human papillomavirus infection among 3416 Tanzanian women.

OBJECTIVE: The objective of the present study was to assess the prevalence and type-specific distribution of cervical high-risk (HR) human papillomavirus (HPV) among women with normal and abnormal cytology, and to describe risk factors for HR HPV among HIV-positive and HIV-negative women in Tanzania. METHODOLOGY: A cross-sectional study was conducted in existing cervical cancer screening clinics in Kilimanjaro and Dar es Salaam. Cervical specimens were obtained from women aged 25-60 years. Samples were shipped to Denmark for cytological examination, and to Germany for HR HPV testing (using Hybrid Capture 2) and genotyping (using LiPaExtra). Risk factors associated with HPV were assessed by multivariable logistic regression analysis. RESULT: Altogether, 4080 women were recruited with 3416 women contributing data for the present paper, including 609 HIV-positive women and 2807 HIV-negative women. The overall HR HPV prevalence was 18.9%, whereas the HR HPV prevalence in women with high-grade squamous intraepithelial lesions (HSILs) was 92.7%. Among HPV-positive women with HSIL, HPV16 (32.5%) and HPV58 (19.3%) were the the most common types followed by HPV18 (16.7%) and HPV52 (16.7%). Factors associated with HR HPV included younger age, increasing number of partners and early age at first intercourse. Similar risk factors were found among HIV-positive and HIV-negative women. In addition, among HIV-positive women, those with CD4 counts <200 cells/mm3 had an increased risk of HR HPV (OR 2.2; 95% CI 1.2 to 4.8) compared with individuals with CD4 count ≥500 cells/mm3. CONCLUSION: Given the HPV distribution among Tanzanian women, the current HPV vaccination in Tanzania using quadrivalent vaccine may be considered replaced by the nonavalent vaccine in the future. In addition, appropriate antiretroviral treatment management including monitoring of viremia may decrease the burden of HR HPV in HIV-positive women.

Significant decline of HPV 6 infection and genital warts despite low HPV vaccination coverage in young women in Germany: a long-term prospective, cohort data analysis.

BACKGROUND: The introduction of human papillomavirus (HPV) vaccination has resulted in a remarkable decline of genital warts in women and men, but in Germany historical rates of vaccination are relatively low. We report long-term surveillance data on changes in HPV 6 and HPV 11 infection and the prevalence of genital warts in young women in the Wolfsburg HPV epidemiological study (WOLVES). METHODS: Women born in 1983/84, 1988/89, and 1993/94 participated in four cohorts between 2009/10 and 2014/15. Quadrivalent vaccination coverage and prevalence of HPV 6/11 infection and genital warts are reported for participants aged 19-22 years and 24-27 years at the time of sample collection. Statistical analyses were done to compare similarly aged participants using 2 × 2 contingency tables (Röhmel-Mansmann unconditional exact test; two-side alpha of 0.05). RESULTS: A total of 2456 women were recruited. Between 2010 and 2015, there was a statistically significant decrease in the prevalence of HPV 6 infection among women aged 24-27 years (2.1% versus 0.0%; P < 0.0001) and women aged 19-22 years (2.0% versus 0.0%; P = 0.0056). There was no significant decline in HPV 11 infection. In total, 52 of 2341 participants were diagnosed with genital warts. There was a statistically significant drop in the risk of developing genital warts in women aged 24-27 years between 2010 and 2015 (4.7% versus 1.7%, respectively; P = 0.0018). The overall risk of developing genital warts in women aged 19-27 years decreased from 3.1% in 2010 to 1.2% in 2015 (P = 0.0022). CONCLUSIONS: An increase in vaccination coverage was associated with a decreased prevalence of genital warts in young women. A protective effect greater than herd immunity alone was seen despite low vaccination rates. Quadrivalent vaccine had a protective effect on genital HPV 6 infection and an almost fully protective effect on the development of genital warts in the youngest population.

Agreement between careHPV and hybrid capture 2 in detecting high-risk HPV in women in Tanzania.

INTRODUCTION: Visual inspection of the cervix with acetic acid is used to control the burden of cervical cancer in low- and middle-income countries. This method has some limitations and HPV DNA testing may be an alternative, but it is expensive and requires a laboratory setup. Cheaper and faster HPV tests have been developed. This study describe the agreement between a fast HPV test (careHPV) and hybrid capture 2 (HC2) in detection of high-risk HPV among Tanzanian women. MATERIAL AND METHODS: The study involved women attending routine cervical cancer screening at the Ocean Road Cancer Institute and Kilimanjaro Christian Medical Centre in Tanzania. The women were offered HIV testing. Two cervical samples were subsequently obtained; the first sample was processed at the clinics using careHPV and the second sample was transported to Denmark and Germany for cytology and HC2 analysis. Kappa statistic was calculated to assess the agreement between careHPV and HC2. The sensitivity, specificity and predictive values of careHPV were calculated using HC2 as reference. The analyses were done for the overall study population and stratified by testing site and HIV status. RESULTS: A total of 4080 women were enrolled, with 437 being excluded due to invalid information, lack of careHPV or HC2 results. Overall agreement between the tests was substantial with a kappa value of 0.69 (95% confidence interval [CI] 0.66-0.72). The sensitivity and specificity of careHPV was 90.7% (95% CI 89.6-91.8) and 84.2% (95% CI 81.2-86.8), respectively. The agreement was similar in the stratified analyses where the kappa values were 0.75 (95% CI 0.70-0.79) in women aged 25-34, 0.66 (95% CI 0.62-0.70) in women aged 35-60, 0.73 (95% CI 0.70-0.77) at the Ocean Road Cancer Institute, 0.64 (95% CI 0.60-0.69) at the Kilimanjaro Christian Medical Center, 0.73 (95% CI 0.68-0.79) in HIV-positive and 0.66 (95% CI 0.63-0.70) in HIV-negative women. The kappa value of 0.64 (95% CI 0.39-0.88) for cervical high-grade lesions indicates a substantial agreement between careHPV and HC2 in detecting HPV among women with cervical high-grade lesions. CONCLUSIONS: A substantial agreement was found between careHPV and HC2 in detecting HPV overall as well as detecting HPV among women with cervical high-grade lesions. However, given the limited resources available in low and middle-income countries, the HPV testing assay should be weighed against the cost-effectiveness of the test.

The association between human papillomavirus and cervical high-grade cytology among HIV-positive and HIV-negative Tanzanian women: A cross-sectional study.

INTRODUCTION: Human papillomavirus (HPV) is the causative agent of precancerous lesions and cervical cancer, cervical cancer being the leading cause of deaths in Tanzanian women. Early detection and treatment of precancerous lesions are important in the prevention of cervical cancer cases. MATERIAL AND METHODS: We conducted a cross-sectional study among 3390 Tanzanian women aged 25-60 years. Information on lifestyle habits was collected, and women underwent gynecological examination with collection of cervical cells for conventional cytological and HPV testing. Blood samples were tested for HIV. The association between cervical high-grade cytology (HGC) and potential risk factors was examined using multivariable logistic regression adjusting for age and high-risk HPV (HR-HPV). RESULTS: The prevalence of HGC was 3.6% and of low-grade cytology was 8.3%. In women who were both HR-HPV-positive and HIV-positive, the prevalence of HGC was 28.3%. It increased by age and was 47% among women aged 50-60 years. Women, who had their sexual debut at age 9-15 years and 16-18 years, respectively, had 2.5 and 2.4 times increased odds of HGC compared with women whose sexual debut was at age 21 years and older. HIV-positive women had increased odds of HGC in comparison with HIV-negative women after adjustment for age (odds ratio [OR] 2.95, 95% CI 1.92-4.54). HR-HPV-positive women had nearly 100-fold increased odds of HGC compared with HR-HPV-negative women (OR 96.6, 95% CI 48.0-194), and this estimate was higher among HIV-positive women (OR 152.2, 95% CI 36.1-642.0). CONCLUSIONS: Increasing age, early age at first intercourse, HR-HPV, and HIV infections were associated with a substantially increased risk of HGC.

[Aerosols and SARS-CoV-2: Statement of the "Expert Group Aerosols" on the development, infectivity, spread and reduction of airborne, virus-containing particles in the air]. / Stellungnahme des "Expertenkreises Aerosole": Aerosole und SARS-CoV-2 ­ Entstehung, Infektiosität, Ausbreitung und Minderung luftgetragener, virenhaltiger Teilchen in der Atemluft.

Aerosols are currently seen as one of the main transmission routes for SARS-CoV-2, but a comprehensive understanding of the processes and appropriate action/adaptation of protection concepts requires the exchange of information across interdisciplinary boundaries. Against this background, the Baden-Württemberg state government launched in October 2020 a multidisciplinary "Expert Group Aerosols" comprising engineers, natural scientists and medical professionals. In its statement, the group has compiled the current state of knowledge in all relevant disciplines in the context of airborne SARS-CoV-2 infection. In addition to the well-known hygiene and social distancing rules, the importance of the correct use of effective masks is emphasized. Furthermore, the necessity for dynamic and correct ventilation is pointed out and illustrated with ventilation intervals and periods for different scenarios as examples. The effectiveness of stationary or mobile cabin air filters as an important component in the protection concept is discussed. The first opinion of the expert group makes it clear that the existing hygiene and social distancing rules offer the best possible protection against SARS-CoV-2 infection only when correctly applied in combination.

Contribution of HDAC3 to transcriptional repression by the human papillomavirus 31 E8

Human papillomaviruses (HPV) such as HPV16 and HPV31 encode an E8^E2 protein that acts as a repressor of viral replication and transcription. E8^E2's repression activities are mediated via the interaction with host-cell NCoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoid and thyroid receptors) corepressor complexes, which consist of NCoR, its homologue SMRT, GPS2 (G-protein pathway suppressor 2), HDAC3 (histone deacetylase 3), TBL1 (transducin b-like protein 1) and its homologue TBLR1 (TBL1-related protein 1). We now provide evidence that transcriptional repression by HPV31 E8^E2 is NCoR/SMRT-dependent but surprisingly always HDAC3-independent when analysing different HPV promoters. This is in contrast to the majority of several cellular transcription factors using NCoR/SMRT complexes whose transcriptional repression activities are both NCoR/SMRT- and HDAC3-dependent. However, NCoR/SMRT-dependent but HDAC3-independent repression has been described for specific cellular genes, suggesting that this may not be specific for HPV promoters but could be a feature of a subset of NCoR/SMRT-HDAC3 regulated genes.

Optimized production strategy of the major capsid protein HPV 16L1 non-assembly variant in E. coli.

The capsid of human papillomavirus (HPV) consists of two capsid proteins - the major capsid protein L1 and the minor capsid protein L2. Assembled virus-like particles, which only consist of L1 proteins, are successfully applied as prophylactic vaccines against HPV infections. The capsid subunits are L1-pentamers, which are also reported to protect efficiently against HPV infections in animals. The recombinant production of L1 has been previously shown in E. coli, yeast, insect cells, plants and mammalian cell culture. Principally, in E. coli-based expression system L1 shows high expression yields but the protein is largely insoluble. In order to overcome this problem reported strategies address fusion proteins and overexpression of bacterial chaperones. However, an insufficient cleavage of the fusion proteins and removal of co-purified chaperones can hamper subsequent down streaming. We report a significant improvement in the production of soluble L1-pentamers by combining (I) a fusion of a N-terminal SUMO-tag to L1, (II) the heterologous co-expression of the chaperon system GroEL/ES and (III) low expression temperature. The fusion construct was purified in a 2-step protein purification including efficient removal of GroEL/ES and complete removal of the N-terminal SUMO-tag. The expression strategy was transferred to process-controlled high-cell-density fermentation with defined media according to the guidelines of good manufacturing practice. The produced L1 protein is highly pure (>95%), free of DNA (260:280 = 0.5) and pentameric. The production strategy yielded 5.73 mg of purified L1-pentamers per gram dry biomass. The optimized strategy is a suitable alternative for high yield L1-pentamer production and purification as a cheaper process for vaccine production.

Risk of vulvar, vaginal and anal high-grade intraepithelial neoplasia and cancer according to cervical human papillomavirus (HPV) status: A population-based prospective cohort study.

OBJECTIVES: All cervical cancers and some vulvar, vaginal and anal cancers are caused by high-risk human papillomavirus (hrHPV). However, little is known about the association between cervical HPV infection and subsequent intraepithelial neoplasia and cancer at other anogenital sites. In this prospective cohort study, we estimated the risk of vulvar, vaginal and anal intraepithelial neoplasia grade 2/3 or cancer (VIN2+, VaIN2+, AIN2+) according to cervical hrHPV status. METHODS: Liquid-based cervical cytology samples were collected from 40,399 women screened against cervical cancer in Copenhagen, Denmark, during 2002-2005. Samples were tested for hrHPV using Hybrid Capture 2 (HC2) and genotyped using INNO-LiPA. We linked the cohort with Danish nationwide registries to identify cases of VIN2+, VaIN2+ and AIN2+ during up to 15 years of follow-up. We estimated age-adjusted hazard ratios (HRs) using Cox regression and cumulative incidences using Aalen-Johansen's estimator. RESULTS: Women with cervical HPV16 infection had increased hazard of VIN2+ (HR = 2.6; 95% confidence interval [CI], 1.2-5.5), VaIN2+ (HR = 23.5; 95% CI, 6.8-81.6) and AIN2+ (HR = 3.7; 95% CI, 1.1-12.2) compared with HC2 negative women. Women with other hrHPV types than HPV16 also had increased hazard of VaIN2+ (HR = 7.1; 95% CI, 2.3-22.3) and a borderline statistically significantly increased risk of AIN2+ (HR = 2.2; 95% CI, 0.9-4.9) compared with HC2 negative women. The 10-year cumulative incidences of VIN2+, VaIN2+ and AIN2+ in women with cervical HPV16 were 0.3% (95% CI, 0.2%-0.7%), 0.2% (95% CI, 0.1%-0.5%) and 0.1% (95 CI, 0.0%-0.4%). CONCLUSIONS: Cervical HPV16 infection is associated with increased risk of VIN2+, VaIN2+ and AIN2+.