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Background Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide and it contributes to considerable maternal and neonatal mortality and morbidity in many low-income countries like Bangladesh. A three-dose regimen of a vaccine against HEV (HEV 239) has shown promising results in China. The effects and safety of this vaccine in other populations and with different dosing regimens remains uncertain. Objectives Investigate the immune response and safety of a two-dose regimen with the HEV 239 vaccine among healthy adults. Examine the feasibility of conducting a larger HEV 239 vaccine trial in rural Bangladesh. Methods One-hundred healthy men and non-pregnant women 16–39 years old were randomized in a 1:1 ratio to receive two doses of either the study (HEV) or control (Hepatitis B virus, HBV) vaccine (at 0, 1 month). Blood samples were collected at day 0, day 60 and 2 years after vaccination. The primary endpoints were the proportion and severity of adverse events up to 2 months after dose one and the longitudinal shift in anti-HEV IgG levels from day 0 to day 60 and 2 years after vaccination. Results Adverse events to HEV 239 were comparable to the control vaccine, mild in severity and resolved within one to nine days. All participants in the study group seroconverted and achieved high levels of HEV IgG antibodies that remained positive for two years in all but one. A T-cell response was detected one month after HEV 239 vaccination. Conclusion Our results show that two doses of the HEV 239 vaccine produces broad and likely functional immune responses against HEV that remain for at least two years. The safety profile was acceptable and a phase four study of HEV 239 in rural Bangladesh is feasible.

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Purpose: We compared the prognostic significance of ectodomain isoforms of the epidermal growth factor receptor (EGFR), which lack the tyrosine kinase (TK) domain, with that of the full-length receptor and its autophosphorylation status in cervical cancers treated with conventional chemoradiotherapy.Experimental Design: Expression of EGFR isoforms was assessed by immunohistochemistry in a prospectively collected cohort of 178 patients with squamous cell cervical carcinoma, and their detection was confirmed with Western blotting and reverse transcriptase PCR. A proximity ligation immunohistochemistry assay was used to assess EGFR-specific autophosphorylation. Pathways associated with the expression of ectodomain isoforms were studied by gene expression analysis with Illumina beadarrays in 110 patients and validated in an independent cohort of 41 patients.Results: Membranous expression of ectodomain isoforms alone, without the coexpression of the full-length receptor, showed correlations to poor clinical outcome that were highly significant for lymph node–negative patients (locoregional control, P = 0.0002; progression-free survival, P < 0.0001; disease-specific survival, P = 0.005 in the log-rank test) and independent of clinical variables. The ectodomain isoforms were primarily 60-kD products of alternative EGFR transcripts. Their membranous expression correlated with transcriptional regulation of oncogenic pathways including activation of MYC and MAX, which was significantly associated with poor outcome. This aggressive phenotype of ectodomain EGFR expressing tumors was confirmed in the independent cohort. Neither total nor full-length EGFR protein level, or autophosphorylation status, showed prognostic significance.Conclusion: Membranous expression of ectodomain EGFR isoforms, and not TK activation, predicts poor outcome after chemoradiotherapy for patients with lymph node–negative cervical cancer. Clin Cancer Res; 17(16); 5501–12. ©2011 AACR.

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PDF file - 23K, Patient and tumor characteristics of the validation cohort and by lymph node status of the basic cohort.

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PDF file - 38K, Gene sets associated with ABrix

PDF file - 97K, Hypoxia treatment of cervical cancer cell lines

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PDF file - 35K, Creating cervical cancer specific hypoxia gene sets

HEV is the most common cause of acute hepatitis globally. This review summarizes the latest knowledge on the epidemiology, clinical characteristics, testing, and treatment of HEV infection. We also focused on Bangladesh to highlight the distinct challenges and the possible remedies. In low-income settings, the virus is mainly transmitted between people by fecal contamination of drinking water causing large outbreaks, and sporadic cases. The disease is usually mild and self-limiting acute hepatitis. Still, pregnant women and their offspring in low-income countries are at particular risk for severe disease, with up to 20% maternal mortality. Despite the high burden of the disease, HEV remains a relatively neglected virus, with detection hampered by costly tests and a lack of suitable treatments. Molecular PCR diagnostics, together with ELISA antibody tests, remain the preferred methods for diagnosis of HEV; however, rapid bedside diagnostics are available and could offer a practical alternative, especially in low-income countries. One vaccine (HEV 239) is only available in China and Pakistan, as efficacy against the other genotypes remains uncertain. The effectiveness trial conducted in Bangladesh might lead the way in gathering more efficacy data and could, together with improved surveillance and raised awareness, dramatically reduce the global burden of HEV. Hepatitis E Virus (HEV) Synopsis: General Aspects and Focus on Bangladesh

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. In many low-income countries it causes large outbreaks and disproportionally affects pregnant women and their offspring. Surveillance studies to find effective preventive interventions are needed but are hampered by the lack of funding and infrastructure. Dried blood spots (DBS) offer an easier and more robust way to collect, transport, and store blood samples compared to plasma/serum samples, and could ease some of the barriers for such studies. In this study we optimize an HEV IgG ELISA for DBS samples and validate it on 300 paired DBS and plasma samples collected in rural areas of Bangladesh from participants in a HEV vaccine study. We demonstrate that HEV IgG in blood stored as DBS is stable for two months at up to 40 °C, and for five freeze-thaw cycles. The specificity was 97% and the overall sensitivity of the DBS assay was 81%. The sensitivity was higher in samples from vaccinated participants (100%) compared to previously infected participants (59%), reflecting a positive correlation between IgG titer and sensitivity. We found a strong correlation between DBS and plasma samples with an r2 of 0.90, but with a higher degree of difference between individual paired samples. Our study shows that DBS offers a stable alternative to plasma/serum for HEV IgG measurements and can facilitate serological studies, particularly in resource limited areas.

ImportanceUnderstanding the susceptibility and infectiousness of children and adolescents in comparison to adults is important to appreciate their role in the COVID-19 pandemic.ObjectiveTo determine SARS-CoV-2 susceptibility and infectiousness of children and adolescents with adults as comparator for three variants (wild-type, Alpha, Delta) in the household setting. We aimed to identify the effects independent of vaccination.Data SourcesWe searched EMBASE, PubMed and medRxiv up to January 2022. Additional studies were identified through contacting subject experts.Study SelectionTwo reviewers independently identified studies providing secondary attack rates (SAR) for SARS-CoV-2 infection in children (0-9 years), adolescents (10-19 years) or both compared with adults (20 years and older) derived from household data.Data Extraction and SynthesisTwo reviewers independently performed data extraction. We assessed risk of bias of included studies using a critical appraisal checklist and a random-effects meta-analysis model to pool association estimates.Main Outcomes and MeasuresOdds ratio (OR) for SARS-CoV-2 infection comparing children and adolescents with adults stratified by wild-type, Alpha, and Delta variant, respectively. Susceptibility was defined as the secondary attack rate (SAR) among susceptible household contacts irrespective of the age of the index case. Infectiousness was defined as the SAR irrespective of the age of household contacts when children/adolescents/adults were the index case.ResultsTwenty-eight studies (308,857 contacts) were included in the susceptibility analysis, for Delta only one (large) study was available. Compared to adults children and adolescents were less susceptible to the wild-type and Delta variant, but equally susceptible to the Alpha variant. In the infectiousness analysis, 21 studies (201,199 index cases) were included. Compared to adults, children and adolescents were less infectious when infected with the wild-type and Delta variant. Alpha variant-related infectiousness remained unclear, 0-9 year old children were at least as infectious as adults. SAR among household contacts was highest during circulation of the Alpha variant, lowest during wild-type circulation and intermediate during Delta circulation.Conclusions and RelevanceWhen considering the potential role of children and adolescents, for each variant susceptibility, infectiousness, age group and overall transmissibility need to be assessed to guide public health policy.KEY POINTSQuestionWhat is the evidence on the susceptibility and infectiousness of wild-type, Alpha and Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among children and adolescents compared with adults in the household setting?FindingsIn this systematic review and meta-analysis of 28 studies that included 308,857 household contacts, children and adolescents were less susceptible to the wild-type and Delta variant and likely equally susceptible to the Alpha variant of SARS-Cov-2. Children aged 0-9 years old infected with the Alpha variant may be more infectious than adults, but for adolescents, Alpha infectiousness is unclear. The overall secondary attack rate (SAR) rose substantially from wild-type to Alpha and dropped somewhat from Alpha to Delta.MeaningThe epidemiological role of children and adolescents towards SARS-CoV-2 may be influenced by susceptibility, infectiousness, variant, age group and overall (relative) contagiousness.

SUMMARYObjectivesWe studied the secondary attack rate (SAR), risk factors, and precautionary practices of household transmission in a prospective, longitudinal study. We further compared transmission between the Alpha (B.1.1.7) variant and non-Variant of Concern (non-VOC) viruses.MethodsWe recruited households of 70 confirmed COVID-19 cases with 146 household contacts from May 2020 to May 2021. Participants donated biological samples 8 times over 6 weeks and answered questionnaires. Whole genome sequencing and droplet digital PCR were used to establish the SARS-CoV-2 variant and viral load.ResultsSARS-CoV-2 transmission occurred in 60% of the households, and the overall SAR for household contacts was 50%. The SAR was significantly higher for the Alpha variant (78%) compared with non-VOC viruses (43%) and was associated with a higher viral load. SAR was higher in household contacts aged ≥40 years (69%) than in younger contacts (40-47%), and for contacts of cases with loss of taste/smell. Children had lower viral loads and were more often asymptomatic than adults. Sleeping separately from the primary case reduced the risk of transmission.ConclusionsWe found substantial household transmission, particularly for the Alpha variant. Precautionary practices seem to reduce SAR, but preventing household transmission may become difficult with more contagious variants.

We studied the secondary attack rate (SAR), risk factors, and precautionary practices of household transmission in a prospective, longitudinal study. We further compared transmission between the Alpha (B.1.1.7) variant and non-Variant of Concern (non-VOC) viruses. From May 2020 throughout April 2021, we recruited 70 confirmed COVID-19 cases with 146 household contacts. Participants donated biological samples eight times over 6 weeks and answered questionnaires. SARS-CoV-2 infection was detected by real-time RT-PCR. Whole genome sequencing and droplet digital PCR were used to establish virus variant and viral load. SARS-CoV-2 transmission occurred in 60% of the households, and the overall SAR for household contacts was 50%. The SAR was significantly higher for the Alpha variant (78%) compared with non-VOC viruses (43%) and was associated with a higher viral load. SAR was higher in household contacts aged ≥40 years (69%) than in younger contacts (40–47%), and for contacts of primary cases with loss of taste/smell. Children had lower viral loads and were more often asymptomatic than adults. Sleeping separately from the primary case reduced the risk of transmission. In conclusion, we found substantial household transmission, particularly for the Alpha variant. Precautionary practices seem to reduce SAR, but preventing household transmission may become difficult with more contagious variants, depending on vaccine use and effectiveness.

IntroductionHepatitis E virus (HEV) is a leading cause of acute viral hepatitis in the developing world and is a public health problem, in particular among pregnant women, where it may lead to severe or fatal complications. A recombinant HEV vaccine, 239 (Hecolin; Xiamen Innovax Biotech, Xiamen, China), is licensed in China, but WHO calls for further studies to evaluate the safety and immunogenicity of this vaccine in vulnerable populations, and to evaluate protection in pregnancy. We are therefore conducting a phase IV trial to assess the effectiveness, safety and immunogenicity of the HEV 239 vaccine when given in women of childbearing age in rural Bangladesh, where HEV infection is endemic.Methods and analysisEnrolment of a target of approximately 20 000 non-pregnant women, aged 16–39 years, started on 2 October 2017 in Matlab, Bangladesh. Sixty-seven villages were randomised by village at a 1:1 ratio to receive either the HEV vaccine or the control vaccine (hepatitis B vaccine). A 3-dose vaccination series at 0, 1 and 6 months is ongoing, and women are followed up for 24 months. The primary outcome is confirmed HEV disease among pregnant women. After vaccination, participants are requested to report information about clinical hepatitis symptoms. Participants who become pregnant are visited at their homes every 2 weeks to collect information about pregnancy outcome and to screen for clinical hepatitis. All suspected hepatitis cases undergo laboratory testing for diagnostic evaluation. The incidence of confirmed HEV disease among pregnant and non-pregnant women will be compared between the HEV vaccinated and control groups, safety and immunogenicity of the vaccine will also be evaluated.Ethics and disseminationThe protocol was reviewed and approved by the International Centre for Diarrhoeal Disease Research, Bangladesh Research Review Committee and Ethical Review Committee, and the Directorate General of Drug Administration in Bangladesh, and by the Regional Ethics Committee in Norway. This article is based on the protocol version 2.2 dated 29 June 2017. We will present the results through peer-reviewed publications and at international conferences.Trial registration numberThe trial is registered at clinicaltrials.gov with the registry name “Effectiveness Trial to Evaluate Protection of Pregnant Women by Hepatitis E Vaccine in Bangladesh” and the identifierNCT02759991.

Purpose of Review Hepatitis E (HEV) is a continuing public health problem in developing countries, causing high mortality in pregnant women. This article reviews the latest knowledge on HEV in pregnancy, the vaccine candidates that have reached clinical trials, and their potential use during pregnancy. Recent Findings New evidence suggests that a genotype-specific tropism to uteroplacental cells contributes to the multifactorial pathology leading to the increased severity of HEV during pregnancy. Data from pregnant women inadvertently vaccinated are limited but show similar rates of adverse events as non-pregnant women, and no evident harm to newborns. Protective anti-HEV IgG antibodies are estimated to last longer after vaccination than natural infection. Accelerated vaccination appears safe and provides protective antibodies similar to a normal dosing regimen. Summary HEV239 is the most extensively studied vaccine and has been shown to be safe and effective, but more data are needed to recommend use of this vaccine on a global basis. Further studies should focus on safety and efficacy in pregnant women, and evaluate a shorter two-dose regime, which is highly warranted in outbreak situations.

Knowledge of the molecular background of functional magnetic resonance (MR) images is required to fully exploit their potential in cancer management. We explored the prognostic impact of dynamic contrast-enhanced MR imaging (DCE-MRI) parameters in cervical cancer combined with global gene expression data to reveal their underlying molecular phenotype and construct a representative gene signature for the relevant parameter. On the basis of 78 patients with cervical cancer subjected to curative chemoradiotherapy, we identified the prognostic DCE-MRI parameter ABrix by pharmacokinetic analysis of pretreatment images based on the Brix model, in which tumors with low ABrix appeared to be most aggressive. Gene set analysis of 46 tumors with pairwise DCE-MRI and gene expression data showed a significant correlation between ABrix and the hypoxia gene sets, whereas gene sets related to other tumor phenotypes were not significant. Hypoxia gene sets specific for cervical cancer created in cell culture experiments, including both targets of the hypoxia inducible factor (HIF1α) and the unfolded protein response, were the most significant. In the remaining 32 tumors, low ABrix was associated with upregulation of HIF1α protein expression, as assessed by immunohistochemistry, consistent with increased hypoxia. On the basis of the hypoxia gene sets, a signature of 31 genes that were upregulated in tumors with low ABrix was constructed. This DCE-MRI hypoxia gene signature showed prognostic impact in an independent validation cohort of 109 patients. Our findings reveal the molecular basis of an aggressive hypoxic phenotype and suggest the use of DCE-MRI to noninvasively identify patients with hypoxia-related chemoradioresistance. Cancer Res; 72(20); 5285–95. ©2012 AACR.

Purpose We have applied the sensitive and specific in situ proximity ligation assay (PLA) to characterize Tyr1068 phosphorylation of the epidermal growth factor receptor (EGFR) in cervical cancer in relation to the protein level and gene dosage. Materials and methods Pretreatment tumor biopsies from 178 patients were analyzed. EGFR protein level was determined by immunohistochemistry, and Tyr1068 phosphorylation was detected with PLA in 97 EGFR positive tumors. EGFR gene dosage was derived from array comparative genomic hybridization of 86 cases. Results EGFR was expressed in most tumors, whereas phosphorylation was seen in about half of the EGFR positive ones. A correlation was found between the expression of EGFR and phosphorylated EGFR ( p =0.016, membrane; p =0.012, cytoplasm). However, tumor regions with high protein level without phosphorylation were occasionally seen and the percentage of EGFR positive cells was higher than the phosphorylated percentage ( p p =0.014) and cytoplasm ( p =0.002) was seen in 11 tumors with gain of EGFR. The protein level was not correlated with gene dosage. Conclusion In contrast to gain of the EGFR chromosomal region, high EGFR protein level may not necessarily indicate Tyr1068 phosphorylation and thereby receptor activation in cervical cancer.

Purpose: We compared the prognostic significance of ectodomain isoforms of the epidermal growth factor receptor (EGFR), which lack the tyrosine kinase (TK) domain, with that of the full-length receptor and its autophosphorylation status in cervical cancers treated with conventional chemoradiotherapy. Experimental Design: Expression of EGFR isoforms was assessed by immunohistochemistry in a prospectively collected cohort of 178 patients with squamous cell cervical carcinoma, and their detection was confirmed with Western blotting and reverse transcriptase PCR. A proximity ligation immunohistochemistry assay was used to assess EGFR-specific autophosphorylation. Pathways associated with the expression of ectodomain isoforms were studied by gene expression analysis with Illumina beadarrays in 110 patients and validated in an independent cohort of 41 patients. Results: Membranous expression of ectodomain isoforms alone, without the coexpression of the full-length receptor, showed correlations to poor clinical outcome that were highly significant for lymph node–negative patients (locoregional control, P = 0.0002; progression-free survival, P < 0.0001; disease-specific survival, P = 0.005 in the log-rank test) and independent of clinical variables. The ectodomain isoforms were primarily 60-kD products of alternative EGFR transcripts. Their membranous expression correlated with transcriptional regulation of oncogenic pathways including activation of MYC and MAX, which was significantly associated with poor outcome. This aggressive phenotype of ectodomain EGFR expressing tumors was confirmed in the independent cohort. Neither total nor full-length EGFR protein level, or autophosphorylation status, showed prognostic significance. Conclusion: Membranous expression of ectodomain EGFR isoforms, and not TK activation, predicts poor outcome after chemoradiotherapy for patients with lymph node–negative cervical cancer. Clin Cancer Res; 17(16); 5501–12. ©2011 AACR.

Tumor hypoxia is an adverse factor in cervical cancer that would be valuable to implement in the clinical decision-making. In a previous study, we identified a hypoxia gene signature associated with aggressiveness in locally advanced cervical cancer. It consisted of 31 genes, and was predictive of progression free survival in an independent cohort treated with chemoradiotherapy. The signature could be useful in the development of a prognostic test to select patients at risk of failure. The aim of the current study was to obtain a deeper understanding of the pathways reflected in the signature. This could provide an increased insight into the hypoxia-related aggressiveness of cervical cancer and be useful to fully exploit the clinical potential of the signature. The gene signature included a total of 12 genes which are known direct targets of HIF1, and 4 genes involved in the unfolded protein response (UPR), including 3 of the 12 HIF1-targets. To obtain a metric for comparison with other relevant factors, a score was calculated from the expression level of the 31 genes in 154 patients. This hypoxia score correlated significantly with HIF1α protein expression (p Cox survival analysis of the hypoxia score was performed on the 154 patients. When stratifying the score on lymph node status, it was predictive of poor outcome only for the patients without metastases to the lymph nodes (89 patients) (p=0.001). When assessing the prognostic value of HIF1α protein expression in the whole patient group, it was not correlated with survival. However, in patients with no lymph node metastases, high levels of HIF1α were found to be a prognostic indicator of poor outcome (p=0.043). Multivariate cox survival analysis was performed on the lymph node negative patients, including the hypoxia score, HIF1α, and clinical parameters. Only the hypoxia score and FIGO stage were found to be independently related to patient survival. In conclusion, it appears that HIF1α is an important mediator of the aggressive phenotype associated with our hypoxia gene signature. However, since the hypoxia score emerged as a significantly better prognostic factor than HIF1α, other signaling pathways may also contribute. Furthermore, this hypoxic phenotype seems to be associated with aggressiveness particularly in patients with no metastases to the lymph nodes. Citation Format: Cathinka Halle, Eva-Katrine Aarnes, Ruth Holm, Gunnar B. Kristensen, Heidi Lyng. HIF1α signaling contributes to an aggressive hypoxic phenotype in cervical cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4037. doi:10.1158/1538-7445.AM2013-4037