Preclinical development of filovirus and flavivirus vaccines based on recombinant insect cell expressed subunits

Ebola Virus Disease (EVD) is the most prominent example of filovirus disease but despite being characterized as a Category A Priority Pathogen by NIH/NIAID over a decade ago, it lacked public and private research resources due to the absence of a commercial market. Transmission from wild animals into the human population typically causes outbreaks of limited scale in endemic areas located in the forested regions of Central Africa and the Philippines (for Reston ebolavirus), therefore other public health threats garnered more attention. This changed when a Zaire Ebolavirus (EBOV) outbreak of increasing size in several West African countries started to reveal the true epidemic potential that filovirus infections can have when entering an urban setting in a highly mobile society. Despite significant progress with the clinical development of several EBOV vaccine candidates during and after the West African outbreak, no EBOV specific therapeutics and vaccines have yet received regulatory approval. Additional research is needed in particular on understanding the mechanism of protection and defining immune correlates of protection for Ebola and other filovirus vaccines. For our multivalent filovirus vaccine candidate, we have produced soluble recombinant filovirus glycoproteins (GP) from EBOV, Marburg marburgvirus (MARV) and Sudan ebolavirus (SUDV) using the Drosophila S2 cell expression system. The immunogenicity of highly purified recombinant subunits and admixtures formulated with or without clinically relevant adjuvants was evaluated in mice, guinea pigs and macaques. Strong antigen-specific IgG titers as well as virus neutralizing titers were observed after administering two or three doses of adjuvanted formulations. In mice and non-human primates subunit proteins were also shown to elicit cell mediated immune responses. Analysis of secreted cytokines in batch-cultured, antigen-stimulated splenocytes or PBMC’s demonstrated antigen-induced Th1 and Th2 type responses. Recombinant vaccine candidates were tested successfully for protection in the mouse model of EBOV. Further studies allowed us to demonstrate that both humoral and cell-mediated immunity are elicited and can mediate protection. Additional immunogenicity and efficacy studies in guinea pigs were focused on optimized antigen dosing, antigenic balance and adjuvantation. Multiple formulations consistently produced strong antibody responses and demonstrated 100% protective efficacy in the EBOV guinea pig model. Results from studies in two species of non-human primates demonstrate that vaccination with formulations of recombinant EBOV subunits and an emulsion-based adjuvant consistently produces high anti-EBOV IgG and virus neutralizing titers. Such vaccination prevents viremia subsequent to live virus challenge and protects animals from terminal EBOV disease. These studies suggest that we have defined a viable Ebola virus vaccine candidate based on non-replicating viral subunits. In addition to updates on efficacy testing against EBOV and MARV, we will discuss current formulation optimization efforts in our laboratory including thermostabilization of recombinant subunits as well as defining correlates of protection. These are prerequisites to enable efficient clinical development of a monovalent vaccine candidate for protection against EVD and a multivalent, recombinant subunit vaccine with protective efficacy against EBOV, SUDV and MARV infection. Recently we have also demonstrated the applicability of our vaccine platform for the rapid development of vaccines against emerging diseases with a focus on Zika virus (ZIKV), a flavivirus, where we were able to demonstrate efficacy in mice and cynomolgus macaques within approximately 13 months from designing the synthetic gene for antigen expression. While a completely different disease from EVD, the recent outbreak of ZIKV in the Americas provided a similar challenge as no vaccine development efforts have been conducted prior to 2016 and an increasing body of evidence suggests that rather than causing a typical, mild form of disease as previously reported, ZIKV infections can cause neurological sequelae as well as fetal and infant malformations. These results demonstrate that the insect cell expression system can be used to rapidly and efficiently produce recombinant viral subunits from a variety of pathogenic viruses that are highly immunogenic in multiple animal species and are capable of providing effective vaccine protection against live virus challenge

Development of a thermostable, multivalent filovirus vaccine based on recombinant subunit proteins

Ebola Virus Disease (EVD) is the most prominent example of filovirus disease but despite being characterized as a Category A Priority Pathogen by NIH/NIAID over a decade ago, it lacked public and private research resources due to the absence of a commercial market. Transmission from wild animals into the human population typically causes outbreaks of limited scale in endemic areas located in the forested regions of Central Africa and the Philippines (for Reston ebolavirus). In the past decade, a Zaire Ebolavirus (EBOV) outbreak causing more than 11,000 deaths in several West African countries started to reveal the true epidemic potential that filovirus infections can have when entering an urban setting in a highly mobile society. In addition a persistent outbreak in the Democratic Republic of the Congo has continued since August 2018 despite significant progress with the clinical development of several EBOV vaccine candidates (one of which recently gained regulatory approvals in Europe, the U.S. and several African countries) and the advanced testing of promising EBOV specific therapeutics. Despite this significant progress, additional research is needed in particular on understanding the mechanism of protection and defining immune correlates of protection for Ebola and other filoviruses do develop fast and efficacious strategies for outbreak control as the incidence of outbreaks and total case numbers has significantly increased over the last decadesPlease click Download on the upper right corner to see the full abstract

A Recombinant Subunit Based Zika Virus Vaccine Is Efficacious in Non-human Primates

Zika Virus (ZIKV), a virus with no severe clinical symptoms or sequelae previously associated with human infection, became a public health threat following an epidemic in French Polynesia 2013–2014 that resulted in neurological complications associated with infection. Although no treatment currently exists, several vaccines using different platforms are in clinical development. These include nucleic acid vaccines based on the prM-E protein from the virus and purified formalin-inactivated ZIKV vaccines (ZPIV) which are in Phase 1/2 clinical trials. Using a recombinant subunit platform consisting of antigens produced in Drosophila melanogaster S2 cells, we have previously shown seroconversion and protection against viremia in an immunocompetent mouse model. Here we demonstrate the efficacy of our recombinant subunits in a non-human primate (NHP) viremia model. High neutralizing antibody titers were seen in all protected macaques and passive transfer demonstrated that plasma from these NHPs was sufficient to protect against viremia in mice subsequently infected with ZIKV. Taken together our data demonstrate the immunogenicity and protective efficacy of the recombinant subunit vaccine candidate in NHPs as well as highlight the importance of neutralizing antibodies in protection against ZIKV infection and their potential implication as a correlate of protection

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015

Forouzanfar MH, Afshin A, Alexander LT, et al. Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. LANCET. 2016;388(10053):1659-1724.Background The Global Burden of Diseases, Injuries, and Risk Factors Study 2015 provides an up-to-date synthesis of the evidence for risk factor exposure and the attributable burden of disease. By providing national and subnational assessments spanning the past 25 years, this study can inform debates on the importance of addressing risks in context. Methods We used the comparative risk assessment framework developed for previous iterations of the Global Burden of Disease Study to estimate attributable deaths, disability-adjusted life-years (DALYs), and trends in exposure by age group, sex, year, and geography for 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks from 1990 to 2015. This study included 388 risk-outcome pairs that met World Cancer Research Fund-defined criteria for convincing or probable evidence. We extracted relative risk and exposure estimates from randomised controlled trials, cohorts, pooled cohorts, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. We developed a metric that allows comparisons of exposure across risk factors-the summary exposure value. Using the counterfactual scenario of theoretical minimum risk level, we estimated the portion of deaths and DALYs that could be attributed to a given risk. We decomposed trends in attributable burden into contributions from population growth, population age structure, risk exposure, and risk-deleted cause-specific DALY rates. We characterised risk exposure in relation to a Socio-demographic Index (SDI). Findings Between 1990 and 2015, global exposure to unsafe sanitation, household air pollution, childhood underweight, childhood stunting, and smoking each decreased by more than 25%. Global exposure for several occupational risks, high body-mass index (BMI), and drug use increased by more than 25% over the same period. All risks jointly evaluated in 2015 accounted for 57.8% (95% CI 56.6-58.8) of global deaths and 41.2% (39.8-42.8) of DALYs. In 2015, the ten largest contributors to global DALYs among Level 3 risks were high systolic blood pressure (211.8 million [192.7 million to 231.1 million] global DALYs), smoking (148.6 million [134.2 million to 163.1 million]), high fasting plasma glucose (143.1 million [125.1 million to 163.5 million]), high BMI (120.1 million [83.8 million to 158.4 million]), childhood undernutrition (113.3 million [103.9 million to 123.4 million]), ambient particulate matter (103.1 million [90.8 million to 115.1 million]), high total cholesterol (88.7 million [74.6 million to 105.7 million]), household air pollution (85.6 million [66.7 million to 106.1 million]), alcohol use (85.0 million [77.2 million to 93.0 million]), and diets high in sodium (83.0 million [49.3 million to 127.5 million]). From 1990 to 2015, attributable DALYs declined for micronutrient deficiencies, childhood undernutrition, unsafe sanitation and water, and household air pollution; reductions in risk-deleted DALY rates rather than reductions in exposure drove these declines. Rising exposure contributed to notable increases in attributable DALYs from high BMI, high fasting plasma glucose, occupational carcinogens, and drug use. Environmental risks and childhood undernutrition declined steadily with SDI; low physical activity, high BMI, and high fasting plasma glucose increased with SDI. In 119 countries, metabolic risks, such as high BMI and fasting plasma glucose, contributed the most attributable DALYs in 2015. Regionally, smoking still ranked among the leading five risk factors for attributable DALYs in 109 countries; childhood underweight and unsafe sex remained primary drivers of early death and disability in much of sub-Saharan Africa. Interpretation Declines in some key environmental risks have contributed to declines in critical infectious diseases. Some risks appear to be invariant to SDI. Increasing risks, including high BMI, high fasting plasma glucose, drug use, and some occupational exposures, contribute to rising burden from some conditions, but also provide opportunities for intervention. Some highly preventable risks, such as smoking, remain major causes of attributable DALYs, even as exposure is declining. Public policy makers need to pay attention to the risks that are increasingly major contributors to global burden. Copyright (C) The Author(s). Published by Elsevier Ltd

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Recombinant Zika Virus Subunits Are Immunogenic and Efficacious in Mice

ABSTRACT Following the 2015 Zika virus (ZIKV) outbreaks in the South Pacific, Caribbean, and Americas, ZIKV has emerged as a serious threat due to its association with infantile microcephaly and other neurologic disorders. Despite an international effort to develop a safe and effective vaccine to combat congenital Zika syndrome and ZIKV infection, only DNA and mRNA vaccines encoding the precursor membrane (prM) and envelope (E) proteins, an inactivated-ZIKV vaccine, and a measles virus-based ZIKV vaccine are currently in phase I or II (prM/E DNA) clinical trials. A ZIKV vaccine based on a nonreplicating, recombinant subunit platform offers a higher safety profile than other ZIKV vaccine candidates but is still highly immunogenic, inducing high virus-neutralizing antibody titers. Here, we describe the production and purification of Drosophila melanogaster S2 insect cell-derived, soluble ZIKV E protein and evaluate its immunogenicity and efficacy in three different mouse strains. As expected, significant virus-specific antibody titers were observed when using formulations containing clinically relevant adjuvants. Immunized mice challenged with live virus demonstrate inhibition of virus replication. Importantly, plaque reduction neutralization tests (PRNTs) indicate the high-titer production of neutralizing antibodies, a correlate of protection in the defense against ZIKV infection. ZIKV challenge of immunocompetent mice led to full protection against viremia with two doses of adjuvanted vaccine candidates. These data demonstrate a proof of concept and establish recombinant subunit immunogens as an effective vaccine candidate against ZIKV infection. IMPORTANCE The recent outbreaks of Zika virus (ZIKV) infection in French Polynesia, the Caribbean, and the Americas have highlighted the severe neuropathological sequelae that such an infection may cause. The development of a safe, effective ZIKV vaccine is critical for several reasons: (i) the difficulty in diagnosing an active infection due to common nonspecific symptoms, (ii) the lack of a specific antiviral therapy, and (iii) the potentially devastating pathological effects of in utero infection. Moreover, a vaccine with an excellent safety profile, such as a nonreplicating, noninfectious vaccine, would be ideal for high-risk people (e.g., pregnant women, immunocompromised patients, and elderly individuals). This report describes the development of a recombinant subunit protein vaccine candidate derived from stably transformed insect cells expressing the ZIKV envelope protein in vitro, the primary antigen to which effective virus-neutralizing antibodies are engendered by immunized animals for several other flaviviruses; the vaccine candidate elicits effective virus-neutralizing antibodies against ZIKV and provides protection against ZIKV infection in mice