Self-Amplifying RNA Vaccines for Venezuelan Equine Encephalitis Virus Induce Robust Protective Immunogenicity in Mice.

Venezuelan equine encephalitis virus (VEEV) is a known biological defense threat. A live-attenuated investigational vaccine, TC-83, is available, but it has a high non-response rate and can also cause severe reactogenicity. We generated two novel VEE vaccine candidates using self-amplifying mRNA (SAM). LAV-CNE is a live-attenuated VEE SAM vaccine formulated with synthetic cationic nanoemulsion (CNE) and carrying the RNA genome of TC-83. IAV-CNE is an irreversibly-attenuated VEE SAM vaccine formulated with CNE, delivering a TC-83 genome lacking the capsid gene. LAV-CNE launches a TC-83 infection cycle in vaccinated subjects but eliminates the need for live-attenuated vaccine production and potentially reduces manufacturing time and complexity. IAV-CNE produces a single cycle of RNA amplification and antigen expression without generating infectious viruses in subjects, thereby creating a potentially safer alternative to live-attenuated vaccine. Here, we demonstrated that mice vaccinated with LAV-CNE elicited immune responses similar to those of TC-83, providing 100% protection against aerosol VEEV challenge. IAV-CNE was also immunogenic, resulting in significant protection against VEEV challenge. These studies demonstrate the proof of concept for using the SAM platform to streamline the development of effective attenuated vaccines against VEEV and closely related alphavirus pathogens such as western and eastern equine encephalitis and Chikungunya viruses.

Effect of Xpert MTB/RIF on clinical outcomes in routine care settings: individual patient data meta-analysis.

BACKGROUND: Xpert MTB/RIF, the most widely used automated nucleic acid amplification test for tuberculosis, is available in more than 130 countries. Although diagnostic accuracy is well documented, anticipated improvements in patient outcomes have not been clearly identified. We performed an individual patient data meta-analysis to examine improvements in patient outcomes associated with Xpert MTB/RIF. METHODS: We searched PubMed, Embase, ClinicalTrials.gov, and the Pan African Clinical Trials Registry from inception to Feb 1, 2018, for randomised controlled trials (RCTs) comparing the use of Xpert MTB/RIF with sputum smear microscopy as tests for tuberculosis diagnosis in adults (aged 18 years or older). We excluded studies of patients with extrapulmonary tuberculosis, and studies in which mortality was not assessed. We used a two-stage approach for our primary analysis and a one-stage approach for the sensitivity analysis. To assess the primary outcome of cumulative 6-month all-cause mortality, we first performed logistic regression models (random effects for cluster randomised trials, with robust SEs for multicentre studies) for each trial, and then pooled the odds ratio (OR) estimates by a fixed-effects (inverse variance) or random-effects (Der Simonian Laird) meta-analysis. We adjusted for age and gender, and stratified by HIV status and previous tuberculosis-treatment history. The study protocol has been registered with PROSPERO, number CRD42014013394. FINDINGS: Our search identified 387 studies, of which five RCTs were eligible for analysis. 8567 adult clinic attendees (4490 [63·5%] of 7074 participants for whom data were available were HIV-positive) were tested for tuberculosis with Xpert MTB/RIF (Xpert group) versus sputum smear microscopy (sputum smear group), across five low-income and middle-income countries (South Africa, Brazil, Zimbabwe, Zambia, and Tanzania). The primary outcome (reported in three studies) occurred in 182 (4·5%) of 4050 patients in the Xpert group and 217 (5·3%) of 4093 patients in the smear group (pooled adjusted OR 0·88, 95% CI 0·68-1·14 [p=0·34]; for HIV-positive individuals OR 0·83, 0·65-1·05 [p=0·12]). Kaplan-Meier estimates showed a lower rate of death (12·73 per 100 person-years in the Xpert group vs 16·38 per 100 person-years in the sputum smear group) for HIV-positive patients (hazard ratio 0·76, 95% CI 0·60-0·97; p=0·03). The risk of bias was assessed as reasonable and the statistical heterogeneity across studies was low (I2<20% for the primary outcome). INTERPRETATION: Despite individual patient data analysis from five RCTs, we were unable to confidently rule in nor rule out an Xpert MTB/RIF-associated reduction in mortality among outpatients tested for tuberculosis. Reduction in mortality among HIV-positive patients in a secondary analysis suggests the possibility of population-level impact. FUNDING: US National Institutes of Health.

Synergistic interactions between the NS3(hel) and E proteins contribute to the virulence of dengue virus type 1.

BACKGROUND: Dengue includes a broad range of symptoms, ranging from fever to hemorrhagic fever and may occasionally have alternative clinical presentations. Many possible viral genetic determinants of the intrinsic virulence of dengue virus (DENV) in the host have been identified, but no conclusive evidence of a correlation between viral genotype and virus transmissibility and pathogenicity has been obtained. METHODOLOGY/PRINCIPAL FINDINGS: We used reverse genetics techniques to engineer DENV-1 viruses with subsets of mutations found in two different neuroadapted derivatives. The mutations were inserted into an infectious clone of DENV-1 not adapted to mice. The replication and viral production capacity of the recombinant viruses were assessed in vitro and in vivo. The results demonstrated that paired mutations in the envelope protein (E) and in the helicase domain of the NS3 (NS3(hel)) protein had a synergistic effect enhancing viral fitness in human and mosquito derived cell lines. E mutations alone generated no detectable virulence in the mouse model; however, the combination of these mutations with NS3(hel) mutations, which were mildly virulent on their own, resulted in a highly neurovirulent phenotype. CONCLUSIONS/SIGNIFICANCE: The generation of recombinant viruses carrying specific E and NS3(hel) proteins mutations increased viral fitness both in vitro and in vivo by increasing RNA synthesis and viral load (these changes being positively correlated with central nervous system damage), the strength of the immune response and animal mortality. The introduction of only pairs of amino acid substitutions into the genome of a non-mouse adapted DENV-1 strain was sufficient to alter viral fitness substantially. Given current limitations to our understanding of the molecular basis of dengue neuropathogenesis, these results could contribute to the development of attenuated strains for use in vaccinations and provide insights into virus/host interactions and new information about the mechanisms of basic dengue biology.

Will the Convention on Biological Diversity put an end to biological control?

Will the Convention on Biological Diversity put an end to biological control? Under the Convention on Biological Diversity countries have sovereign rights over their genetic resources. Agreements governing the access to these resources and the sharing of the benefits arising from their use need to be established between involved parties. This also applies to species collected for potential use in biological control. Recent applications of access and benefit sharing principles have already made it difficult or impossible to collect and export natural enemies for biological control research in several countries. If such an approach is widely applied it would impede this very successful and environmentally safe pest management method based on the use of biological diversity. The International Organization for Biological Control of Noxious Animals and Plants has, therefore, created the "Commission on Biological Control and Access and Benefit Sharing". This commission is carrying out national and international activities to make clear how a benefit sharing regime might seriously frustrate the future of biological control. In addition, the IOBC Commission members published information on current regulations and perceptions concerning exploration for natural enemies and drafted some 30 case studies selected to illustrate a variety of points relevant to access and benefit sharing. In this article, we summarize our concern about the effects of access and benefit sharing systems on the future of biological control.

Poderá a Convenção em Diversidade Biológica por um fim no Controle Biológico? Baseando-se na Convenção sobre Diversidade Biológica, os países têm soberania sobre seus recursos genéticos. Acordos que governam o acesso a tais recursos e o compartilhamento dos benefícios provenientes do seu uso precisam ser estabelecidos de comum acordo com as partes envolvidas. Isto também é aplicável a espécies coletadas com uso potencial em controle biológico. Recentes aplicações dos princípios de introdução e compartilhamento dos benefícios têm tornado difícil, ou mesmo impossível, coletar e exportar inimigos naturais em muitos paises para pesquisas em controle biológico em muitos países. Como esta é uma medida amplamente utilizada, tais procedimentos poderão impedir este bem sucedido e ambientalmente seguro método de manejo de pragas, baseado no uso da diversidade biológica. A Organização Internacional para Controle Biológico de Plantas e Animais Nocivos (IOBC) criou a "Comissão em Controle Biológico e Introdução e Benefícios Mútuos" para estudar o assunto. Tal comissão está desenvolvendo atividades nacionais e internacionais para esclarecer como o regime de compartilhamento de benefícios pode prejudicar seriamente o futuro do controle biológico. Além disto, membros da Comissão da IOBC publicaram informações sobre regulamentos atuais e suas opiniões relacionadas à exploração de inimigos naturais, listando cerca de 30 casos para ilustrar os pontos relevantes para a introdução e compartilhamento de benefícios. No presente artigo, é sumarizado o ponto de vista dessa comissão na IOBC sobre os efeitos dos sistemas de introdução e compartilhamento para o futuro do Controle Biológico.

Construction and characterization of a stable subgenomic replicon system of a Brazilian dengue virus type 3 strain (BR DEN3 290-02).

Dengue viruses (DENV) cause the most common arboviral disease afflicting men. Clinical manifestations range from asymptomatic to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The mechanisms involved in the disease pathogenesis are not fully understood. The severity of the disease seems to be influenced by both viral and host factors. Subgenomic replicons of DENV can be used to study viral replication mechanisms and evaluate the effects of antiviral drugs on viral replication. The objective was to generate and characterize biologically a replicon from a clinical isolate of DENV-3, as part of our studies to understand how this new isolate interacts with cells. To obtain this replicon several RT-PCR fragments encoding the non-structural proteins genes were cloned in high-copy vectors, and used to assemble the replicon in a BAC plasmid vector containing a synthetic DNA molecule encoding the 5' and 3' ends of a viral cDNA with a T7 DNA-dependent RNA polymerase promoter and a ribozyme. In vitro transcribed RNA recovered from this BAC plasmid was transfected into C6/36 mosquito cells, and dengue virus protein expression was assessed by indirect immunofluorescence using polyclonal antibodies. The results showed that the replicon was replicated efficiently in cells, demonstrating successful assembly of a DENV-3 replicon.

Construction of an infectious cDNA clone for a Brazilian prototype strain of dengue virus type 1: characterization of a temperature-sensitive mutation in NS1.

To help understand the mechanism of pathogenesis of dengue virus (DV), we set out to create an infectious cDNA of the Brazilian prototype strain of DV serotype 1 (DV1-BR/90). PCR-amplified fragments of DV1-BR/90 cDNA were readily assembled into a subgenomic cDNA that could be used to produce replicating RNAs (replicons), lacking the structural protein-encoding regions of the genome. However, assembly of a cDNA capable of producing infectious virus was only possible using a bacterial artificial chromosome plasmid, indicating that DV1 sequences were especially difficult to propagate in E. coli. While characterizing our cDNA we discovered a fortuitous temperature-sensitive mutation in the NS1 encoding region. Using our infectious cDNA and a renilla luciferase-expressing replicon we were able to demonstrate that this mutation produced a defect in RNA replication at 37 degrees C, demonstrating that the DV1 NS1 protein plays an essential role in RNA replication.