Shedding of soluble glycoprotein 1 detected during acute Lassa virus infection in human subjects

<p>Abstract</p> <p>Background</p> <p>Lassa hemorrhagic fever (LHF) is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. With a high rate of infection that may lead to morbidity and mortality, understanding how the virus interacts with the host's immune system is of great importance for generating vaccines and therapeutics. Previous work by our group identified a soluble isoform of the Lassa virus (LASV) GP1 (sGP1) <it>in vitro </it>resulting from the expression of the glycoprotein complex (GPC) gene <abbrgrp><abbr bid="B1">1</abbr><abbr bid="B2">2</abbr></abbrgrp>. Though no work has directly been done to demonstrate the function of this soluble isoform in arenaviral infections, evidence points to immunomodulatory effects against the host's immune system mediated by a secreted glycoprotein component in filoviruses, another class of hemorrhagic fever-causing viruses. A significant fraction of shed glycoprotein isoforms during viral infection and biogenesis may attenuate the host's inflammatory response, thereby enhancing viral replication and tissue damage. Such shed glycoprotein mediated effects were previously reported for Ebola virus (EBOV), a filovirus that also causes hemorrhagic fever with nearly 90% fatality rates <abbrgrp><abbr bid="B3">3</abbr><abbr bid="B4">4</abbr><abbr bid="B5">5</abbr></abbrgrp>. The identification of an analogous phenomenon <it>in vivo </it>could establish a new correlate of LHF infection leading to the development of sensitive diagnostics targeting the earliest molecular events of the disease. Additionally, the reversal of potentially untoward immunomodulatory functions mediated by sGP1 could potentiate the development of novel therapeutic intervention. To this end, we investigated the presence of sGP1 in the serum of suspected LASV patients admitted to the Kenema Government Hospital (KGH) Lassa Fever Ward (LFW), in Kenema, Sierra Leone that tested positive for viral antigen or displayed classical signs of Lassa fever.</p> <p>Results</p> <p>It is reasonable to expect that a narrow window exists for detection of sGP1 as the sole protein shed during early arenaviral biogenesis. This phenomenon was clearly distinguishable from virion-associated GP1 only prior to the emergence of <it>de novo </it>viral particles. Despite this restricted time frame, in 2/46 suspected cases in two studies performed in late 2009 and early 2010, soluble glycoprotein component shedding was identified. Differential detection of viral antigens GP1, GP2, and NP by western blot yielded five different scenarios: whole LASV virions (GP1, GP2, NP; i.e. active viremia), different combinations of these three proteins, sGP1 only, NP only, and absence of all three proteins. Four additional samples showed inconclusive evidence for sGP1 shedding due to lack of detection of GP2 and NP by western blot; however, a sensitive LASV NP antigen capture ELISA generated marginally positive signals</p> <p>Conclusions</p> <p>During a narrow window following active infection with LASV, soluble GP1 can be detected in patient sera. This phenomenon parallels other VHF infection profiles, with the actual role of a soluble viral glycoprotein component <it>in vivo </it>remaining largely speculative. The expenditure of energy and cellular resources toward secretion of a critical protein during viral biogenesis without apparent specific function requires further investigation. Future studies will be aimed at systematically identifying the role of LASV sGP1 in the infection process and outcome <it>in vitro </it>and <it>in vivo</it>.</p

Crimean-Congo Hemorrhagic Fever, Afghanistan, 2009

In response to an outbreak of Crimean-Congo hemorrhagic fever in western Afghanistan, we measured immunoglobulin G seroprevalence among household members and their animals. Seroprevalence was 11.2% and 75.0% in humans (n = 330) and livestock (n = 132), respectively. Persons with frequent exposure to cattle had an elevated risk of being immunoglobulin G positive

Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever

<p>Abstract</p> <p>Background</p> <p>Lassa fever is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. Treatment of acute Lassa fever infections has successfully utilized intravenous administration of ribavirin, a nucleotide analogue drug, but this is not an approved use; efficacy of oral administration has not been demonstrated. To date, several potential new vaccine platforms have been explored, but none have progressed toward clinical trials and commercialization. Therefore, the development of a robust vaccine platform that could be generated in sufficient quantities and at a low cost per dose could herald a subcontinent-wide vaccination program. This would move Lassa endemic areas toward the control and reduction of major outbreaks and endemic infections. To this end, we have employed efficient mammalian expression systems to generate a Lassa virus (LASV)-like particle (VLP)-based modular vaccine platform.</p> <p>Results</p> <p>A mammalian expression system that generated large quantities of LASV VLP in human cells at small scale settings was developed. These VLP contained the major immunological determinants of the virus: glycoprotein complex, nucleoprotein, and Z matrix protein, with known post-translational modifications. The viral proteins packaged into LASV VLP were characterized, including glycosylation profiles of glycoprotein subunits GP1 and GP2, and structural compartmentalization of each polypeptide. The host cell protein component of LASV VLP was also partially analyzed, namely glycoprotein incorporation, though the identity of these proteins remain unknown. All combinations of LASV Z, GPC, and NP proteins that generated VLP did not incorporate host cell ribosomes, a known component of native arenaviral particles, despite detection of small RNA species packaged into pseudoparticles. Although VLP did not contain the same host cell components as the native virion, electron microscopy analysis demonstrated that LASV VLP appeared structurally similar to native virions, with pleiomorphic distribution in size and shape. LASV VLP that displayed GPC or GPC+NP were immunogenic in mice, and generated a significant IgG response to individual viral proteins over the course of three immunizations, in the absence of adjuvants. Furthermore, sera from convalescent Lassa fever patients recognized VLP in ELISA format, thus affirming the presence of native epitopes displayed by the recombinant pseudoparticles.</p> <p>Conclusions</p> <p>These results established that modular LASV VLP can be generated displaying high levels of immunogenic viral proteins, and that small laboratory scale mammalian expression systems are capable of producing multi-milligram quantities of pseudoparticles. These VLP are structurally and morphologically similar to native LASV virions, but lack replicative functions, and thus can be safely generated in low biosafety level settings. LASV VLP were immunogenic in mice in the absence of adjuvants, with mature IgG responses developing within a few weeks after the first immunization. These studies highlight the relevance of a VLP platform for designing an optimal vaccine candidate against Lassa hemorrhagic fever, and warrant further investigation in lethal challenge animal models to establish their protective potential.</p

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

<p>Abstract</p> <p>Background</p> <p>There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2).</p> <p>Results</p> <p>Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of <it>Escherichia coli </it>(<it>E. coli</it>) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA).</p> <p>Conclusion</p> <p>These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.</p

Capacity building permitting comprehensive monitoring of a severe case of Lassa hemorrhagic fever in Sierra Leone with a positive outcome: Case Report

Lassa fever is a neglected tropical disease with a significant impact on the health care system of endemic West African nations. To date, case reports of Lassa fever have focused on laboratory characterisation of serological, biochemical and molecular aspects of the disease imported by infected individuals from Western Africa to the United States, Canada, Europe, Japan and Israel. Our report presents the first comprehensive real time diagnosis and characterization of a severe, hemorrhagic Lassa fever case in a Sierra Leonean individual admitted to the Kenema Government Hospital Lassa Fever Ward. Fever, malaise, unresponsiveness to anti-malarial and antibiotic drugs, followed by worsening symptoms and onset of haemorrhaging prompted medical officials to suspect Lassa fever. A recombinant Lassa virus protein based diagnostic was employed in diagnosing Lassa fever upon admission. This patient experienced a severe case of Lassa hemorrhagic fever with dysregulation of overall homeostasis, significant liver and renal system involvement, the interplay of pro- and anti-inflammatory cytokines during the course of hospitalization and an eventual successful outcome. These studies provide new insights into the pathophysiology and management of this viral illness and outline the improved infrastructure, research and real-time diagnostic capabilities within LASV endemic areas

Seroprevalence and distribution of arboviral infections among rural Kenyan adults: A cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Arthorpod-borne viruses (arboviruses) cause wide-spread morbidity in sub-Saharan Africa, but little research has documented the burden and distribution of these pathogens.</p> <p>Methods</p> <p>Using a population-based, cross-sectional study design, we administered a detailed questionnaire and used ELISA to test the blood of 1,141 healthy Kenyan adults from three districts for the presence of anti-viral Immunoglobulin G (IgG) antibodies to the following viruses: dengue (DENV), West Nile (WNV), yellow fever (YFV), Chikungunya (CHIKV), and Rift Valley fever (RVFV).</p> <p>Results</p> <p>Of these, 14.4% were positive for DENV, 9.5% were WNV positive, 9.2% were YFV positive, 34.0% were positive for CHIKV and 0.7% were RVFV positive. In total, 46.6% had antibodies to at least one of these arboviruses.</p> <p>Conclusions</p> <p>For all arboviruses, district of residence was strongly associated with seropositivity. Seroprevalence to YFV, DENV and WNV increased with age, while there was no correlation between age and seropositivity for CHIKV, suggesting that much of the seropositivity to CHIKV is due to sporadic epidemics. Paradoxically, literacy was associated with increased seropositivity of CHIKV and DENV.</p

Nomenclature- and Database-Compatible Names for the Two Ebola Virus Variants that Emerged in Guinea and the Democratic Republic of the Congo in 2014

In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: “Makona”, Middle Africa: “Lomela”) and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures

Evolution and Spread of Ebola Virus in Liberia, 2014–2015

The 2013–present Western African Ebola virus disease (EVD) outbreak is the largest ever recorded with >28,000 reported cases. Ebola virus (EBOV) genome sequencing has played an important role throughout this outbreak; however, relatively few sequences have been determined from patients in Liberia, the second worst-affected country. Here, we report 140 EBOV genome sequences from the second wave of the Liberian outbreak and analyze them in combination with 782 previously published sequences from throughout the Western African outbreak. While multiple early introductions of EBOV to Liberia are evident, the majority of Liberian EVD cases are consistent with a single introduction, followed by spread and diversification within the country. Movement of the virus within Liberia was widespread and reintroductions from Liberia served as an important source for the continuation of the already ongoing EVD outbreak in Guinea. Overall, little evidence was found for incremental adaptation of EBOV to the human host.Organismic and Evolutionary Biolog

Undiagnosed Acute Viral Febrile Illnesses, Sierra Leone

Sierra Leone in West Africa is in a Lassa fever–hyperendemic region that also includes Guinea and Liberia. Each year, suspected Lassa fever cases result in submission of ≈500–700 samples to the Kenema Government Hospital Lassa Diagnostic Laboratory in eastern Sierra Leone. Generally only 30%–40% of samples tested are positive for Lassa virus (LASV) antigen and/or LASV-specific IgM; thus, 60%–70% of these patients have acute diseases of unknown origin. To investigate what other arthropod-borne and hemorrhagic fever viral diseases might cause serious illness in this region and mimic Lassa fever, we tested patient serum samples that were negative for malaria parasites and LASV. Using IgM-capture ELISAs, we evaluated samples for antibodies to arthropod-borne and other hemorrhagic fever viruses. Approximately 25% of LASV-negative patients had IgM to dengue, West Nile, yellow fever, Rift Valley fever, chikungunya, Ebola, and Marburg viruses but not to Crimean-Congo hemorrhagic fever virus