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

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

Correction: Lassa hemorrhagic fever in a late term pregnancy from northern Sierra Leone with a positive maternal outcome: case report

Lassa fever (LF) is a devastating viral disease prevalent in West Africa. Efforts to take on this public health crisis have been hindered by lack of infrastructure and rapid field deployable diagnosis in areas where the disease is prevalent. Recent capacity building at the Kenema Government Hospital Lassa Fever Ward (KGH LFW) in Sierra Leone has lead to a major turning point in the diagnosis, treatment and study of LF. Herein we present the first comprehensive rapid diagnosis and real time characterization of an acute hemorrhagic LF case at KGH LFW. This case report focuses on a third trimester pregnant Sierra Leonean woman from the historically non-endemic Northern district of Tonkolili who survived the illness despite fetal demise. Employed in this study were newly developed recombinant LASV Antigen Rapid Test cassettes and dipstick lateral flow immunoassays (LFI) that enabled the diagnosis of LF within twenty minutes of sample collection. Deregulation of overall homeostasis, significant hepatic and renal system involvement, and immunity profiles were extensively characterized during the course of hospitalization. Rapid diagnosis, prompt treatment with a full course of intravenous (IV) ribavirin, IV fluids management, and real time monitoring of clinical parameters resulted in a positive maternal outcome despite admission to the LFW seven days post onset of symptoms, fetal demise, and a natural still birth delivery. These studies solidify the growing rapid diagnostic, treatment, and surveillance capabilities at the KGH LF Laboratory, and the potential to significantly improve the current high mortality rate caused by LF. As a result of the growing capacity, we were also able to isolate Lassa virus (LASV) RNA from the patient and perform Sanger sequencing where we found significant genetic divergence from commonly circulating Sierra Leonean strains, showing potential for the discovery of a newly emerged LASV strain with expanded geographic distribution. Furthermore, recent emergence of LF cases in Northern Sierra Leone highlights the need for superior diagnostics to aid in the monitoring of LASV strain divergence with potentially increased geographic expansion.Organismic and Evolutionary BiologyOther Research Uni

Clinical Illness and Outcomes in Patients with Ebola in Sierra Leone

Background Limited clinical and laboratory data are available on patients with Ebola virus disease (EVD). The Kenema Government Hospital in Sierra Leone, which had an existing infrastructure for research regarding viral hemorrhagic fever, has received and cared for patients with EVD since the beginning of the outbreak in Sierra Leone in May 2014. Methods We reviewed available epidemiologic, clinical, and laboratory records of patients in whom EVD was diagnosed between May 25 and June 18, 2014. We used quantitative reverse-transcriptase–polymerase-chain-reaction assays to assess the load of Ebola virus (EBOV, Zaire species) in a subgroup of patients. Results Of 106 patients in whom EVD was diagnosed, 87 had a known outcome, and 44 had detailed clinical information available. The incubation period was estimated to be 6 to 12 days, and the case fatality rate was 74%. Common findings at presentation included fever (in 89% of the patients), headache (in 80%), weakness (in 66%), dizziness (in 60%), diarrhea (in 51%), abdominal pain (in 40%), and vomiting (in 34%). Clinical and laboratory factors at presentation that were associated with a fatal outcome included fever, weakness, dizziness, diarrhea, and elevated levels of blood urea nitrogen, aspartate aminotransferase, and creatinine. Exploratory analyses indicated that patients under the age of 21 years had a lower case fatality rate than those over the age of 45 years (57% vs. 94%, P=0.03), and patients presenting with fewer than 100,000 EBOV copies per milliliter had a lower case fatality rate than those with 10 million EBOV copies per milliliter or more (33% vs. 94%, P=0.003). Bleeding occurred in only 1 patient. Conclusions The incubation period and case fatality rate among patients with EVD in Sierra Leone are similar to those observed elsewhere in the 2014 outbreak and in previous outbreaks. Although bleeding was an infrequent finding, diarrhea and other gastrointestinal manifestations were common. (Funded by the National Institutes of Health and others.

Lassa Fever in Post-Conflict Sierra Leone

Background: Lassa fever (LF), an often-fatal hemorrhagic disease caused by Lassa virus (LASV), is a major public health threat in West Africa. When the violent civil conflict in Sierra Leone (1991 to 2002) ended, an international consortium assisted in restoration of the LF program at Kenema Government Hospital (KGH) in an area with the world's highest incidence of the disease. Methodology/Principal Findings Clinical and laboratory records of patients presenting to the KGH Lassa Ward in the post-conflict period were organized electronically. Recombinant antigen-based LF immunoassays were used to assess LASV antigenemia and LASV-specific antibodies in patients who met criteria for suspected LF. KGH has been reestablished as a center for LF treatment and research, with over 500 suspected cases now presenting yearly. Higher case fatality rates (CFRs) in LF patients were observed compared to studies conducted prior to the civil conflict. Different criteria for defining LF stages and differences in sensitivity of assays likely account for these differences. The highest incidence of LF in Sierra Leone was observed during the dry season. LF cases were observed in ten of Sierra Leone's thirteen districts, with numerous cases from outside the traditional endemic zone. Deaths in patients presenting with LASV antigenemia were skewed towards individuals less than 29 years of age. Women self-reporting as pregnant were significantly overrepresented among LASV antigenemic patients. The CFR of ribavirin-treated patients presenting early in acute infection was lower than in untreated subjects. Conclusions/Significance: Lassa fever remains a major public health threat in Sierra Leone. Outreach activities should expand because LF may be more widespread in Sierra Leone than previously recognized. Enhanced case finding to ensure rapid diagnosis and treatment is imperative to reduce mortality. Even with ribavirin treatment, there was a high rate of fatalities underscoring the need to develop more effective and/or supplemental treatments for LF

Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design

Emerging trends in Lassa fever: redefining the role of immunoglobulin M and inflammation in diagnosing acute infection

Abstract Background Lassa fever (LF) is a devastating hemorrhagic viral disease that is endemic to West Africa and responsible for thousands of human deaths each year. Analysis of humoral immune responses (IgM and IgG) by antibody-capture ELISA (Ab-capture ELISA) and Lassa virus (LASV) viremia by antigen-capture ELISA (Ag-capture ELISA) in suspected patients admitted to the Kenema Government Hospital (KGH) Lassa Fever Ward (LFW) in Sierra Leone over the past five years is reshaping our understanding of acute LF. Results Analyses in LF survivors indicated that LASV-specific IgM persists for months to years after initial infection. Furthermore, exposure to LASV appeared to be more prevalent in historically non-endemic areas of West Africa with significant percentages of reportedly healthy donors IgM and IgG positive in LASV-specific Ab-capture ELISA. We found that LF patients who were Ag positive were more likely to die than suspected cases who were only IgM positive. Analysis of metabolic and immunological parameters in Ag positive LF patients revealed a strong correlation between survival and low levels of IL-6, -8, -10, CD40L, BUN, ALP, ALT, and AST. Despite presenting to the hospital with fever and in some instances other symptoms consistent with LF, the profiles of Ag negative IgM positive individuals were similar to those of normal donors and nonfatal (NF) LF cases, suggesting that IgM status cannot necessarily be considered a diagnostic marker of acute LF in suspected cases living in endemic areas of West Africa. Conclusion Only LASV viremia assessed by Ag-capture immunoassay, nucleic acid detection or virus isolation should be used to diagnose acute LASV infection in West Africans. LASV-specific IgM serostatus cannot be considered a diagnostic marker of acute LF in suspected cases living in endemic areas of West Africa. By applying these criteria, we identified a dysregulated metabolic and pro-inflammatory response profile conferring a poor prognosis in acute LF. In addition to suggesting that the current diagnostic paradigm for acute LF should be reconsidered, these studies present new opportunities for therapeutic interventions based on potential prognostic markers in LF.</p