Matt L. Boisen

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 worlds 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 Leones 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.

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

BackgroundLassa 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.ResultsA 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.ConclusionsThese 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.

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

BackgroundLassa 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.ResultsAnalyses 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.ConclusionOnly 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.

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.

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.

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.

Development of Prototype Filovirus Recombinant Antigen Immunoassays

BACKGROUND Throughout the 2014-2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. METHODS Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. RESULTS Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus-specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. CONCLUSIONS The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins.

Lassa Virus Seroprevalence in Sibirilia Commune, Bougouni District, Southern Mali.

The high rate documented in this study highlights the need for increased surveillance. Lassa Virus Seroprevalence, Mali

Vectorborne Infections, Mali.

To the Editor: As in many West Africa nations, vectorborne diseases represent a substantial health burden in Mali; however, beyond malaria, the incidence and etiology of many of these diseases is poorly understood. Of the estimated 14.1 million persons living in sub-Saharan Mali, ≈70% live in remote rural settings with an ecologic landscape that puts inhabitants at an increased risk for contact with rodent and arthropodborne diseases. We retrospectively analyzed serum samples for evidence of recent (IgM+) and previous (IgG+) infection with chikungunya (CHIKV), dengue (DENV), West Nile (WNV), Lassa (LASV), Crimean-Congo hemorrhagic fever (CCHFV), and Ebola (EBOV) virus, as well as Old World hantaviruses (OW-HANV) and Leptospira spp., which is regularly misdiagnosed as an acute viral infection. We tested 376 deidentified serum samples collected from acutely ill patients who had a history of fever and hemorrhagic, diarrheal, or icteric syndromes (Technical Appendix Figure). Research on samples from humans was conducted in accordance with the policies and regulations of the National Institutes of Health and adhered to the principles of the Belmont Report (1979) (http://www.hhs.gov/ohrp/humansubjects/guidance/belmont.html). This research was conducted under an institutional review board–approved document. Samples had previously tested negative for acute Plasmodium falciparum malaria and yellow fever virus infections. Commercially available IgM capture and conventional IgG ELISAs were used for serologic testing for CHIKV (GenWay Biotech, San Diego, CA, USA); DENV (all four serotypes) and WNV (both from Focus Diagnostics, Cypress, CA, USA); OW-HANVs (Euroimmun, Luebeck, Germany); and Leptospira spp. (Abnova, Taipei City, Taiwan). Conventional IgM/IgG ELISAs were used for LASV (Corgenix, Broomfield, CO, USA) and CCHFV (Vector-Best, Novosibirsk, Russia), and reagents for the EBOV IgM/IgG ELISA (infected/uninfected cell lysates) were prepared at the Rocky Mountain Laboratories (Hamilton, MT, USA) and validated with serum from experimentally infected monkeys. With the exception of the CHIKV, Leptospira spp., and in-house EBOV assays, the tests conducted in this study are under preclinical development for human diagnostic assays. Samples were tested at a 1:100 dilution according to manufacturer specifications (CHIKV, CCHFV, WNV, DENV, OW-HANVs, LASV, and Leptospira spp.) or in-house quality-control assessments (EBOV), in a blinded fashion. Serologic reactivity was assessed according to manufacturer recommendations. For the EBOV ELISA, samples were deemed positive if optical density at 405 nm was >3 SD above that of the average of known negative samples. Serologic evidence suggestive of acute infection (IgM+) with 1 of the pathogens tested for was observed for 39.9% of samples (Table). At 14.4%, Leptospira spp. was the most prevalent probable etiologic agent of acute disease identified. Of mosquitoborne viruses tested, DENV had the highest prevalence at 7.7%, followed by CHIKV (5.3%) and WNV (0.27%). Of rodentborne pathogens, OW-HANVs had a seroprevalence of 7.2%, whereas LASV was considerably lower (0.27%). CCHFV IgM was documented in 4.8% of samples. Overall, little annual variation in the IgM seroprevalence was noted, except for Leptospira spp., for which 2 obvious peaks in seroprevalence were observed (Table). Table IgM and IgG seroprevalence rates of selected vectorborne pathogens in samples submitted from suspected yellow fever cases in Mali, 2009–2013* Most IgM+ samples demonstrated serologic reactivity in only 1 assay. The exception was 2 samples that were IgM+ for hantaviruses and Leptospira spp., an acute dual infection that might be underrecognized (1). With the exception of DENV, few samples were both IgM+ and IgG+, suggesting the results were not attributable to IgM persistence. The DENV IgM+/IgG+ results might represent IgM persistence. However, because the ELISA detected all 4 serotypes, it is plausible that some results represent recent infection with DENV in the presence of IgG reactive with a different serotype. The relatively high IgG seroprevalence for most of the pathogens tested supports the findings of the IgM assays and further suggest the circulation of and potential for human exposure to these agents in Mali (Table). Geographically, serologic evidence of infections with Leptospira spp., DENV, WNV, OW-HANVs, and CHIKV was observed throughout Mali (online Technical Appendix). No samples were reactive with EBOV, and the low incidence of LASV infection is not surprising because the samples analyzed here were collected outside of the 1 documented LASV-endemic region in Mali (2). We used commercially available diagnostic platforms, primarily IgM capture and conventional IgG ELISAs, many of which are validated for human diagnostics. Ideally, diagnostics for zoonotic diseases would not rely on IgM/IgG serologic analysis because of caveats including IgM persistence and cross-reactivity between closely related pathogens (3,4). In the industrialized world, as well as in several countries throughout Africa, molecular approaches are often used to genetically identify pathogens, or follow-up convalescent-phase serum samples are collected to determine seroconversion or increased antibody titers or to conduct plaque reduction neutralization assays. Unfortunately, because of the nature of the samples available, including time of collection, storage history, and remaining volume, many of these tests were not feasible for our study. Despite these limitations, these serologic findings indicate that flaviviruses, bunyaviruses, and togaviruses, as well as Leptospira spp., are contributing to human illness in Mali. These results add to those recently documented in studies conducted in Sierra Leone, implying that several of these zoonotic pathogens are widely distributed yet underreported throughout West Africa (5,6). Technical Appendix. Distribution of serum samples tested, by region and year. IgM and IgG seroprevalence rates of selected vectorborne pathogens, by region of sample collection. Click here to view.(192K, pdf)

Annual Incidence of Lassa Virus Infection in Southern Mali

AbstractPreviously, we reported a high seroprevalence rate of Lassa virus antibodies in inhabitants of three villages in southern Mali where infected rodents have been demonstrated. Herein, we report a 1-year follow-up study in which we were able to collect a second blood samples from 88.7% of participants of the same cohort. We identified 23 seroconversions for IgG antibodies reactive against Lassa virus, representing an incidence of 6.3% (95% confidence interval = 3.8-8.8%). Seroconversion was frequently seen in preteenage children (12/23, 51.7%) and two household/familial clusters were identified. These results confirm active transmission of Lassa virus is occurring in southern Mali and appropriate diagnostic testing should be established for this etiological agent of severe viral hemorrhagic fever.