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BackgroundLassa fever (LF) is a rodent-borne disease endemic to West Africa. In the absence of licensed therapeutics or vaccines, rodent exclusion from living spaces remains the primary method of preventing LF. Zoonotic surveillance of Lassa virus (LASV), the etiologic agent of LF, can assess the burden of LASV in a region and guide public health measures against LF.MethodsIn this study, we adapted commercially available LASV human diagnostics to assess the prevalence of LASV in peri-domestic rodents in Eastern Sierra Leone. Small mammal trapping was conducted in Kenema district, Sierra Leone between November 2018-July 2019. LASV antigen was detected using a commercially available LASV NP antigen rapid diagnostic test. LASV IgG antibodies against LASV nucleoprotein (NP) and glycoprotein (GP) were tested by adapting a commercially available semi-quantitative enzyme linked immunosorbent assay (ELISA) for detection of mouse-related and rat-related species IgG.FindingsOf the 373 tested specimens, 74 (20%) tested positive for LASV antigen. 40 (11%) specimens tested positive for LASV NP IgG, while an additional 12 (3%) specimens only tested positive for LASV GP IgG. Simultaneous antigen presence and IgG antibody presence was linked inMastomys sp. specimens (p< 0.01), but notRattus sp. specimens (p= 1). Despite the link between antigen presence and IgG antibody presence inMastomys sp., the strength of antigen response did not correlate with the strength of IgG response to either GP IgG or NP IgG.InterpretationThe tools developed in this study can aid in the generation of valuable public health data for rapid field assessment of LASV burden during outbreak investigations and general LASV surveillance.FundingFunding for this work was supported by the National Institute of Allergy and Infectious Diseases National Institute of Health, Department of Health and Human Services under the following grants: International Collaboration in Infectious Disease Research on Lassa fever and Ebola - ICIDR - U19 AI115589, Consortium for Viral Systems Biology - CViSB - 5U19AI135995, West African Emerging Infectious Disease Research Center - WARN-ID - U01AI151812, West African Center for Emerging Infectious Diseases: U01AI151801.

Lassa fever (LF) causes multisystem disease and has a fatality rate

Pediatric Lassa fever (LF) usually presents as a nonspecific febrile illness, similar to other endemic diseases in countries like Sierra Leone, where LF is considered to be hyperendemic. The nonspecificity of presentation and lack of research have made it difficult to fully understand best practices for pediatric management. We aim to describe clinical characteristics of hospitalized pediatric patients suspected or diagnosed with LF and assess factors associated with hospital outcomes among those with LF antigen–positive results. We conducted a 7-year retrospective cohort study using routine data for all children younger than 18 years admitted at the Kenema Government Hospital’s LF ward. A total of 292 children with suspected or confirmed LF were analyzed. Overall, mortality was high (21%). Children with antigen-positive results had a high case fatality rate of 63% (P < 0.01). In univariate analyses, children who presented with unexplained bleeding (odds ratio [OR]: 3.58; 95% CI: 1.08–11.86; P = 0.040) and confusion (altered sensorium) (OR: 5.37; 95% CI: 1.34–21.48; P = 0.020) had increased odds of death. Abnormal serum levels of alanine aminotransferase (P = 0.001), creatinine (P = 0.004), and potassium (P = 0.003) were associated with increased likelihood of death in these children. Treatment with ribavirin was not significantly associated with survival (P = 0.916). Our findings provide insights into current pediatric LF clinical presentation and management. More evidence-based, high-quality research in creating predictive algorithms of antigen-positivity and hospital outcomes is needed in the management of pediatric LF.

Lassa virus (LASV) is the causative agent of Lassa fever, an often-fatal hemorrhagic disease that is endemic in West Africa. Seven genetically distinct LASV lineages have been identified. As part of CEPI’s (Coalition for Epidemic Preparedness Innovations) Lassa vaccine development program, we assessed the potential of the human immune system to mount cross-reactive and cross-protective humoral immune responses to antigens from the most prevalent LASV lineages, which are lineages II and III in Nigeria and lineage IV in Sierra Leone. IgG and IgM present in the blood of Lassa fever survivors from Nigeria or Sierra Leone exhibited substantial cross-reactivity for binding to LASV nucleoprotein and two engineered (linked and prefusion) versions of the glycoproteins (GP) of lineages II–IV. There was less cross-reactivity for the Zinc protein. Serum or plasma from Nigerian Lassa fever survivors neutralized LASV pseudoviruses expressing lineage II GP better than they neutralized lineage III or IV GP expressing pseudoviruses. Sierra Leonean survivors did not exhibit a lineage bias. Neutralization titres determined using LASV pseudovirus assays showed significant correlation with titres determined by plaque reduction with infectious LASV. These studies provide guidance for comparison of humoral immunity to LASV of distinct lineages following natural infection or immunization.

Lassa virus (LASV) is the causative agent of Lassa fever (LF), an often-fatal hemorrhagic disease. LF is endemic in Nigeria, Sierra Leone and other West African countries. Diagnosis of LASV infection is challenged by the genetic diversity of the virus, which is greatest in Nigeria. The ReLASV Pan-Lassa Antigen Rapid Test (Pan-Lassa RDT) is a point-of-care, in vitro diagnostic test that utilizes a mixture of polyclonal antibodies raised against recombinant nucleoproteins of representative strains from the three most prevalent LASV lineages (II, III and IV). We compared the performance of the Pan-LASV RDT to available quantitative PCR (qPCR) assays during the 2018 LF outbreak in Nigeria. For patients with acute LF (RDT positive, IgG/IgM negative) during initial screening, RDT performance was 83.3% sensitivity and 92.8% specificity when compared to composite results of two qPCR assays. 100% of samples that gave Ct values below 22 on both qPCR assays were positive on the Pan-Lassa RDT. There were significantly elevated case fatality rates and elevated liver transaminase levels in subjects whose samples were RDT positive compared to RDT negative.

Many countries in sub-Saharan Africa have experienced lower COVID-19 caseloads and fewer deaths than countries in other regions worldwide. Under-reporting of cases and a younger population could partly account for these differences, but pre-existing immunity to coronaviruses is another potential factor. Blood samples from Sierra Leonean Lassa fever and Ebola survivors and their contacts collected before the first reported COVID-19 cases were assessed using enzyme-linked immunosorbent assays for the presence of antibodies binding to proteins of coronaviruses that infect humans. Results were compared to COVID-19 subjects and healthy blood donors from the United States. Prior to the pandemic, Sierra Leoneans had more frequent exposures than Americans to coronaviruses with epitopes that cross-react with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), SARS-CoV, and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). The percentage of Sierra Leoneans with antibodies reacting to seasonal coronaviruses was also higher than for American blood donors. Serological responses to coronaviruses by Sierra Leoneans did not differ by age or sex. Approximately a quarter of Sierra Leonian pre-pandemic blood samples had neutralizing antibodies against SARS-CoV-2 pseudovirus, while about a third neutralized MERS-CoV pseudovirus. Prior exposures to coronaviruses that induce cross-protective immunity may contribute to reduced COVID-19 cases and deaths in Sierra Leone.

Lassa fever is listed among the diseases that pose the greatest risks to public health by the World Health Organization. This severe viral hemorrhagic fever is caused by Lassa virus, a zoonotic pathogen that repeatedly spills over to humans from its rodent reservoirs. It is currently not known how climate change, transformations in land use, and human population growth could affect the endemic area of this virus, currently limited to parts of West Africa. By exploring the environmental data associated with virus occurrence, we show how temperature, precipitation and the presence of pastures determine ecological suitability for virus circulation. We project that regions in Central and East Africa will likely become suitable for Lassa virus over the next decades and estimate that the total population living in areas suitable for Lassa virus may grow from about 100 million to 700 million by 2070. By analysing geotagged viral genomes, we find that in the event of Lassa virus being introduced into a new suitable region, its spread might remain spatially limited over the first decades. Our results highlight how the endemic area of Lassa virus may expand well beyond West Africa in the next decades due to human impact on the environment, putting hundreds of million more people at risk of infection.

Monoclonal antibodies can mediate protection against Ebola virus (EBOV) infection through direct neutralization as well as through the recruitment of innate immune effector functions. However, the antibody functional response following survival of acute EBOV disease has not been well characterized. In this study, serum antibodies from Ebola virus disease (EVD) survivors from Sierra Leone were profiled to capture variation in overall subclass/isotype abundance, neutralizing activity, and innate immune effector functions. Antibodies from EVD survivors exhibited robust innate immune effector functions, mediated primarily by IgG1 and IgA1. In conclusion, development of functional antibodies follows survival of acute EVD., The humoral immune response in human survivors of Ebola virus disease includes the development of neutralizing antibodies and polyfunctional IgG1 and IgA antibodies that can mediate effector functions against the Ebola virus glycoprotein via multiple innate immune effector cell types.

Summary During 2018, an unusual increase in Lassa fever cases occurred in Nigeria, raising concern among national and international public health agencies. We analyzed 220 Lassa virus genomes from infected patients, including 129 from the 2017–2018 transmission season, to understand the viral populations underpinning the increase. A total of 14 initial genomes from 2018 samples were generated at Redeemer’s University in Nigeria, and the findings were shared with the Nigerian Center for Disease Control in real time. We found that the increase in cases was not attributable to a particular Lassa virus strain or sustained by human-to-human transmission. Instead, the data were consistent with ongoing cross-species transmission from local rodent populations. Phylogenetic analysis also revealed extensive viral diversity that was structured according to geography, with major rivers appearing to act as barriers to migration of the rodent reservoir.

Recent outbreaks of viral hemorrhagic fevers (VHFs), including Ebola virus disease (EVD) and Lassa fever (LF), highlight the urgent need for sensitive, deployable tests to diagnose these devastating human diseases. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. We perform safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We develop a user-friendly protocol and mobile application (HandLens) to report results, facilitating SHERLOCK’s use in endemic regions. Finally, we successfully deploy our tests in Sierra Leone and Nigeria in response to recent outbreaks., Outbreaks of viral hemorrhagic fevers highlight the need for sensitive, field-deployable diagnostics. Here the authors present a CRISPR-based SHERLOCK platform with field protocol and mobile app for Ebola and Lassa fever outbreaks.

The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine., Early and robust T cell responses have been associated with survival from Lassa fever (LF), but the Lassa virus-specific memory responses have not been well characterized. Regions within the virus surface glycoprotein (GPC) and nucleoprotein (NP) are the main targets of the Lassa virus-specific T cell responses, but, to date, only a few T cell epitopes within these proteins have been identified. We identified GPC and NP regions containing T cell epitopes and HLA haplotypes from LF survivors and used predictive HLA-binding algorithms to identify putative epitopes, which were then experimentally tested using autologous survivor samples. We identified 12 CD8-positive (CD8+) T cell epitopes, including epitopes common to both Nigerian and Sierra Leonean survivors. These data should be useful for the identification of dominant Lassa virus-specific T cell responses in Lassa fever survivors and vaccinated individuals as well as for designing vaccines that elicit cell-mediated immunity. IMPORTANCE The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine.

Viral hemorrhagic fevers (VHFs) remain some of the most devastating human diseases, and recent outbreaks of Ebola virus disease (EVD) 1,2 and Lassa fever (LF) 3,4 highlight the urgent need for sensitive, field-deployable tests to diagnose them 5,6. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. Our EBOV diagnostic detects both the L and NP genes, thereby eliminating the potential for false positive results caused by the rVSVΔG-ZEBOV-GP live attenuated vaccine. Our two LASV diagnostics together capture 90% of known viral diversity and demonstrate that CRISPR-RNAs (crRNAs) can be effectively multiplexed to provide greater coverage of known viral diversity. We performed safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating and chemically treating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We developed a user-friendly field protocol and mobile application (HandLens) to report results, facilitating SHERLOCK’s use in endemic regions. Finally, we successfully deployed our tests in Sierra Leone and Nigeria in response to recent outbreaks.

BackgroundEbola virus (EBOV) disease has killed thousands of West and Central Africans over the past several decades. Many who survive the acute disease later experience post-Ebola syndrome, a constellation of symptoms whose causative pathogenesis is unclear.MethodsWe investigated EBOV-specific CD8+ and CD4+ T-cell responses in 37 Sierra Leonean EBOV disease survivors with (n = 19) or without (n = 18) sequelae of arthralgia and ocular symptoms. Peripheral blood mononuclear cells were infected with recombinant vesicular stomatitis virus encoding EBOV antigens. We also studied the presence of EBOV-specific immunoglobulin G, antinuclear antibodies, anti–cyclic citrullinated peptide antibodies, rheumatoid factor, complement levels, and cytokine levels in these 2 groups.ResultsSurvivors with sequelae had a significantly higher EBOV-specific CD8+ and CD4+ T-cell response. No differences in EBOV-specific immunoglobulin G, antinuclear antibody, or anti–cyclic citrullinated peptide antibody levels were found. Survivors with sequelae showed significantly higher rheumatoid factor levels.ConclusionEBOV-specific CD8+ and CD4+ T-cell responses were significantly higher in Ebola survivors with post-Ebola syndrome. These findings suggest that pathogenesis may occur as an immune-mediated disease via virus-specific T-cell immune response or that persistent antigen exposure leads to increased and sustained T-cell responses.

Summary Protective Ebola virus (EBOV) antibodies have neutralizing activity and induction of antibody constant domain (Fc)-mediated innate immune effector functions. Efforts to enhance Fc effector functionality often focus on maximizing antibody-dependent cellular cytotoxicity, yet distinct combinations of functions could be critical for antibody-mediated protection. As neutralizing antibodies have been cloned from EBOV disease survivors, we sought to identify survivor Fc effector profiles to help guide Fc optimization strategies. Survivors developed a range of functional antibody responses, and we therefore applied a rapid, high-throughput Fc engineering platform to define the most protective profiles. We generated a library of Fc variants with identical antigen-binding fragments (Fabs) from an EBOV neutralizing antibody. Fc variants with antibody-mediated complement deposition and moderate natural killer (NK) cell activity demonstrated complete protective activity in a stringent in vivo mouse model. Our findings highlight the importance of specific effector functions in antibody-mediated protection, and the experimental platform presents a generalizable resource for identifying correlates of immunity to guide therapeutic antibody design.

Background Ebola virus disease (EVD) survivors are at risk for uveitis during convalescence. Vision loss has been observed following uveitis due to cataracts. Since Ebola virus (EBOV) may persist in the ocular fluid of EVD survivors for an unknown duration, there are questions about the safety and feasibility of vision restorative cataract surgery in EVD survivors. Methods We conducted a cross-sectional study of EVD survivors anticipating cataract surgery and patients with active uveitis to evaluate EBOV RNA persistence in ocular fluid, as well as vision outcomes post cataract surgery. Patients with aqueous humor that tested negative for EBOV RNA were eligible to proceed with manual small incision cataract surgery (MSICS). Findings We screened 137 EVD survivors from June 2016 – August 2017 for enrolment. We enrolled 50 EVD survivors; 46 with visually significant cataract, 1 with a subluxated lens, 2 with active uveitis and 1 with a blind painful eye due to uveitis. The median age was 24.0 years (IQR 17–35) and 35 patients (70%) were female. The median logMAR visual acuity (VA) was 3.0 (Snellen VA Hand motions; Interquartile Range, IQR: 1.2-3.0, Snellen VA 20/320 – Hand motions). All patients tested negative for EBOV RNA by RT-PCR in aqueous humor/vitreous fluid and conjunctiva at a median of 19 months (IQR 18-20) from EVD diagnosis in Phase 1 of ocular fluid sampling and 34 months (IQR 32-36) from EVD diagnosis in Phase 2 of ocular fluid sampling. Thirty-four patients underwent MSICS, with a preoperative median VA improvement from hand motions to 20/30 at three-month postoperative follow-up (P, Highlights • Ebola virus disease (EVD) survivors are at-risk for severe vision impairment due to uveitis and subsequent cataract development. • Fifty EVD survivors underwent ocular fluid sampling and tested negative for Ebola virus by RT-PCR. • Thirty-four survivors underwent cataract surgery with excellent safety measures and vision restorative outcomes. Ebola virus disease (EVD) survivors are at high risk for uveitis, an inflammatory condition affecting the eye that may lead to severe vision impairment, often from cataract. Because persistent Ebola virus (EBOV) has been identified within the immune privileged eye, understanding the prevalence of intraocular EBOV persistence is significant for patients, providers, and public health policy. In this study, 50 EVD survivors tested negative for EBOV by RT-PCR of their intraocular fluid. Thirty-four underwent cataract surgery with vision restorative outcomes. These findings improve our ability to impact vision care and quality-of-life for thousands of EVD survivors at-risk for eye disease.

While investigating a signal of adaptive evolution in humans at the gene LARGE, we encountered an intriguing finding by Dr. Stefan Kunz that the gene plays a critical role in Lassa virus binding and entry. This led us to pursue field work to test our hypothesis that natural selection acting on LARGE—detected in the Yoruba population of Nigeria—conferred resistance to Lassa Fever in some West African populations. As we delved further, we conjectured that the “emerging” nature of recently discovered diseases like Lassa fever is related to a newfound capacity for detection, rather than a novel viral presence, and that humans have in fact been exposed to the viruses that cause such diseases for much longer than previously suspected. Dr. Stefan Kunz’s critical efforts not only laid the groundwork for this discovery, but also inspired and catalyzed a series of events that birthed Sentinel, an ambitious and large-scale pandemic prevention effort in West Africa. Sentinel aims to detect and characterize deadly pathogens before they spread across the globe, through implementation of its three fundamental pillars: Detect, Connect, and Empower. More specifically, Sentinel is designed to detect known and novel infections rapidly, connect and share information in real time to identify emerging threats, and empower the public health community to improve pandemic preparedness and response anywhere in the world. We are proud to dedicate this work to Stefan Kunz, and eagerly invite new collaborators, experts, and others to join us in our efforts.

There are no approved treatments for Lassa fever, which is endemic to the same regions of West Africa that were recently devastated by Ebola. Here we show that a combination of human monoclonal antibodies that cross-react with the glycoproteins of all four clades of Lassa virus is able to rescue 100% of cynomolgus macaques when treatment is initiated at advanced stages of disease, including up to 8 d after challenge.

Lassa fever is a rodent-borne illness that is endemic to parts of sub-Saharan Africa, including Sierra Leone, Nigeria, and Guinea. The disease is named after the town of Lassa, Nigeria where it was discovered in 1969. This data article focuses on the epidemiology of Lassa fever in Sierra Leone following a decade-long civil war that ended in 2002. The data were collected at Kenema Government Hospital (KGH) in Kenema, Sierra Leone, which maintains the country׳s only Lassa fever treatment facility and a biosafety level 3 (BSL-3) laboratory. The key data set variables include Lassa fever serostatus determined using antigen (Ag), immunoglobulin M (IgM), and immunoglobulin G (IgG) ELISA diagnostic techniques; and patient demographics, survival outcome, and treatment (ribavirin) status. The individual data used to generate the graphs and tables in the corresponding research manuscript published in PLOS Neglected Tropical Diseases in 2014 and its coding guide are provided as Supplementary material (Shaffer et al., 2014) [1]. Keywords: Infectious disease, Lassa fever, Epidemiology, Enzyme-linked immunosorbent assay (ELISA), Sierra Leone

Lassa fever is a significant health threat to West African human populations with hundreds of thousands of annual cases. There are no approved medical countermeasures currently available. Compassionate use of the antiviral drug ribavirin or transfusion of convalescent serum has resulted in mixed success depending on when administered or the donor source, respectively. We previously identified several recombinant human monoclonal antibodies targeting the glycoprotein of Lassa virus with strong neutralization profiles in vitro. Here, we demonstrate remarkable therapeutic efficacy using first-in-class human antibodies in a guinea pig model of Lassa infection thereby presenting a promising treatment alternative.

The 2013-2016 West African Ebola virus disease (EVD) epidemic is the largest recorded. Triage on the basis of clinical signs had limited success, and the time to diagnosis by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) could exceed 5 days. Here we describe the development and field validation of the ReEBOV Antigen Rapid Test (ReEBOV RDT) to aid triage of individuals with suspected EVD.Samples from patients with suspected EVD were submitted to Kenema Government Hospital, Sierra Leone, for Lassa fever and EVD screening throughout 2014. Banked residual clinical samples were tested in November 2014 and January 2015 in a blinded field trial to estimate the clinical effectiveness of the ReEBOV RDT, compared with EBOV-specific qRT-PCR.Preliminary ReEBOV RDT performance demonstrated a positive percentage agreement (PPA) of 91.1% (195 of 214 results; 95% confidence interval [CI], 86.5%-94.6%) and a negative percentage agreement (NPA) of 90.2% (175 of 194; 95% CI, 85.1%-94.0%). The final estimates used by the Food and Drug Administration to determine whether to grant emergency use authorization for the test, which excluded a qRT-PCR reference method threshold cutoff, were a PPA of 62.1% (72 of 116 results; 95% CI, 52.6%-70.9%) and a NPA of 96.7% (58 of 60; 95% CI, 88.5%-99.6%), with a diagnostic likelihood of 18.6. A subsequent, independent evaluation by the World Health Organization generated results consistent with the preliminary performance estimates.The ReEBOV RDT demonstrated the potential to provide clinically effective rapid and accurate point-of-care test results and, thus, to be a powerful tool for increasing triage efficiency.

Background.Kenema Government Hospital (KGH) has developed an advanced clinical and laboratory research capacity to manage the threat of Lassa fever, a viral hemorrhagic fever (VHF). The 2013–2016 Ebola virus (EBOV) disease (EVD) outbreak is the first to have occurred in an area close to a facility with established clinical and laboratory capacity for study of VHFs. Methods.Because of its proximity to the epicenter of the EVD outbreak, which began in Guinea in March 2014, the KGH Lassa fever Team mobilized to establish EBOV surveillance and diagnostic capabilities. Results.Augustine Goba, director of the KGH Lassa laboratory, diagnosed the first documented case of EVD in Sierra Leone, on 25 May 2014. Thereafter, KGH received and cared for numbers of patients with EVD that quickly overwhelmed the capacity for safe management. Numerous healthcare workers contracted and lost their lives to EVD. The vast majority of subsequent EVD cases in West Africa can be traced back to a single transmission chain that includes this first diagnosed case. Conclusions.Responding to the challenges of confronting 2 hemorrhagic fever viruses will require continued investments in the development of countermeasures (vaccines, therapeutic agents, and diagnostic assays), infrastructure, and human resources.

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 virus can cause haemorrhagic fever for which no specific treatment currently exists. Here the authors have cloned 113 monoclonal antibodies from the survivors of Lassa infection and show that the majority of neutralizing antibodies target a complex of GP1 and GP2 viral proteins.

Many factors contribute to the selection of epitope-specific T cells. Often, candidate epitopes are generated through in silico prediction of various biological processes, mainly peptide generation and MHC binding, that are then tested in relevant biological assays. This is especially true of epitopes derived from BSL-4 pathogens or where relevant patient samples are difficult to obtain. Two previous studies identified CD8+ T cell epitopes from the Lassa virus glycoprotein through in silico prediction, experimental MHC binding assays, and epitope generation in HLA-A2 transgenic mice. Using samples from ten HLA-A*02:01 Lassa fever survivors, we tested whether these previously described epitope-specific CD8+ T cells were present and their relation to the broader Lassa virus-specific T cell response. Using overnight stimulation assays, we detected robust LASV-specific responses to the glycoprotein and nucleoprotein, but only one of the three epitopes (GP60–68)shown to be present and protective in mice made a substantial contribution to the overall LASV-specific response. Using a more sensitive proliferation assay, we detected the remaining two epitopes in some individuals at a very low frequency. Overall, this study shows the limitations of epitope discovery through in silico prediction, MHC binding and transgenic mouse models and highlights the complex nature of T cell selection during natural infection of humans.

Situated in southeastern Sierra Leone, Kenema Government Hospital (KGH) maintains one of the world's only Lassa fever isolation wards and was a strategic Ebola virus disease (EVD) treatment facility during the 2014 EVD outbreak. Since 2006, the Viral Hemorrhagic Fever Consortium (VHFC) has carried out research activities at KGH, capturing clinical and laboratory data for suspected cases of Lassa fever. Here we describe the approach, progress, and challenges in designing and maintaining a data capture and management system (DCMS) at KGH to assist infectious disease researchers in building and sustaining DCMS in low-resource environments. Results on screening patterns and case-fatality rates are provided to illustrate the context and scope of the DCMS covered in this study. A medical records system and DCMS was designed and implemented between 2010 and 2016 linking historical and prospective Lassa fever data sources across KGH Lassa fever units and its peripheral health units. Data were captured using a case report form (CRF) system, enzyme-linked immunosorbent assay (ELISA) plate readers, polymerase chain reaction (PCR) machines, blood chemistry analyzers, and data auditing procedures. Between 2008 and 2016, blood samples for 4,229 suspected Lassa fever cases were screened at KGH, ranging from 219 samples in 2008 to a peak of 760 samples in 2011. Lassa fever case-fatality rates before and following the Ebola outbreak were 65.5% (148/226) and 89.5% (17/19), respectively, suggesting that fewer, but more seriously ill subjects with Lassa fever presented to KGH following the 2014 EVD outbreak (p = .040). DCMS challenges included weak specificity of the Lassa fever suspected case definition, limited capture of patient survival outcome data, internet costs, lapses in internet connectivity, low bandwidth, equipment and software maintenance, lack of computer teaching laboratories, and workload fluctuations due to variable screening activity. DCMS are the backbone of international research efforts and additional literature is needed on the topic for establishing benchmarks and driving goal-based approaches for its advancement in developing countries.

The recent Ebola epidemic exemplified the importance of understanding and controlling emerging infections. Despite the importance of T cells in clearing virus during acute infection, little is known about Ebola-specific CD8+ T cell responses. We investigated immune responses of individuals infected with Ebola virus (EBOV) during the 2013–2016 West Africa epidemic in Sierra Leone, where the majority of the >28,000 EBOV disease (EVD) cases occurred. We examined T cell memory responses to seven of the eight Ebola proteins (GP, sGP, NP, VP24, VP30, VP35, and VP40) and associated HLA expression in survivors. Of the 30 subjects included in our analysis, CD8+ T cells from 26 survivors responded to at least one EBOV antigen. A minority, 10 of 26 responders (38%), made CD8+ T cell responses to the viral GP or sGP. In contrast, 25 of the 26 responders (96%) made response to viral NP, 77% to VP24 (20 of 26), 69% to VP40 (18 of 26), 42% (11 of 26) to VP35, with no response to VP30. Individuals making CD8+ T cells to EBOV VP24, VP35, and VP40 also made CD8+ T cells to NP, but rarely to GP. We identified 34 CD8+ T cell epitopes for Ebola. Our data indicate the immunodominance of the EBOV NP-specific T cell response and suggest that its inclusion in a vaccine along with the EBOV GP would best mimic survivor responses and help boost cell-mediated immunity during vaccination.

Lassa fever, a hemorrhagic fever caused by Lassa virus (LASV), is endemic in West Africa. It is difficult to distinguish febrile illnesses that are common in West Africa from Lassa fever based solely on a patient’s clinical presentation. The field performance of recombinant antigen-based Lassa fever immunoassays was compared to that of quantitative polymerase chain assays (qPCRs) using samples from subjects meeting the case definition of Lassa fever presenting to Kenema Government Hospital in Sierra Leone. The recombinant Lassa virus (ReLASV) enzyme-linked immunosorbant assay (ELISA) for detection of viral antigen in blood performed with 95% sensitivity and 97% specificity using a diagnostic standard that combined results of the immunoassays and qPCR. The ReLASV rapid diagnostic test (RDT), a lateral flow immunoassay based on paired monoclonal antibodies to the Josiah strain of LASV (lineage IV), performed with 90% sensitivity and 100% specificity. ReLASV immunoassays performed better than the most robust qPCR currently available, which had 82% sensitivity and 95% specificity. The performance characteristics of recombinant antigen-based Lassa virus immunoassays indicate that they can aid in the diagnosis of LASV Infection and inform the clinical management of Lassa fever patients.

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. RecombinantEbola and Marburg virus matrixVP40and glycoprotein (GP)antigenswere usedtoderive 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 fever (LF) is a potentially fatal disease that affects an estimated 300,000–500,000 people in endemic areas of west Africa each year. Though past studies have identified fatality rates of 5–20% in patients suspected to have contracted Lassa virus (LASV), new studies using more precise clinical diagnoses and modern diagnostic assays show fatalities rates above 60% in acutely ill patients from endemic regions. Currently, there are no approved vaccines or therapeutics, and only one Comformité Européenne (CE) marked rapid immunodiagnostic for acute LASV infection. Therefore, preventing LASV transmission is the primary goal in endemic regions. Development of rapid immunodiagnostics and research into the efficacy of current treatment options continues toward saving lives in west Africa as well as creating a line of defense against the nefarious use of LASV in bioterrorism settings.

Lassa fever (LF) is a severe viral hemorrhagic fever caused by Lassa virus (LASV). The LF program at the Kenema Government Hospital (KGH) in Eastern Sierra Leone currently provides diagnostic services and clinical care for more than 500 suspected LF cases per year. Nearly two-thirds of suspected LF patients presenting to the LF Ward test negative for either LASV antigen or anti-LASV immunoglobulin M (IgM), and therefore are considered to have a non-Lassa febrile illness (NLFI). The NLFI patients in this study were generally severely ill, which accounts for their high case fatality rate of 36%. The current studies were aimed at determining possible causes of severe febrile illnesses in non-LF cases presenting to the KGH, including possible involvement of filoviruses. A seroprevalence survey employing commercial enzyme-linked immunosorbent assay tests revealed significant IgM and IgG reactivity against dengue virus, chikungunya virus, West Nile virus (WNV), Leptospira, and typhus. A polymerase chain reaction-based survey using sera from subjects with acute LF, evidence of prior LASV exposure, or NLFI revealed widespread infection with Plasmodium falciparum malaria in febrile patients. WNV RNA was detected in a subset of patients, and a 419 nt amplicon specific to filoviral L segment RNA was detected at low levels in a single patient. However, 22% of the patients presenting at the KGH between 2011 and 2014 who were included in this survey registered anti-Ebola virus (EBOV) IgG or IgM, suggesting prior exposure to this agent. The 2014 Ebola virus disease (EVD) outbreak is already the deadliest and most widely dispersed outbreak of its kind on record. Serological evidence reported here for possible human exposure to filoviruses in Sierra Leone prior to the current EVD outbreak supports genetic analysis that EBOV may have been present in West Africa for some time prior to the 2014 outbreak.

Background\ud \ud 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.\ud \ud Methods\ud \ud 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.\ud \ud Results\ud \ud 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.\ud \ud Conclusions\ud \ud 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.)

Evolution of Ebola virus over time The high rate of mortality in the current Ebola epidemic has made it difficult for researchers to collect samples of the virus and study its evolution. Gire et al. describe Ebola epidemiology on the basis of 99 whole-genome sequences, including samples from 78 affected individuals. The authors analyzed changes in the viral sequence and conclude that the current outbreak probably resulted from the spread of the virus from central Africa in the past decade. The outbreak started from a single transmission event from an unknown animal reservoir into the human population. Two viral lineages from Guinea then spread from person to person into Sierra Leone. Science , this issue p. 1369

BACKGROUND The 2013-2016 West African Ebola virus disease (EVD) epidemic is the largest recorded. Triage on the basis of clinical signs had limited success, and the time to diagnosis by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) could exceed 5 days. Here we describe the development and field validation of the ReEBOV Antigen Rapid Test (ReEBOV RDT) to aid triage of individuals with suspected EVD. METHODS Samples from patients with suspected EVD were submitted to Kenema Government Hospital, Sierra Leone, for Lassa fever and EVD screening throughout 2014. Banked residual clinical samples were tested in November 2014 and January 2015 in a blinded field trial to estimate the clinical effectiveness of the ReEBOV RDT, compared with EBOV-specific qRT-PCR. RESULTS Preliminary ReEBOV RDT performance demonstrated a positive percentage agreement (PPA) of 91.1% (195 of 214 results; 95% confidence interval [CI], 86.5%-94.6%) and a negative percentage agreement (NPA) of 90.2% (175 of 194; 95% CI, 85.1%-94.0%). The final estimates used by the Food and Drug Administration to determine whether to grant emergency use authorization for the test, which excluded a qRT-PCR reference method threshold cutoff, were a PPA of 62.1% (72 of 116 results; 95% CI, 52.6%-70.9%) and a NPA of 96.7% (58 of 60; 95% CI, 88.5%-99.6%), with a diagnostic likelihood of 18.6. A subsequent, independent evaluation by the World Health Organization generated results consistent with the preliminary performance estimates. CONCLUSIONS The ReEBOV RDT demonstrated the potential to provide clinically effective rapid and accurate point-of-care test results and, thus, to be a powerful tool for increasing triage efficiency.

Primary infection with Ebola virus protects against subsequent challenge, thus an optimal vaccine should generate immune responses similar to those found in disease survivors. To this end, we characterized CD8 and CD4 T cell responses in Ebola survivors to uncover the immunogenicity of seven of eight Ebola virus proteins. T cell responses of 33 Sierra Leonean Ebola survivors were examined. Nearly all individuals studied harbored nucleoprotein (NP)-specific CD8 T cell responses. Moreover, NP-specific T cells responded more strongly and were more numerous in 65% of NP responders compared to responses to other EBOV proteins in the same individual. Conversely, we found glycoprotein (GP) CD8+ T cell responses in only 37% of EBOV responders. GP responses were also weaker and less numerous than other responses in the same individual. Specificity of the CD4+ T cell responses were more evenly distributed. Altogether, our data indicate that NP is among the most immunogenic EBOV proteins while only VP30 was less immunogenic than GP in our cohort of Sierra Leonean survivors. An optimal vaccine designed to elicit both humoral and cell mediated immune responses in a broad human population should contain both NP and the GP.

The 2013–2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013–2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics., National Institutes of Health (U.S.), United States. Dept. of Health and Human Services (Award 1DP2OD006514-01), National Institute of Allergy and Infectious Diseases (U.S.) (Contracts HHSN272200900049C, HHSN272201000022C, HHSN272200900018C, and U19AI110818), United States Army Medical Acquisition Activity (Grant W81XWH-10-1-0098), David & Lucile Packard Foundation (Fellowship for Science and Engineering), Broad Institute of MIT and Harvard (SPARC Award), German Research Foundation (Grant GU 883/1-1), National Science Foundation (U.S.). Graduate Research Fellowship Program