The arenavirus Lassa causes severe hemorrhagic fever and a significant disease burden in West Africa every year. The glycoprotein, GPC, is the sole antigen expressed on the viral surface and the ...critical target for antibody-mediated neutralization. Here we present the crystal structure of the trimeric, prefusion ectodomain of Lassa GP bound to a neutralizing antibody from a human survivor at 3.2-angstrom resolution. The antibody extensively anchors two monomers together at the base of the trimer, and biochemical analysis suggests that it neutralizes by inhibiting conformational changes required for entry. This work illuminates pH-driven conformational changes in both receptor-binding and fusion subunits of Lassa virus, illustrates the unique assembly of the arenavirus glycoprotein spike, and provides a much-needed template for vaccine design against these threats to global health.
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.
Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major ...contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit's potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines against ETEC.
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.
Lassa virus (LASV) is the causative agent of the deadly Lassa fever (LF). Seven distinct LASV lineages circulate through western Africa, among which lineage I (LI), the first to be identified, is ...particularly resistant to antibody neutralization. Lineage I LASV evades neutralization by half of known antibodies in the GPC-A antibody competition group and all but one of the antibodies in the GPC-B competition group. Here, we solve two cryo-electron microscopy (cryo-EM) structures of LI GP in complex with a GPC-A and a GPC-B antibody. We used complementary structural and biochemical techniques to identify single-amino-acid substitutions in LI that are responsible for immune evasion by each antibody group. Further, we show that LI infection is more dependent on the endosomal receptor lysosome-associated membrane protein 1 (LAMP1) for viral entry relative to LIV. In the absence of LAMP1, LI requires a more acidic fusion pH to initiate membrane fusion with the host cell relative to LIV.
No vaccine or therapeutics are approved to prevent LASV infection or treat LF. All vaccine platforms currently under development present only the LIV GP sequence. However, our data suggest that the high genetic diversity of LASV may be problematic for designing both a broadly reactive immunogen and therapeutic. Here, we examine antibodies that are highly potent against LIV yet are ineffective against LI. By pinpointing LI mutations responsible for this decrease in antibody efficacy, we suggest that future vaccine platforms may need to incorporate specific LI-like mutations in order to generate a broadly neutralizing antibody response against all LASV lineages.
A 59-year-old male with follicular lymphoma treated by anti-CD20-mediated B-cell depletion and ablative chemotherapy was hospitalized with a COVID-19 infection. Although the patient did not develop ...specific humoral immunity, he had a mild clinical course overall. The failure of all therapeutic options allowed infection to persist nearly 300 days with active accumulation of SARS-CoV-2 virus mutations. As a rescue therapy, an infusion of REGEN-COV (10933 and 10987) anti-spike monoclonal antibodies was performed 270 days from initial diagnosis. Due to partial clearance after the first dose (2.4 g), a consolidation dose (8 g) was infused six weeks later. Complete virus clearance could then be observed over the following month, after he was vaccinated with the Pfizer-BioNTech anti-COVID-19 vaccination. The successful management of this patient required prolonged enhanced quarantine, monitoring of virus mutations, pioneering clinical decisions based upon close consultation, and the coordination of multidisciplinary experts in virology, immunology, pharmacology, input from REGN, the FDA, the IRB, the health care team, the patient, and the patient's family. Current decisions to take revolve around patient's follicular lymphoma management, and monitoring for virus clearance persistence beyond disappearance of REGEN-COV monoclonal antibodies after anti-SARS-CoV-2 vaccination. Overall, specific guidelines for similar cases should be established.
Developing potent therapeutics and effective vaccines are the ultimate goals in controlling infectious diseases. Lassa virus (LASV), the causative pathogen of Lassa fever (LF), infects hundreds of ...thousands annually, but effective antivirals or vaccines against LASV infection are still lacking. Furthermore, neutralizing antibodies against LASV are rare. Here, we describe biochemical analyses and high-resolution cryo-electron microscopy structures of a therapeutic cocktail of three broadly protective antibodies that target the LASV glycoprotein complex (GPC), previously identified from survivors of multiple LASV infections. Structural and mechanistic analyses reveal compatible neutralizing epitopes and complementary neutralization mechanisms that offer high potency, broad range, and resistance to escape. These antibodies either circumvent or exploit specific glycans comprising the extensive glycan shield of GPC. Further, they require mammalian glycosylation, native GPC cleavage, and proper GPC trimerization. These findings guided engineering of a next-generation GPC antigen suitable for future neutralizing antibody and vaccine discovery. Together, these results explain protective mechanisms of rare, broad, and potent antibodies and identify a strategy for the rational design of therapeutic modalities against LF and related infectious diseases.
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.
There are no approved treatments for Lassa fever (LF), which is responsible for thousands of deaths each year in West Africa. A major challenge in developing effective medical countermeasures against ...LF is the high diversity of circulating Lassa virus (LASV) strains with four recognized lineages and four proposed lineages. The recent resurgence of LASV in Nigeria caused by genetically distinct strains underscores this concern. Two LASV lineages (II and III) are dominant in Nigeria. Here, we show that combinations of two or three pan-lineage neutralizing human monoclonal antibodies (8.9F, 12.1F, 37.D) known as Arevirumab-2 or Arevirumab-3 can protect up to 100% of cynomolgus macaques against challenge with both lineage II and III LASV isolates when treatment is initiated at advanced stages of disease on day 8 after LASV exposure. This work demonstrates that it may be possible to develop postexposure interventions that can broadly protect against most strains of LASV.
Recently, castellated columns have been increasingly used in buildings because of their flexibility, easy compatibility and potential savings due to less steel consumption. However, there is a lack ...of research related to the subject. In view of that, this work evaluates the influence of the opening type and size on the resistant capacity of the column, considering a nonlinear plastic analysis by the finite element method (792 simulations). For this, columns without openings were modeled to validate the methodology proposed. After validation, a nonlinear buckling analysis of castellated columns was performed. In this study, different cut patterns of the beam web are evaluated, as well as residual stresses, material and geometric imperfections, based on a statistical design. The loads obtained by castellated columns and columns without web are compared. Also, the resistance reduction factors associated with global buckling (χ) for castellated profiles are compared to the values proposed by ABNR NBR 8800 (2008). Furthermore, statistically, there are no differences between the opening types evaluated. Finally, in order to contribute to the design of castellated columns, equations based on regression models were found to predict the load capacity of these elements.
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