Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.

Without an approved vaccine or treatments, Ebola outbreak management has been limited to palliative care and barrier methods to prevent transmission. These approaches, however, have yet to end the 2014 outbreak of Ebola after its prolonged presence in West Africa. Here we show that a combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge. High fever, viraemia and abnormalities in blood count and blood chemistry were evident in many animals before ZMapp intervention. Advanced disease, as indicated by elevated liver enzymes, mucosal haemorrhages and generalized petechia could be reversed, leading to full recovery. ELISA and neutralizing antibody assays indicate that ZMapp is cross-reactive with the Guinean variant of Ebola. ZMapp exceeds the efficacy of any other therapeutics described so far, and results warrant further development of this cocktail for clinical use.

Pan-Filovirus Serum Neutralizing Antibodies in a Subset of Congolese Ebolavirus Infection Survivors.

One year after a Zaire ebolavirus (EBOV) outbreak occurred in the Boende Health Zone of the Democratic Republic of the Congo during 2014, we sought to determine the breadth of immune response against diverse filoviruses including EBOV, Bundibugyo (BDBV), Sudan (SUDV), and Marburg (MARV) viruses. After assessing the 15 survivors, 5 individuals demonstrated some degree of reactivity to multiple ebolavirus species and, in some instances, Marburg virus. All 5 of these survivors had immunoreactivity to EBOV glycoprotein (GP) and EBOV VP40, and 4 had reactivity to EBOV nucleoprotein (NP). Three of these survivors showed serologic responses to the 3 species of ebolavirus GPs tested (EBOV, BDBV, SUDV). All 5 samples also exhibited ability to neutralize EBOV using live virus, in a plaque reduction neutralization test. Remarkably, 3 of these EBOV survivors had plasma antibody responses to MARV GP. In pseudovirus neutralization assays, serum antibodies from a subset of these survivors also neutralized EBOV, BDBV, SUDV, and Taï Forest virus as well as MARV. Collectively, these findings suggest that some survivors of naturally acquired ebolavirus infection mount not only a pan-ebolavirus response, but also in less frequent cases, a pan-filovirus neutralizing response.

Ebola Virus Neutralizing Antibodies Detectable in Survivors of theYambuku, Zaire Outbreak 40 Years after Infection.

The first reported outbreak of Ebola virus disease occurred in 1976 in Yambuku, Democratic Republic of Congo. Antibody responses in survivors 11 years after infection have been documented. However, this report is the first characterization of anti-Ebola virus antibody persistence and neutralization capacity 40 years after infection. Using ELISAs we measured survivor's immunological response to Ebola virus Zaire (EBOV) glycoprotein and nucleoprotein, and assessed VP40 reactivity. Neutralization of EBOV was measured using a pseudovirus approach and plaque reduction neutralization test with live EBOV. Some survivors from the original EBOV outbreak still harbor antibodies against all 3 measures. Interestingly, a subset of these survivors' serum antibodies could still neutralize live virus 40 years postinitial infection. These data provide the longest documentation of both anti-Ebola serological response and neutralization capacity within any survivor cohort, extending the known duration of response from 11 years postinfection to at least 40 years after symptomatic infection.

Molecular Evidence of Sexual Transmission of Ebola Virus.

A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiologic data, the genomic analysis provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD. (Funded by the Defense Threat Reduction Agency and others.).

Detection of Ebola Virus RNA Through Aerosol Sampling of Animal Biosafety Level 4 Rooms Housing Challenged Nonhuman Primates.

Ebola virus disease is a serious illness of humans and nonhuman primates (NHPs). Direct contact has been shown to be the primary source of Ebola (EBOV) transmission. We used a high-volume air sampler to determine whether EBOV could be detected during 3 independent studies with EBOV-challenged NHPs. Viral RNA was recovered during days 9 and 10 of Study I and days 7 and 8 of Study III. Viral RNA levels were below limits of detection during all other collections. The results demonstrate that the biosafety level 4 (BSL-4) suit protects workers from aerosols in a BSL-4 environment using proper engineering and administrative controls.

Assessment and Optimization of the GeneXpert Diagnostic Platform for Detection of Ebola Virus RNA in Seminal Fluid.

Recent studies have suggested that Ebola virus (EBOV) ribonucleic acid (RNA) potentially present in the semen of a large number of survivors of Ebola virus disease (EVD) in Western Africa may contribute to sexual transmission of EVD and generate new clusters of cases in regions previously declared EVD-free. These findings drive the immediate need for a reliable, rapid, user-friendly assay for detection of EBOV RNA in semen that is deployable to multiple sites across Western Africa. In this study, we optimized the Xpert EBOV assay for semen samples by adding dithiothreitol. Compared to the assays currently in use in Liberia (including Ebola Zaire Target 1, major groove binder real-time-polymerase chain reaction assays, and original Xpert EBOV assay), the modified Xpert EBOV assay demonstrated greater sensitivity than the comparator assays. Thus, the modified Xpert EBOV assay is optimal for large-scale monitoring of EBOV RNA persistence in male survivors.

Use of Existing Diagnostic Reverse-Transcription Polymerase Chain Reaction Assays for Detection of Ebola Virus RNA in Semen.

Sexual transmission of Ebola virus in Liberia has now been documented and associated with new clusters in regions previously declared Ebola free. Assays that have Emergency Use Authorization (EUA) and are routinely used to detect Ebola virus RNA in whole blood and plasma specimens at the Liberian Institute for Biomedical Research were tested for their suitability in detecting the presence of Ebola virus RNA in semen. Qiagen AVL extraction protocols, as well as the Ebola Zaire Target 1 and major groove binder quantitative reverse-transcription polymerase chain reaction assays, were demonstrably suitable for this purpose and should facilitate epidemiologic investigations, including those involving long-term survivors of Ebola.

Lateral Flow Immunoassays for Ebola Virus Disease Detection in Liberia.

BACKGROUND: Lateral flow immunoassays (LFIs) are point-of-care diagnostic assays that are designed for single use outside a formal laboratory, with in-home pregnancy tests the best-known example of these tests. Although the LFI has some limitations over more-complex immunoassay procedures, such as reduced sensitivity and the potential for false-positive results when using complex sample matrices, the assay has the benefits of a rapid time to result and ease of use. These benefits make it an attractive option for obtaining rapid results in an austere environment. In an outbreak of any magnitude, a field-based rapid diagnostic assay would allow proper patient transport and for safe burials to be conducted without the delay caused by transport of samples between remote villages and testing facilities. Use of such point-of-care instruments in the ongoing Ebola virus disease (EVD) outbreak in West Africa would have distinct advantages in control and prevention of local outbreaks, but proper understanding of the technology and interpretation of results are important. METHODS: In this study, a LFI, originally developed by the Naval Medical Research Center for Ebola virus environmental testing, was evaluated for its ability to detect the virus in clinical samples in Liberia. Clinical blood and plasma samples and post mortem oral swabs submitted to the Liberian Institute for Biomedical Research, the National Public Health Reference Laboratory for EVD testing, were tested and compared to results of real-time reverse transcription-polymerase chain reaction (rRT-PCR), using assays targeting Ebola virus glycoprotein and nucleoprotein. RESULTS: The LFI findings correlated well with those of the real-time RT-PCR assays used as benchmarks. CONCLUSIONS: Rapid antigen-detection tests such as LFIs are attractive alternatives to traditional immunoassays but have reduced sensitivity and specificity, resulting in increases in false-positive and false-negative results. An understanding of the strengths, weaknesses, and limitations of a particular assay lets the diagnostician choose the correct situation to use the correct assay and properly interpret the results.

Development, Evaluation, and Integration of a Quantitative Reverse-Transcription Polymerase Chain Reaction Diagnostic Test for Ebola Virus on a Molecular Diagnostics Platform.

BACKGROUND: The 2013-2016 Ebola epidemic in West Africa resulted in accelerated development of rapid diagnostic tests for emergency outbreak preparedness. We describe the development and evaluation of the Idylla™ prototype Ebola virus test, a fully automated sample-to-result molecular diagnostic test for rapid detection of Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV). METHODS: The Idylla™ prototype Ebola virus test can simultaneously detect EBOV and SUDV in 200 µL of whole blood. The sample is directly added to a disposable cartridge containing all reagents for sample preparation, RNA extraction, and amplification by reverse-transcription polymerase chain reaction analysis. The performance was evaluated with a variety of sample types, including synthetic constructs and whole blood samples from healthy volunteers spiked with viral RNA, inactivated virus, and infectious virus. RESULTS: The 95% limits of detection for EBOV and SUDV were 465 plaque-forming units (PFU)/mL (1010 copies/mL) and 324 PFU/mL (8204 copies/mL), respectively. In silico and in vitro analyses demonstrated 100% correct reactivity for EBOV and SUDV and no cross-reactivity with relevant pathogens. The diagnostic sensitivity was 97.4% (for EBOV) and 91.7% (for SUDV), the specificity was 100%, and the diagnostic accuracy was 95.9%. CONCLUSIONS: The Idylla™ prototype Ebola virus test is a fast, safe, easy-to-use, and near-patient test that meets the performance criteria to detect EBOV in patients with suspected Ebola.

Monitoring of Ebola Virus Makona Evolution through Establishment of Advanced Genomic Capability in Liberia.

To support Liberia's response to the ongoing Ebola virus (EBOV) disease epidemic in Western Africa, we established in-country advanced genomic capabilities to monitor EBOV evolution. Twenty-five EBOV genomes were sequenced at the Liberian Institute for Biomedical Research, which provided an in-depth view of EBOV diversity in Liberia during September 2014-February 2015. These sequences were consistent with a single virus introduction to Liberia; however, shared ancestry with isolates from Mali indicated at least 1 additional instance of movement into or out of Liberia. The pace of change is generally consistent with previous estimates of mutation rate. We observed 23 nonsynonymous mutations and 1 nonsense mutation. Six of these changes are within known binding sites for sequence-based EBOV medical countermeasures; however, the diagnostic and therapeutic impact of EBOV evolution within Liberia appears to be low.

Possible sexual transmission of Ebola virus - Liberia, 2015.

On March 20, 2015, 30 days after the most recent confirmed Ebola Virus Disease (Ebola) patient in Liberia was isolated, Ebola was laboratory confirmed in a woman in Monrovia. The investigation identified only one epidemiologic link to Ebola: unprotected vaginal intercourse with a survivor. Published reports from previous outbreaks have demonstrated Ebola survivors can continue to harbor virus in immunologically privileged sites for a period of time after convalescence. Ebola virus has been isolated from semen as long as 82 days after symptom onset and viral RNA has been detected in semen up to 101 days after symptom onset. One instance of possible sexual transmission of Ebola has been reported, although the accompanying evidence was inconclusive. In addition, possible sexual transmission of Marburg virus, a filovirus related to Ebola, was documented in 1968. This report describes the investigation by the Government of Liberia and international response partners of the source of Liberia's latest Ebola case and discusses the public health implications of possible sexual transmission of Ebola virus. Based on information gathered in this investigation, CDC now recommends that contact with semen from male Ebola survivors be avoided until more information regarding the duration and infectiousness of viral shedding in body fluids is known. If male survivors have sex (oral, vaginal, or anal), a condom should be used correctly and consistently every time.

Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques.

Filovirus infections can cause a severe and often fatal disease in humans and nonhuman primates, including great apes. Here, three anti-Ebola virus mouse/human chimeric mAbs (c13C6, h-13F6, and c6D8) were produced in Chinese hamster ovary and in whole plant (Nicotiana benthamiana) cells. In pilot experiments testing a mixture of the three mAbs (MB-003), we found that MB-003 produced in both manufacturing systems protected rhesus macaques from lethal challenge when administered 1 h postinfection. In a pivotal follow-up experiment, we found significant protection (P < 0.05) when MB-003 treatment began 24 or 48 h postinfection (four of six survived vs. zero of two controls). In all experiments, surviving animals that received MB-003 experienced little to no viremia and had few, if any, of the clinical symptoms observed in the controls. The results represent successful postexposure in vivo efficacy by a mAb mixture and suggest that this immunoprotectant should be further pursued as a postexposure and potential therapeutic for Ebola virus exposure.

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant.

No countermeasures currently exist for the prevention or treatment of the severe sequelae of Filovirus (such as Ebola virus; EBOV) infection. To overcome this limitation in our biodefense preparedness, we have designed monoclonal antibodies (mAbs) which could be used in humans as immunoprotectants for EBOV, starting with a murine mAb (13F6) that recognizes the heavily glycosylated mucin-like domain of the virion-attached glycoprotein (GP). Point mutations were introduced into the variable region of the murine mAb to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a mAb (h-13F6) appropriate for development for human use. We have evaluated the efficacy of three variants of h-13F6 carrying different glycosylation patterns in a lethal mouse EBOV challenge model. The pattern of glycosylation of the various mAbs was found to correlate to level of protection, with aglycosylated h-13F6 providing the least potent efficacy (ED(50) = 33 µg). A version with typical heterogenous mammalian glycoforms (ED(50) = 11 µg) had similar potency to the original murine mAb. However, h-13F6 carrying complex N-glycosylation lacking core fucose exhibited superior potency (ED(50) = 3 µg). Binding studies using Fcγ receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcγRIII. Together the results indicate the presence of Fc N-glycans enhances the protective efficacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as biodefense therapeutics.

Therapeutic administration of a cross-reactive mAb targeting the fusion glycoprotein of Nipah virus protects nonhuman primates.

No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.

Acute Late-Stage Myocarditis in the Crab-Eating Macaque Model of Hemorrhagic Smallpox.

Hemorrhagic smallpox, caused by variola virus (VARV), was a rare but nearly 100% lethal human disease manifestation. Hemorrhagic smallpox is frequently characterized by secondary bacterial infection, coagulopathy, and myocardial and subendocardial hemorrhages. Previous experiments have demonstrated that intravenous (IV) cowpox virus (CPXV) exposure of macaques mimics human hemorrhagic smallpox. The goal of this experiment was to further understand the onset, nature, and severity of cardiac pathology and how it may contribute to disease. The findings support an acute late-stage myocarditis with lymphohistiocytic infiltrates in the CPXV model of hemorrhagic smallpox.

Efficacy and Safety Outcomes of Cataract Surgery in Survivors of Ebola Virus Disease: 12-Month Results From the PREVAIL VII Study.

Purpose: In survivors of Ebola virus disease (EVD), intraocular viral persistence raises questions about the timing and safety of cataract surgery. To the best of our knowledge, this is the first controlled study evaluating Ebola virus persistence and cataract surgery safety and outcomes in EVD survivors. Methods: Seropositive EVD survivors and seronegative controls with vision worse than 20/40 from cataract and without active intraocular inflammation were enrolled. Aqueous humor from survivors was tested with reverse transcription-polymerase chain reaction for Ebola viral RNA. Participants underwent manual small-incision cataract surgery and 1 year of follow-up examinations. Results: Twenty-two eyes of 22 survivors and 12 eyes of eight controls underwent cataract surgery. All of the aqueous samples tested negative for Ebola viral RNA. Median visual acuity improved from 20/200 at baseline to 20/25 at 1 year in survivors and from count fingers to 20/50 in controls (overall, P < 0.001; between groups, P = 0.07). After a 1-month course of topical corticosteroids, 55% of survivors and 67% of controls demonstrated at least 1+ anterior chamber cell. Twelve months after surgery, optical coherence tomography revealed a median increase in macular central subfield thickness of 42 µm compared with baseline (overall, P = 0.029; between groups, P = 0.995). Conclusions: EVD survivors and controls demonstrated significant visual improvement from cataract surgery. The persistence of intraocular inflammation highlights the importance of follow-up. The absence of detectable intraocular Ebola viral RNA provides guidance regarding the safety of eye surgery in Ebola survivors. Translational Relevance: These findings demonstrate the safety and efficacy of cataract surgery in Ebola survivors and will inform ocular surgery guidelines in this population.

Scalable, semi-automated fluorescence reduction neutralization assay for qualitative assessment of Ebola virus-neutralizing antibodies in human clinical samples.

Antibody titers against a viral pathogen are typically measured using an antigen binding assay, such as an enzyme-linked immunosorbent assay (ELISA), which only measures the ability of antibodies to identify a viral antigen of interest. Neutralization assays measure the presence of virus-neutralizing antibodies in a sample. Traditional neutralization assays, such as the plaque reduction neutralization test (PRNT), are often difficult to use on a large scale due to being both labor and resource intensive. Here we describe an Ebola virus fluorescence reduction neutralization assay (FRNA), which tests for neutralizing antibodies, that requires only a small volume of sample in a 96-well format and is easy to automate. The readout of the FRNA is the percentage of Ebola virus-infected cells measured with an optical reader or overall chemiluminescence that can be generated by multiple reading platforms. Using blinded human clinical samples (EVD survivors or contacts) obtained in Liberia during the 2013-2016 Ebola virus disease outbreak, we demonstrate there was a high degree of agreement between the FRNA-measured antibody titers and the Filovirus Animal Non-clinical Group (FANG) ELISA titers with the FRNA providing information on the neutralizing capabilities of the antibodies.

Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis.

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. Unlike the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viral RNA was measurable in circulating blood 2 days after exposure, and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of livers from terminal monkeys and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host response suggests that relatively small subsets of a host's response to infection may be responsible for driving hemorrhagic fever pathogenesis. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host⁻response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures.

Use of the Filovirus Animal Non-Clinical Group (FANG) Ebola virus immuno-assay requires fewer study participants to power a study than the Alpha Diagnostic International assay.

As part of the scientific community's development of medical countermeasures against Ebola virus disease, optimization of standardized assays for product evaluation is paramount. The recent outbreak heightened awareness to the scarcity of available assays and limited information on performance and reproducibility. To evaluate the immunogenicity of vaccines entering Phase I-III trials and to identify survivors, two enzyme-linked immunosorbent assays, the Filovirus Animal Non-Clinical Group assay and the Alpha Diagnostics International assay, were evaluated for detection of immunoglobulin G against Ebola virus glycoprotein. We found that the Filovirus Animal Nonclinical Group assay produced a wider range of relative antibody concentrations, higher assay precision, larger relative accuracy range, and lower regional background. Additionally, to sufficiently power a vaccine trial, use of the Filovirus Animal Nonclinical Group assay would require one third the number of participants than the Alpha Diagnostics International assay. This reduction in needed study participants will require less money, fewer man hours, and much less time to evaluate vaccine immunogenicity.

3B11-N, a monoclonal antibody against MERS-CoV, reduces lung pathology in rhesus monkeys following intratracheal inoculation of MERS-CoV Jordan-n3/2012.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was identified in 2012 as the causative agent of a severe, lethal respiratory disease occurring across several countries in the Middle East. To date there have been over 1600 laboratory confirmed cases of MERS-CoV in 26 countries with a case fatality rate of 36%. Given the endemic region, it is possible that MERS-CoV could spread during the annual Hajj pilgrimage, necessitating countermeasure development. In this report, we describe the clinical and radiographic changes of rhesus monkeys following infection with 5×10(6) PFU MERS-CoV Jordan-n3/2012. Two groups of NHPs were treated with either a human anti-MERS monoclonal antibody 3B11-N or E410-N, an anti-HIV antibody. MERS-CoV Jordan-n3/2012 infection resulted in quantifiable changes by computed tomography, but limited other clinical signs of disease. 3B11-N treated subjects developed significantly reduced lung pathology when compared to infected, untreated subjects, indicating that this antibody may be a suitable MERS-CoV treatment.