Perspectives on Advancing Countermeasures for Filovirus Disease: Report From a Multisector Meeting.

Although there are now approved treatments and vaccines for Ebola virus disease, the case fatality rate remains unacceptably high even when patients are treated with the newly approved therapeutics. Furthermore, these countermeasures are not expected to be effective against disease caused by other filoviruses. A meeting of subject-matter experts was held during the 10th International Filovirus Symposium to discuss strategies to address these gaps. Several investigational therapeutics, vaccine candidates, and combination strategies were presented. The greatest challenge was identified to be the implementation of well-designed clinical trials of safety and efficacy during filovirus disease outbreaks. Preparing for this will require agreed-upon common protocols for trials intended to bridge multiple outbreaks across all at-risk countries. A multinational research consortium including at-risk countries would be an ideal mechanism to negotiate agreement on protocol design and coordinate preparation. Discussion participants recommended a follow-up meeting be held in Africa to establish such a consortium.

Intensive Care Unit-Like Care of Nonhuman Primates with Ebola Virus Disease.

BACKGROUND: Ebola virus disease (EVD) supportive care strategies are largely guided by retrospective observational research. This study investigated the effect of EVD supportive care algorithms on duration of survival in a controlled nonhuman primate (NHP) model. METHODS: Fourteen rhesus macaques were challenged intramuscularly with a target dose of Ebola virus (1000 plaque-forming units; Kikwit). NHPs were allocated to intensive care unit (ICU)-like algorithms (n = 7), intravenous fluids plus levofloxacin (n = 2), or a control group (n = 5). The primary outcome measure was duration of survival, and secondary outcomes included changes in clinical laboratory values. RESULTS: Duration of survival was not significantly different between the pooled ICU-like algorithm and control groups (8.2 vs 6.9 days of survival; hazard ratio; 0.50; P = .25). Norepinephrine was effective in transiently maintaining baseline blood pressure. NHPs treated with ICU-like algorithms had delayed onset of liver and kidney injury. CONCLUSIONS: While an obvious survival difference was not observed with ICU-like care, clinical observations from this model may aid in EVD supportive care NHP model refinement.

Will There Be a Cure for Ebola?

Despite the unprecedented Ebola virus outbreak response in West Africa, no Ebola medical countermeasures have been approved by the US Food and Drug Administration. However, multiple valuable lessons have been learned about the conduct of clinical research in a resource-poor, high risk-pathogen setting. Numerous therapeutics were explored or developed during the outbreak, including repurposed drugs, nucleoside and nucleotide analogues (BCX4430, brincidofovir, favipiravir, and GS-5734), nucleic acid-based drugs (TKM-Ebola and AVI-7537), and immunotherapeutics (convalescent plasma and ZMapp). We review Ebola therapeutics progress in the aftermath of the West Africa Ebola virus outbreak and attempt to offer a glimpse of a path forward.

Development of a liquid chromatography high resolution mass spectrometry method for the quantitation of viral envelope glycoprotein in Ebola virus-like particle vaccine preparations.

BACKGROUND: Ebola virus like particles (EBOV VLPs, eVLPs), are produced by expressing the viral transmembrane glycoprotein (GP) and structural matrix protein VP40 in mammalian cells. When expressed, these proteins self-assemble and bud from 'host' cells displaying morphology similar to infectious virions. Several studies have shown that rodents and non-human primates vaccinated with eVLPs are protected from lethal EBOV challenge. The mucin-like domain of envelope glycoprotein GP1 serves as the major target for a productive humoral immune response. Therefore GP1 concentration is a critical quality attribute of EBOV vaccines and accurate measurement of the amount of GP1 present in eVLP lots is crucial to understanding variability in vaccine efficacy. METHODS: After production, eVLPs are characterized by determining total protein concentration and by western blotting, which only provides semi-quantitative information for GP1. Therefore, a liquid chromatography high resolution mass spectrometry (LC-HRMS) approach for accurately measuring GP1 concentration in eVLPs was developed. The method employs an isotope dilution strategy using four target peptides from two regions of the GP1 protein. Purified recombinant GP1 was generated to serve as an assay standard. GP1 quantitation in 5 eVLP lots was performed on an LTQ-Orbitrap Elite and the final quantitation was derived by comparing the relative response of 200 fmol AQUA peptide standards to the analyte response at 4 ppm. RESULTS: Conditions were optimized to ensure complete tryptic digestion of eVLP, however, persistent missed cleavages were observed in target peptides. Additionally, N-terminal truncated forms of the GP1 protein were observed in all eVLP lots, making peptide selection crucial. The LC-HRMS strategy resulted in quantitation of GP1 with a lower limit of quantitation of 1 fmol and an average percent coefficient of variation (CV) of 7.6 %. Unlike western blot values, the LC-HRMS quantitation of GP1 in 5 eVLP vaccine lots exhibited a strong linear relationship (positive correlation) with survival (after EBOV challenge) in mice. CONCLUSIONS: This method provides a means to rapidly determine eVLP batch quality based upon quantitation of antigenic GP1. By monitoring variability in GP1 content, the eVLP production process can be optimized, and the total amount of GP1 needed to confer protection accurately determined.

A thermostable, chromatographically purified Ebola nano-VLP vaccine.

BACKGROUND: Filovirus virus-like particles (VLP) are strong immunogens with the potential for development into a safe, non-infectious vaccine. However, the large size and filamentous structure of this virus has heretofore made production of such a vaccine difficult. Herein, we present new assays and a purification procedure to yield a better characterized and more stable product. METHODS: Sonication of VLP was used to produce smaller "nano-VLP", which were purified by membrane chromatography. The sizes and lengths of VLP particles were analyzed using electron microscopy and an assay based on transient occlusion of a nanopore. Using conformationally-sensitive antibodies, we developed an in vitro assay for measuring GP conformational integrity in the context of VLP, and used it to profile thermal stability. RESULTS: We developed a new procedure for rapid isolation of Ebola VLP using membrane chromatography that yields a filterable and immunogenic product. Disruption of VLP filaments by sonication followed by filtration produced smaller particles of more uniform size, having a mean diameter close to 230 nm. These reduced-size VLP retained GP conformation and were protective against mouse-adapted Ebola challenge in mice. The "nano-VLP" consists of GP-coated particles in a mixture of morphologies including circular, branched, "6"-shaped, and filamentous ones up to ~1,500 nm in length. Lyophilization conferred a high level of thermostability on the nano-VLP. Unlike Ebola VLP in solution, which underwent denaturation of GP upon moderate heating, the lyophilized nano-VLP can withstand at least 1 h at 75°C, while retaining conformational integrity of GP and the ability to confer protective immunity in a mouse model. CONCLUSIONS: We showed that Ebola virus-like particles can be reduced in size to a more amenable range for manipulation, and that these smaller particles retained their temperature stability, the structure of the GP antigen, and the ability to stimulate a protective immune response in mice. We developed a new purification scheme for "nano-VLP" that is more easily scaled up and filterable. The product could also be made thermostable by lyophilization, which is highly significant for vaccines used in tropical countries without a reliable "cold-chain" of refrigeration.

The Evolution of Medical Countermeasures for Ebola Virus Disease: Lessons Learned and Next Steps.

The Ebola virus disease outbreak that occurred in Western Africa from 2013-2016, and subsequent smaller but increasingly frequent outbreaks of Ebola virus disease in recent years, spurred an unprecedented effort to develop and deploy effective vaccines, therapeutics, and diagnostics. This effort led to the U.S. regulatory approval of a diagnostic test, two vaccines, and two therapeutics for Ebola virus disease indications. Moreover, the establishment of fieldable diagnostic tests improved the speed with which patients can be diagnosed and public health resources mobilized. The United States government has played and continues to play a key role in funding and coordinating these medical countermeasure efforts. Here, we describe the coordinated U.S. government response to develop medical countermeasures for Ebola virus disease and we identify lessons learned that may improve future efforts to develop and deploy effective countermeasures against other filoviruses, such as Sudan virus and Marburg virus.

Adjuvant selection impacts the correlates of vaccine protection against Ebola infection.

The establishment of correlates of protection is particularly relevant in the context of rare, highly lethal pathogens such as filoviruses. We previously demonstrated that an Ebola glycoprotein virus-like particle (VLP) vaccine, when given as two intramuscular doses, conferred protection from challenge in a murine challenge model. In this study, we compared the ability of Advax inulin-based adjuvant formulations (Advax1-4) to enhance Ebola VLP vaccine protection in mice. After two immunizations, Advax-adjuvants that included a TLR9 agonist component induced high IgG responses, with complete protection against Ebola virus challenge. Although anti-Ebola IgG levels waned over time, protection was durable and was still evident 150 days post-immunization. Mice were protected after just a single VLP immunization with Advax-2 or -4 adjuvants. Advax-adjuvanted VLPs induced a stronger IFN-γ, TNF and IL-12 signature and serum transferred from Advax-adjuvanted vaccinees was able to transfer protection to naïve animals, showing that Ebola protection can be achieved by antibodies in the absence of cellular immunity. By contrast, serum from vaccinees incorporating a pICLC adjuvant did not transfer protection despite high IgG levels on ELISA. These data highlight the importance of adjuvant selection for development of a successful Ebola VLP vaccine.

Pre-positioned Outbreak Research: The Joint Medical Emerging Diseases Intervention Clinical Capability Experience in Uganda.

The West Africa Ebola virus disease outbreak of 2014-2016 demonstrated that responses to viral hemorrhagic fever epidemics must go beyond emergency stopgap measures and should incorporate high-quality medical care and clinical research. Optimal patient management is essential to improving outcomes, and it must be implemented regardless of geographical location or patient socioeconomic status. Coupling clinical research with improved care has a significant added benefit: Improved data quality and management can guide the development of more effective supportive care algorithms and can support regulatory approvals of investigational medical countermeasures (MCMs), which can alter the cycle of emergency response to reemerging pathogens. However, executing clinical research during outbreaks of high-consequence pathogens is complicated and comes with ethical and research regulatory challenges. Aggressive care and excellent quality control must be balanced by the requirements of an appropriate infection prevention and control posture for healthcare workers and by overcoming the resource limitations inherent in many outbreak settings. The Joint Mobile Emerging Disease Intervention Clinical Capability was established in 2015 to develop a high-quality clinical trial capability in Uganda to support rigorous evaluation of MCMs targeting high-consequence pathogens like Ebola virus. This capability assembles clinicians, laboratorians, clinical researchers, logisticians, and regulatory professionals trained in infection prevention and control and in good clinical and good clinical laboratory practices. The resulting team is prepared to provide high-quality medical care and clinical research during high-consequence outbreaks.

Recent successes in therapeutics for Ebola virus disease: no time for complacency.

The PALM trial in the Democratic Republic of the Congo identified a statistically significant survival benefit for two monoclonal antibody-based therapeutics in the treatment of acute Ebola virus disease; however, substantial gaps remain in improving the outcomes of acute Ebola virus disease and for the survivors. Ongoing efforts are needed to develop more effective strategies, particularly for individuals with severe disease, for prevention and treatment of viral persistence in immune-privileged sites, for optimisation of post-exposure prophylaxis, and to increase therapeutic breadth. As antibody-based approaches are identified and advanced, promising small-molecule antivirals currently in clinical stage development should continue to be evaluated for filovirus diseases, with consideration of their added value in combination approaches with bundled supportive care, their penetration in tissues of interest, the absence of interaction with glycoprotein-based vaccines, and filoviral breadth.

Glucopyranosyl lipid adjuvant enhances immune response to Ebola virus-like particle vaccine in mice.

The identification of adjuvants that promote lasting antigen-specific immunity and augment vaccine efficacy are integral to the development of new protein-based vaccines. The Ebola virus-like particle (VLP) vaccine expressing Ebola virus glycoprotein (GP) and matrix protein (VP40) was used in this study to evaluate the ability of TLR4 agonist glucopyranosyl lipid adjuvant (GLA) formulated in a stable emulsion (SE) to enhance immunogenicity and promote durable protection against mouse-adapted Ebola virus (ma-EBOV). Antibody responses and Ebola-specific T cell responses were evaluated post vaccination. Survival analysis after lethal ma-EBOV challenge was performed 4 weeks and 22 weeks following final vaccination. GLA-SE enhanced EBOV-specific immunity and resulted in long-term protection against challenge with ma-EBOV infection in a mouse model. Specifically, GLA-SE elicited Th1-skewed antibodies and promoted the generation of EBOV GP-specific polyfunctional T cells. These results provide further support for the utility of TLR4 activating GLA-SE-adjuvanted vaccines.

Neutralizing Antibodies from Convalescent Chikungunya Virus Patients Can Cross-Neutralize Mayaro and Una Viruses.

Most alphaviruses are mosquito-borne and can cause severe disease in domesticated animals and humans. The most notable recent outbreak in the Americas was the 2014 chikungunya virus (CHIKV) outbreak affecting millions and producing disease highlighted by rash and arthralgia. Chikungunya virus is a member of the Semliki Forest (SF) serocomplex, and before its arrival in the Americas, two other member of the SF complex, Una (UNAV) and Mayaro (MAYV) viruses, were circulating in Central and South America. This study examined whether antibodies from convalescent CHIKV patients could cross-neutralize UNAV and MAYV. Considerable cross-neutralization of both viruses was observed, suggesting that exposure to CHIKV can produce antibodies that may mitigate infection with UNAV or MAYV. Understanding the impact of CHIKV exposure on population susceptibility to other emerging viruses may help predict outbreaks; moreover, identification of cross-reactive immune responses among alphaviruses may lead to the development of vaccines targeting multiple viruses.

The Joint Mobile Emerging Disease Clinical Capability (JMEDICC) laboratory approach: Capabilities for high-consequence pathogen clinical research.

Following the 2013-2016 Ebola virus outbreak in West Africa, numerous groups advocated for the importance of executing clinical trials in outbreak settings. The difficulties associated with obtaining reliable data to support regulatory approval of investigational vaccines and therapeutics during that outbreak were a disappointment on a research and product development level, as well as on a humanitarian level. In response to lessons learned from the outbreak, the United States Department of Defense established a multi-institute project called the Joint Mobile Emerging Disease Intervention Clinical Capability (JMEDICC). JMEDICC's primary objective is to establish the technical capability in western Uganda to execute clinical trials during outbreaks of high-consequence pathogens such as the Ebola virus. A critical component of clinical trial execution is the establishment of laboratory operations. Technical, logistical, and political challenges complicate laboratory operations, and these challenges have been mitigated by JMEDICC to enable readiness for laboratory outbreak response operations.

Treatment-focused Ebola trials, supportive care and future of filovirus care.

INTRODUCTION: During the 2014-2016 Ebolavirus (EBOV) outbreak, several candidate therapeutics were used in EBOV-infected patients in clinical trials and under expanded access for emergency use. This review will focus briefly on medications used during the outbreak. We will discuss current therapeutic candidates and their status and will then turn to a related and essential topic: supportive care and the standard of care for filovirus infected patients. Potential benefits and pitfalls of combination therapies for filoviruses will be discussed. Areas covered: Clinical trials of therapeutics targeting EBOV; clinical usage of therapeutics during recent EBOV outbreak; potential need for combination therapy; role of supportive care in treatment of Ebola virus disease (EVD). Expert commentary: In the absence of another large scale EBOV outbreak, the path to therapeutic product licensure in the United States of America (USA) would need to be via the FDA Animal Rule. However, human data may be needed to supplement animal data. The future of filovirus therapeutics may therefore benefit by establishing the ability to implement clinical trials in an outbreak setting in a timely fashion. Supportive care guidelines for filovirus infection should be defined and established as standard of care for treatment of EVD.

Distinguishing Respiratory Features of Category A/B Potential Bioterrorism Agents from Community-Acquired Pneumonia.

Differentiating between illness caused by community-acquired respiratory pathogens versus infection by biothreat agents is a challenge. This review highlights respiratory and clinical features of category A and B potential biothreat agents that have respiratory features as their primary presenting signs and symptoms. Recent world events make such a reminder that the possibility of rare diseases and unlikely events can occur timely for clinicians, policymakers, and public health authorities. Despite some distinguishing features, nothing can replace good clinical acumen and a strong index of suspicion in the diagnosis of uncommon infectious diseases.

Frequency of Chronic Joint Pain Following Chikungunya Virus Infection: A Colombian Cohort Study.

OBJECTIVE: To estimate the frequency of chronic joint pain after infection with chikungunya virus in a Latin American cohort. METHODS: A cross-sectional follow-up of a prospective cohort of 500 patients from the Atlántico Department, Colombia who were clinically diagnosed as having chikungunya virus during the 2014-2015 epidemic was conducted. Baseline symptoms and follow-up symptoms at 20 months were evaluated in serologically confirmed cases. RESULTS: Among the 500 patients enrolled, 485 had serologically confirmed chikungunya virus and reported joint pain status. Patients were predominantly adults (mean ± SD age 49 ± 16 years) and female, had an education level of high school or less, and were of Mestizo ethnicity. The most commonly affected joints were the small joints, including the wrists, ankles, and fingers. The initial virus symptoms lasted a median of 4 days (interquartile range [IQR] 3-8 days). Sixteen percent of the participants reported missing school or work (median 4 days [IQR 2-7 days]). After 20 months, one-fourth of the participants had persistent joint pain. A multivariable analysis indicated that significant predictors of persistent joint pain included college graduate status, initial symptoms of headache or knee pain, missed work, normal activities affected, ≥4 days of initial symptoms, and ≥4 weeks of initial joint pain. CONCLUSION: This is the first report to describe the frequency of chikungunya virus-related arthritis in the Americas after a 20-month follow-up. The high frequency of chronic disease highlights the need for the development of prevention and treatment methods.

Chikungunya Arthritis Mechanisms in the Americas: A Cross-Sectional Analysis of Chikungunya Arthritis Patients Twenty-Two Months After Infection Demonstrating No Detectable Viral Persistence in Synovial Fluid.

OBJECTIVE: To determine if chikungunya virus persists in synovial fluid after infection, potentially acting as a causative mechanism of persistent arthritis. METHODS: We conducted a cross-sectional study of 38 Colombian participants with clinical chikungunya virus infection during the 2014-2015 epidemic who reported chronic arthritis and 10 location-matched controls without chikungunya virus or arthritis. Prior chikungunya virus infection status was serologically confirmed, and the presence of synovial fluid chikungunya virus, viral RNA, and viral proteins was determined by viral culture, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and mass spectrometry, respectively. Biomarkers were assessed by multiplex analysis. RESULTS: Patients with serologically confirmed chikungunya arthritis (33 of 38 [87%]) were predominantly female (82%) and African Colombian (55%) or white Colombian (33%), with moderate disease activity (mean ± SD Disease Activity Score in 28 joints 4.52 ± 0.77) a median of 22 months after infection (interquartile range 21-23 months). Initial symptoms of chikungunya virus infection included joint pain (97%), swelling (97%), stiffness (91%), and fever (91%). The most commonly affected joints were the knees (87%), elbows (76%), wrists (75%), ankles (56%), fingers (56%), and toes (56%). Synovial fluid samples from all patients with chikungunya arthritis were negative for chikungunya virus on qRT-PCR, showed no viral proteins on mass spectrometry, and cultures were negative. Case and control plasma cytokine and chemokine concentrations did not differ significantly. CONCLUSION: This is one of the largest observational studies involving analysis of the synovial fluid of chikungunya arthritis patients. Synovial fluid analysis revealed no detectable chikungunya virus. This finding suggests that chikungunya virus may cause arthritis through induction of potential host autoimmunity, suggesting a role for immunomodulating agents in the treatment of chikungunya arthritis, or that low-level viral persistence exists in synovial tissue only and is undetectable in synovial fluid.

T-cell-dependent mechanisms promote Ebola VLP-induced antibody responses, but are dispensable for vaccine-mediated protection.

Humoral responses are essential for the protective efficacy of most Ebola virus (EBOV) candidate vaccines; however, the in vivo development of protective anti-EBOV B-cell responses is poorly defined. Here, by using the virus-like particle (VLP) as a model antigen, we demonstrate that humoral responses are generated through follicular B-cell and T-cell-dependent mechanisms in a mouse model of EBOV infection. In addition, we show that the inclusion of the clinical-grade dsRNA adjuvant known as poly-ICLC in VLP vaccinations both augments and sustains germinal center B-cell reactions, antigen-specific B-cell frequencies and anti-EBOV serum titers. Finally, we used mice that were deficient in either B-cells or T-cell-dependent antibody production to distinguish the contributing roles of EBOV humoral responses. We demonstrate that while anti-EBOV antibody responses promote protection, VLP-vaccinated mice can survive EBOV infection in the absence of detectable anti-EBOV antibodies. Moreover, we found that adjuvant signaling could circumvent the complete requirement for B-cell immunity in protection against EBOV. Collectively, these studies may prove valuable for the characterization and future development of additional EBOV vaccine candidates.

Ebola virus disease candidate vaccines under evaluation in clinical trials.

Filoviruses are the etiological agents of two human illnesses: Ebola virus disease and Marburg virus disease. Until 2013, medical countermeasure development against these afflictions was limited to only a few research institutes worldwide as both infections were considered exotic due to very low case numbers. Together with the high case-fatality rate of both diseases, evaluation of any candidate countermeasure in properly controlled clinical trials seemed impossible. However, in 2013, Ebola virus was identified as the etiological agent of a large disease outbreak in Western Africa including almost 30,000 infections and more than 11,000 deaths, including case exportations to Europe and North America. These large case numbers resulted in medical countermeasure development against Ebola virus disease becoming a global public-health priority. This review summarizes the status quo of candidate vaccines against Ebola virus disease, with a focus on those that are currently under evaluation in clinical trials.

Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity.

Protein-based vaccines offer a safer alternative to live-attenuated or inactivated vaccines but have limited immunogenicity. The identification of adjuvants that augment immunogenicity, specifically in a manner that is durable and antigen-specific, is therefore critical for advanced development. In this study, we use the filovirus virus-like particle (VLP) as a model protein-based vaccine in order to evaluate the impact of four candidate vaccine adjuvants on enhancing long term protection from Ebola virus challenge. Adjuvants tested include poly-ICLC (Hiltonol), MPLA, CpG 2395, and alhydrogel. We compared and contrasted antibody responses, neutralizing antibody responses, effector T cell responses, and T follicular helper (Tfh) cell frequencies with each adjuvant's impact on durable protection. We demonstrate that in this system, the most effective adjuvant elicits a Th1-skewed antibody response and strong CD4 T cell responses, including an increase in Tfh frequency. Using immune-deficient animals and adoptive transfer of serum and cells from vaccinated animals into naïve animals, we further demonstrate that serum and CD4 T cells play a critical role in conferring protection within effective vaccination regimens. These studies inform on the requirements of long term immune protection, which can potentially be used to guide screening of clinical-grade adjuvants for vaccine clinical development.

Vaccine adjuvant uses of poly-IC and derivatives.

Pathogen-associated molecular patterns (PAMPs) are stand-alone immunomodulators or 'danger signals,' that are increasingly recognized as critical components of many modern vaccines. Polyinosinic-polycytidylic acid (poly-IC) is a synthetic dsRNA that can activate multiple elements of the host defense in a pattern that parallels that of a viral infection. When properly combined with an antigen, it can be utilized as a PAMP-adjuvant, resulting in modulation and optimization of the antigen-specific immune response. We briefly review the preclinical and clinical uses of poly-IC and two poly-IC derivatives, poly-IC12U (Ampligen) and poly-ICLC (Hiltonol), as vaccine adjuvants.