Structural Biology Illuminates Molecular Determinants of Broad Ebolavirus Neutralization by Human Antibodies for Pan-Ebolavirus Therapeutic Development

Monoclonal antibodies (mAbs) have proven effective for the treatment of ebolavirus infection in humans, with two mAb-based drugs Inmazeb™ and Ebanga™ receiving FDA approval in 2020. While these drugs represent a major advance in the field of filoviral therapeutics, they are composed of antibodies with single-species specificity for Zaire ebolavirus. The Ebolavirus genus includes five additional species, two of which, Bundibugyo ebolavirus and Sudan ebolavirus, have caused severe disease and significant outbreaks in the past. There are several recently identified broadly neutralizing ebolavirus antibodies, including some in the clinical development pipeline, that have demonstrated broad protection in preclinical studies. In this review, we describe how structural biology has illuminated the molecular basis of broad ebolavirus neutralization, including details of common antigenic sites of vulnerability on the glycoprotein surface. We begin with a discussion outlining the history of monoclonal antibody therapeutics for ebolaviruses, with an emphasis on how structural biology has contributed to these efforts. Next, we highlight key structural studies that have advanced our understanding of ebolavirus glycoprotein structures and mechanisms of antibody-mediated neutralization. Finally, we offer examples of how structural biology has contributed to advances in anti-viral medicines and discuss what opportunities the future holds, including rationally designed next-generation therapeutics with increased potency, breadth, and specificity against ebolaviruses.

Flow cytometry evaluation of the rhesus monkey cellular immunity following the Zaire ebolavirus (Filoviridae; Ebolavirus: Zaire ebolavirus) experimental infection

Introduction. The outbreaks of the Zaire ebolavirus (ZE) disease (ZED) that have arisen in the last decade determine the need to study the infection pathogenesis, the formation of specific immunity forming as well as the development of effective preventive and therapeutic means. All stages of fight against the ZED spread require the experimental infection in sensitive laboratory animals, which are rhesus monkeys in case of this disease .The aim of the study is to evaluate the rhesus monkey cellular immunity following the ZE experimental infection by the means of flow cytometry (cytofluorimetry).Material and methods. Male rhesus monkeys were intramuscularly infected by the dose of 15 LD50 (dose of the pathogen that causes 50% mortality of infected animals) of the ZE, the Zaire strain (ZEBOV). Levels of 18 peripheral blood lymphocyte populations of the animals before the ZE experimental infection and at the terminal stage of the disease were assessed using flow cytometry.Results and discussion. The certain changes in the levels of the lymphocyte populations were observed following infection, indicating simultaneous activation and suppression of the immune system during ZED. The increase in content was observed for T-lymphocytes, T-helper and cytotoxic T-lymphocytes expressing the corresponding markers of early activation. The decrease was recorded for T-lymphocytes and double-positive T-lymphocytes expressing corresponding markers of late activation, as well as natural killer cells expressing CD8 (p < 0.05).Conclusion. For the first time in the Russian Federation, the rhesus monkey cellular immunity before and after the ZE experimental infection was assessed using flow cytometry.

Comparison of Zaire and Bundibugyo ebolavirus polymerase complexes and susceptibility to antivirals through a newly developed Bundibugyo minigenome system

Members of the genus Ebolavirus cause lethal disease in humans with Zaire ebolavirus (EBOV) being the most pathogenic (up to 90% morality) and Bundibugyo ebolavirus (BDBV) the least pathogenic (∼37% mortality). Historically, there has been a lack of research on BDBV and there is no means to study BDBV outside of a high-containment laboratory. Here, we describe a minigenome replication system to study BDBV transcription and compare the efficacy of small molecule inhibtors between EBOV and BDBV. Using this system, we examined the ability of the polymerase complex proteins from EBOV and BDBV to interact and form a functional unit as well as the impact of the genomic untranslated ends, known to contain important signals for transcription (3’-untranslated region) and replication (5’-untranslated region). Varying levels of compatibility were observed between proteins of the polymerase complex from each ebolavirus resulting in differences in genome transcription efficiency. Most pronounced was the effect of the nucleoprotein and the 3’-untranslated region. These data suggest that there are intrinsic specificities in the polymerase complex and untranslated signaling regions that could offer insight regarding observed pathogenic differences. Further adding to the differences in the polymerase complexes, post-transfection/infection treatment with the compound remdesivir (GS-5734) showed a greater inhibitory effect against BDBV compared to EBOV. The delayed growth kinetics of BDBV and the greater susceptibility to polymerase inhibitors indicate that disruption of the polymerase complex may be a viable target for therapeutics. Importance Ebolavirus disease is a viral infection and is fatal in 25-90% of cases, depending on the viral species and the amount of supportive care available. Two species have caused outbreaks in the Democratic Republic of the Congo, Zaire ebolavirus (EBOV) and Bundibugyo ebolavirus (BDBV). Pathogenesis and clinical outcome differ between these two species but there is still limited information regarding the viral mechanism for these differences. Previous studies suggest that BDBV replicates slower than EBOV but it is unknown if this is due to differences in the polymerase complex and its role in transcription and replication. This study details the construction of a minigenome replication system which can be used in a biosafety level (BSL) 2 laboartory. This system will be important for studying the polymerase complex of BDBV and comparing it with other filoviruses and can be used as a tool for screening inhibitors of viral growth.

STAT-1 Knockout Mice as a Model for Wild-Type Sudan Virus (SUDV)

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014–2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 101 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures.

P300-mediated NEDD4 acetylation drives ebolavirus VP40 egress by enhancing NEDD4 ligase activity

The final stage of Ebola virus (EBOV) replication is budding from host cells, where the matrix protein VP40 is essential for driving this process. Many post-translational modifications such as ubiquitination are involved in VP40 egress, but acetylation has not been studied yet. Here, we characterize NEDD4 is acetylated at a conserved Lys667 mediated by the acetyltransferase P300 which drives VP40 egress process. Importantly, P300-mediated NEDD4 acetylation promotes NEDD4-VP40 interaction which enhances NEDD4 E3 ligase activity and is essential for the activation of VP40 ubiquitination and subsequent egress. Finally, we find that Zaire ebolavirus production is dramatically reduced in P300 knockout cell lines, suggesting that P300-mediated NEDD4 acetylation may have a physiological effect on Ebola virus life cycle. Thus, our study identifies an acetylation-dependent regulatory mechanism that governs VP40 ubiquitination and provides insights into how acetylation controls EBOV VP40 egress.

Development of Pandemic Vaccines: ERVEBO Case Study

Preventative vaccines are considered one of the most cost-effective and efficient means to contain outbreaks and prevent pandemics. However, the requirements to gain licensure and manufacture a vaccine for human use are complex, costly, and time-consuming. The 2013–2016 Ebola virus disease (EVD) outbreak was the largest EVD outbreak to date and the third Public Health Emergency of International Concern in history, so to prevent a pandemic, numerous partners from the public and private sectors combined efforts and resources to develop an investigational Zaire ebolavirus (EBOV) vaccine candidate (rVSVΔG-ZEBOV-GP) as quickly as possible. The rVSVΔG-ZEBOV-GP vaccine was approved as ERVEBOTM by the European Medicines Authority (EMA) and the United States Food and Drug Administration (FDA) in December 2019 after five years of development. This review describes the development program of this EBOV vaccine, summarizes what is known about safety, immunogenicity, and efficacy, describes ongoing work in the program, and highlights learnings applicable to the development of pandemic vaccines.