Prototype of a DNA Vaccine against Marburg Virus.

The preparation and study of the biological properties of the pVAKS-GPVM DNA immunogen containing a gene encoding Marburgvirus glycoprotein are described. The specificity of blood serum antibodies of guinea pigs immunized with DNA immunogen was analyzed by ELISA. Inactivated viral preparation, recombinant glycoprotein (GP) obtained in the prokaryotic system and virus-like particles based on the recombinant vesicular stomatitis virus exhibiting Marburgvirus GP were used as the antigens. The neutralizing activity of antibodies of immunized animals was tested in vitro using a pseudovirus system. It was demonstrated that the developed immunogen administered to guinea pigs induced the production of specific antibodies that neutralize virus-like particles and Marburgvirus in cultured Vero cells.

Discovery of Marburg virus neutralizing antibodies from virus-naïve human antibody repertoires using large-scale structural predictions.

Marburg virus (MARV) disease is lethal, with fatality rates up to 90%. Neutralizing antibodies (Abs) are promising drug candidates to prevent or treat the disease. Current efforts are focused in part on vaccine development to induce such MARV-neutralizing Abs. We analyzed the antibody repertoire from healthy unexposed and previously MARV-infected individuals to assess if naïve repertoires contain suitable precursor antibodies that could become neutralizing with a limited set of somatic mutations. We computationally searched the human Ab variable gene repertoire for predicted structural homologs of the neutralizing Ab MR78 that is specific to the receptor binding site (RBS) of MARV glycoprotein (GP). Eight Ab heavy-chain complementarity determining region 3 (HCDR3) loops from MARV-naïve individuals and one from a previously MARV-infected individual were selected for testing as HCDR3 loop chimeras on the MR78 Ab framework. Three of these chimerized antibodies bound to MARV GP. We then tested a full-length native Ab heavy chain encoding the same 17-residue-long HCDR3 loop that bound to the MARV GP the best among the chimeric Abs tested. Despite only 57% amino acid sequence identity, the Ab from a MARV-naïve donor recognized MARV GP and possessed neutralizing activity against the virus. Crystallization of both chimeric and full-length native heavy chain-containing Abs provided structural insights into the mechanism of binding for these types of Abs. Our work suggests that the MARV GP RBS is a promising candidate for epitope-focused vaccine design to induce neutralizing Abs against MARV.

Modeling the efficiency of filovirus entry into cells in vitro: Effects of SNP mutations in the receptor molecule.

Interaction between filovirus glycoprotein (GP) and the Niemann-Pick C1 (NPC1) protein is essential for membrane fusion during virus entry. Some single-nucleotide polymorphism (SNPs) in two surface-exposed loops of NPC1 are known to reduce viral infectivity. However, the dependence of differences in entry efficiency on SNPs remains unclear. Using vesicular stomatitis virus pseudotyped with Ebola and Marburg virus GPs, we investigated the cell-to-cell spread of viruses in cultured cells expressing NPC1 or SNP derivatives. Eclipse and virus-producing phases were assessed by in vitro infection experiments, and we developed a mathematical model describing spatial-temporal virus spread. This mathematical model fit the plaque radius data well from day 2 to day 6. Based on the estimated parameters, we found that SNPs causing the P424A and D508N substitutions in NPC1 most effectively reduced the entry efficiency of Ebola and Marburg viruses, respectively. Our novel approach could be broadly applied to other virus plaque assays.

The Role of Reference Materials in the Research and Development of Diagnostic Tools and Treatments for Haemorrhagic Fever Viruses.

Following the Ebola outbreak in Western Africa in 2013-16, a global effort has taken place for preparedness for future outbreaks. As part of this response, the development of vaccines, treatments and diagnostic tools has been accelerated, especially towards pathogens listed as likely to cause an epidemic and for which there are no current treatments. Several of the priority pathogens identified by the World Health Organisation are haemorrhagic fever viruses. This review provides information on the role of reference materials as an enabling tool for the development and evaluation of assays, and ultimately vaccines and treatments. The types of standards available are described, along with how they can be applied for assay harmonisation through calibration as a relative potency to a common arbitrary unitage system (WHO International Unit). This assures that assay metrology is accurate and robust. We describe reference materials that have been or are being developed for haemorrhagic fever viruses and consider the issues surrounding their production, particularly that of biosafety where the viruses require specialised containment facilities. Finally, we advocate the use of reference materials at early stages, including research and development, as this helps produce reliable assays and can smooth the path to regulatory approval.

Recent advances in marburgvirus research.

Marburgviruses are closely related to ebolaviruses and cause a devastating disease in humans. In 2012, we published a comprehensive review of the first 45 years of research on marburgviruses and the disease they cause, ranging from molecular biology to ecology. Spurred in part by the deadly Ebola virus outbreak in West Africa in 2013-2016, research on all filoviruses has intensified. Not meant as an introduction to marburgviruses, this article instead provides a synopsis of recent progress in marburgvirus research with a particular focus on molecular biology, advances in animal modeling, and the use of Egyptian fruit bats in infection experiments.

Complete protection of the BALB/c and C57BL/6J mice against Ebola and Marburg virus lethal challenges by pan-filovirus T-cell epigraph vaccine.

There are a number of vaccine candidates under development against a small number of the most common outbreak filoviruses all employing the virus glycoprotein (GP) as the vaccine immunogen. However, antibodies induced by such GP vaccines are typically autologous and limited to the other members of the same species. In contrast, T-cell vaccines offer a possibility to design a single pan-filovirus vaccine protecting against all known and even likely existing, but as yet unencountered members of the family. Here, we used a cross-filovirus immunogen based on conserved regions of the filovirus nucleoprotein, matrix and polymerase to construct simian adenovirus- and poxvirus MVA-vectored vaccines, and in a proof-of-concept study demonstrated a protection of the BALB/c and C57BL/6J mice against high, lethal challenges with Ebola and Marburg viruses, two distant members of the family, by vaccine-elicited T cells in the absence of GP antibodies.

Marburg virus pathogenesis - differences and similarities in humans and animal models.

Marburg virus (MARV) is a highly pathogenic virus associated with severe disease and mortality rates as high as 90%. Outbreaks of MARV are sporadic, deadly, and often characterized by a lack of resources and facilities to diagnose and treat patients. There are currently no approved vaccines or treatments, and the chaotic and infrequent nature of outbreaks, among other factors, makes testing new countermeasures during outbreaks ethically and logistically challenging. Without field efficacy studies, researchers must rely on animal models of MARV infection to assess the efficacy of vaccines and treatments, with the limitations being the accuracy of the animal model in recapitulating human pathogenesis. This review will compare various animal models to the available descriptions of human pathogenesis and aims to evaluate their effectiveness in modeling important aspects of Marburg virus disease.

Marburgviruses: An Update.

Ebolaviruses have gained much attention recently due to the outbreak from 2014 through 2016. The related marburgviruses also have been responsible for large outbreaks with high case fatality rates. The purpose of this article is to provide the clinical laboratory scientist with a review of the most current developments in marburgvirus research. The PubMed database was reviewed using the keywords "Marburg virus," "Ravn virus," and "marburgviruses," with publication dates from January 1, 2015 through June 20, 2017. The search yielded 345 articles. In total, 52 articles met the inclusion criteria and were reviewed. Advances have been made in the areas of ecology and host reservoir studies, seroprevalence studies, pathology and pathogenesis studies, laboratory assay development, and treatment and vaccine development. Marburgviruses are highly lethal viruses that pose a significant threat to the human population. Although numerous advances have been made, there are still large gaps in knowledge, and it is imperative that scientists gain more information to fully understand virus/host interactions. An approved vaccine and treatment remain elusive.

Marburg haemorrhagic fever in returning travellers: an overview aimed at clinicians.

Marburg virus haemorrhagic fever (MARV HF) is a dramatic disease that can occur in a traveller returning from an area where the virus is endemic. In this article, we provide an overview of MARV HF as an imported infection with an emphasis on clinical aspects. Although late features such as rash, signs of haemorrhagic diathesis and liver necrosis may point to the diagnosis, the initial clinical picture is non-specific. If in this early phase the patient's epidemiological exposure history is compatible with MARV HF, the patient should be isolated and managed according to viral haemorrhagic fever protocol and RT-PCR should be performed on the patient's blood as soon as possible to rule out MARV HF (or other possible viral haemorrhagic fevers). In severe cases, direct electron microscopy of blood in specialized centres (e.g. Bernhard-Nocht Institute in Hamburg, Germany) may be considered if the result of the RT-PCR is not readily available. Adequate diagnostics and empirical treatment for other acute life-threatening illnesses should not be withheld while test results are awaited, but all management and diagnostics should be weighed against the risks of nosocomial transmission.

Virulence of Marburg Virus Angola Compared to Mt. Elgon (Musoke) in Macaques: A Pooled Survival Analysis.

Angola variant (MARV/Ang) has replaced Mt. Elgon variant Musoke isolate (MARV/MtE-Mus) as the consensus standard variant for Marburg virus research and is regarded as causing a more aggressive phenotype of disease in animal models; however, there is a dearth of published evidence supporting the higher virulence of MARV/Ang. In this retrospective study, we used data pooled from eight separate studies in nonhuman primates experimentally exposed with either 1000 pfu intramuscular (IM) MARV/Ang or MARV/MtE-Mus between 2012 and 2017 at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). Multivariable Cox proportional hazards regression was used to evaluate the association of variant type with time to death, the development of anorexia, rash, viremia, and 10 select clinical laboratory values. A total of 47 cynomolgus monkeys were included, of which 18 were exposed to MARV/Ang in three separate studies and 29 to MARV/MtE-Mus in five studies. Following universally fatal Marburg virus exposure, compared to MARV/MtE-Mus, MARV/Ang was associated with an increased risk of death (HR = 22.10; 95% CI: 7.08, 68.93), rash (HR = 5.87; 95% CI: 2.76, 12.51) and loss of appetite (HR = 35.10; 95% CI: 7.60, 162.18). Our data demonstrate an increased virulence of MARV/Ang compared to MARV/MtE-Mus variant in the 1000 pfu IM cynomolgus macaque model.

Distinct Biological Phenotypes of Marburg and Ravn Virus Infection in Macaques.

Filoviruses are among the most pathogenic infectious agents known to human, with high destructive potential, as evidenced by the recent Ebola virus epidemic in West Africa. As members of the filovirus family, marburgviruses have caused similar devastating outbreaks, albeit with lower case numbers. In this study we compare the pathogenesis of Ravn virus (RAVV) and Marburg virus (MARV) strains Angola, Musoke, and Ozolin in rhesus and cynomolgus macaques, the 2 nonhuman primate species most commonly used in filovirus research. Our results reveal the most pathogenic MARV strain to be Angola, followed by Musoke, whereas Ozolin is the least pathogenic. We also demonstrate that RAVV is highly pathogenic in cynomolgus macaques but less pathogenic in rhesus macaques. Our results demonstrate a preferential infection of endothelial cells by MARVs; in addition, analysis of tissue samples suggests that lymphocyte and hepatocyte apoptosis might play a role in MARV pathogenicity. This information expands our knowledge about pathogenicity and virulence of marburgviruses.

A Chimeric Lloviu Virus Minigenome System Reveals that the Bat-Derived Filovirus Replicates More Similarly to Ebolaviruses than Marburgviruses.

Recently, traces of zoonotic viruses have been discovered in bats and other species around the world, but despite repeated attempts, full viral genomes have not been rescued. The absence of critical genetic sequences from these viruses and the difficulties to isolate infectious virus from specimens prevent research on their pathogenic potential for humans. One example of these zoonotic pathogens is Lloviu virus (LLOV), a filovirus that is closely related to Ebola virus. Here, we established LLOV minigenome systems based on sequence complementation from other filoviruses. Our results show that the LLOV replication and transcription mechanisms are, in general, more similar to ebolaviruses than to marburgviruses. We also show that a single nucleotide at the 3' genome end determines species specificity of the LLOV polymerase. The data obtained here will be instrumental for the rescue of infectious LLOV clones for pathogenesis studies.

Catching Chances: The Movement to Be on the Ground and Research Ready before an Outbreak.

After more than 28,000 Ebola virus disease cases and at least 11,000 deaths in West Africa during the 2014⁻2016 epidemic, the world remains without a licensed vaccine or therapeutic broadly available and demonstrated to alleviate suffering. This deficiency has been felt acutely in the two, short, following years with two Ebola virus outbreaks in the Democratic Republic of Congo (DRC), and a Marburg virus outbreak in Uganda. Despite billions of U.S. dollars invested in developing medical countermeasures for filoviruses in the antecedent decades, resulting in an array of preventative, diagnostic, and therapeutic products, none are available on commercial shelves. This paper explores why just-in-time research efforts in the field during the West Africa epidemic failed, as well as some recent initiatives to prevent similarly lost opportunities.

Antibody-Dependent Enhancement of Ebola Virus Infection by Human Antibodies Isolated from Survivors.

Some monoclonal antibodies (mAbs) recovered from survivors of filovirus infections can protect against infection. It is currently unknown whether natural infection also induces some antibodies with the capacity for antibody-dependent enhancement (ADE). A panel of mAbs obtained from human survivors of filovirus infection caused by Ebola, Bundibugyo, or Marburg viruses was evaluated for their ability to facilitate ADE. ADE was observed readily with all mAbs examined at sub-neutralizing concentrations, and this effect was not restricted to mAbs with a particular epitope specificity, neutralizing capacity, or subclass. Blocking of specific Fcγ receptors reduced but did not abolish ADE that was associated with high-affinity binding antibodies, suggesting that lower-affinity interactions still cause ADE. Mutations of Fc fragments of an mAb that altered its interaction with Fc receptors rendered the antibody partially protective in vivo at a low dose, suggesting that ADE counteracts antibody-mediated protection and facilitates dissemination of filovirus infections.

Pathogenicity of Ebola and Marburg Viruses Is Associated With Differential Activation of the Myeloid Compartment in Humanized Triple Knockout-Bone Marrow, Liver, and Thymus Mice.

Ebola virus (EBOV) and Marburg virus (MARV) outbreaks are highly lethal, and infection results in a hemorrhagic fever with complex etiology. These zoonotic viruses dysregulate the immune system to cause disease, in part by replicating within myeloid cells that would normally innately control viral infection and shape the adaptive immune response. We used triple knockout (TKO)-bone marrow, liver, thymus (BLT) humanized mice to recapitulate the early in vivo human immune response to filovirus infection. Disease severity in TKO-BLT mice was dissimilar between EBOV and MARV with greater severity observed during EBOV infection. Disease severity was related to increased Kupffer cell infection in the liver, higher levels of myeloid dysfunction, and skewing of macrophage subtypes in EBOV compared with MARV-infected mice. Overall, the TKO-BLT model provided a practical in vivo platform to study the human immune response to filovirus infection and generated a better understanding of how these viruses modulate specific components of the immune system.

Marburg- and Ebolaviruses: A Look Back and Lessons for the Future.

Since the discovery of Marburg virus 50 years ago, filoviruses have reemerged in the human population more than 40 times. Already the first episode was as dramatic as most of the subsequent ones, but none of them was as devastating as the West-African Ebola virus outbreak in 2013-2015. Although progress toward a better understanding of the viruses is impressive, there is clearly a need to improve and strengthen the measures to detect and control these deadly infections.

A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system.

Marburg virus (MARV) can cause lethal hemorrhagic fever in humans. Handling of MARV is restricted to high-containment biosafety level 4 (BSL-4) facilities, which greatly impedes research into this virus. In this study, a high titer of MARV pseudovirus was generated through optimization of the HIV backbone vectors, the ratio of backbone vector to MARV glycoprotein expression vector, and the transfection reagents. An in vitro neutralization assay and an in vivo bioluminescent imaging mouse model for MARV were developed based on the pseudovirus. Protective serum against MARV was successfully induced in guinea pigs, which showed high neutralization activity in vitro and could also protect Balb/c mice from MARV pseudovirus infection in vivo. This system could be a convenient tool to enable the evaluation of vaccines and therapeutic drugs against MARV in non-BSL-4 laboratories.

A hamster model for Marburg virus infection accurately recapitulates Marburg hemorrhagic fever.

Marburg virus (MARV), a close relative of Ebola virus, is the causative agent of a severe human disease known as Marburg hemorrhagic fever (MHF). No licensed vaccine or therapeutic exists to treat MHF, and MARV is therefore classified as a Tier 1 select agent and a category A bioterrorism agent. In order to develop countermeasures against this severe disease, animal models that accurately recapitulate human disease are required. Here we describe the development of a novel, uniformly lethal Syrian golden hamster model of MHF using a hamster-adapted MARV variant Angola. Remarkably, this model displayed almost all of the clinical features of MHF seen in humans and non-human primates, including coagulation abnormalities, hemorrhagic manifestations, petechial rash, and a severely dysregulated immune response. This MHF hamster model represents a powerful tool for further dissecting MARV pathogenesis and accelerating the development of effective medical countermeasures against human MHF.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses.

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.

Distribution of Marburg virus in Africa: An evolutionary approach.

The aim of this study was to investigate the origin and geographical dispersion of Marburg virus, the first member of the Filoviridae family to be discovered. Seventy-three complete genome sequences of Marburg virus isolated from animals and humans were retrieved from public databases and analysed using a Bayesian phylogeographical framework. The phylogenetic tree of the Marburg virus data set showed two significant evolutionary lineages: Ravn virus (RAVV) and Marburg virus (MARV). MARV divided into two main clades; clade A included isolates from Uganda (five from the European epidemic in 1967), Kenya (1980) and Angola (from the epidemic of 2004-2005); clade B included most of the isolates obtained during the 1999-2000 epidemic in the Democratic Republic of the Congo (DRC) and a group of Ugandan isolates obtained in 2007-2009. The estimated mean evolutionary rate of the whole genome was 3.3×10(-4) substitutions/site/year (credibility interval 2.0-4.8). The MARV strain had a mean root time of the most recent common ancestor of 177.9years ago (YA) (95% highest posterior density 87-284), thus indicating that it probably originated in the mid-XIX century, whereas the RAVV strain had a later origin dating back to a mean 33.8 YA. The most probable location of the MARV ancestor was Uganda (state posterior probability, spp=0.41), whereas that of the RAVV ancestor was Kenya (spp=0.71). There were significant migration rates from Uganda to the DRC (Bayes Factor, BF=42.0) and in the opposite direction (BF=5.7). Our data suggest that Uganda may have been the cradle of Marburg virus in Africa.