Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses.

Vaccines and therapies are urgently needed to address public health needs stemming from emerging pathogens and biological threat agents such as the filoviruses Ebola virus (EBOV) and Marburg virus (MARV). Here, we developed replication-competent vaccines against EBOV and MARV based on attenuated recombinant vesicular stomatitis virus vectors expressing either the EBOV glycoprotein or MARV glycoprotein. A single intramuscular injection of the EBOV or MARV vaccine elicited completely protective immune responses in nonhuman primates against lethal EBOV or MARV challenges. Notably, vaccine vector shedding was not detectable in the monkeys and none of the animals developed fever or other symptoms of illness associated with vaccination. The EBOV vaccine induced humoral and apparent cellular immune responses in all vaccinated monkeys, whereas the MARV vaccine induced a stronger humoral than cellular immune response. No evidence of EBOV or MARV replication was detected in any of the protected animals after challenge. Our data suggest that these vaccine candidates are safe and highly efficacious in a relevant animal model.

Pathogenesis of Marburg hemorrhagic fever in cynomolgus macaques.

BACKGROUND: Marburg virus (MARV) infection causes a severe and often fatal hemorrhagic disease in primates; however, little is known about the development of MARV hemorrhagic fever. In this study we evaluated the progression of MARV infection in nonhuman primates. METHODS: Eighteen cynomolgus monkeys were infected with MARV; blood and tissues were examined sequentially over an 8-day period to investigate disease pathogenesis. RESULTS: Disease caused by MARV in cynomolgus macaques was very similar to disease previously described for Ebola virus-infected macaques. Monocytes, macrophages, Kupffer cells, and dendritic cells (DCs) were identified as the initial targets of MARV infection. Bystander lymphocyte apoptosis occurred at early stages in the disease course in intravascular and extravascular locations. The loss of splenic and lymph node DCs or downregulation of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) on DCs as early as day 2 and continuing through day 8 after MARV infection was a prominent finding. Evidence of disseminated intravascular coagulation was noted; however, the degree of fibrin deposition in tissues was less prominent than was reported in Ebola-infected macaques. CONCLUSIONS: The sequence of pathogenic events identified in this study provides an understanding of the development of disease processes and also may provide new targets for rational prophylactic and chemotherapeutic interventions.

Pathogenesis of Lassa fever in cynomolgus macaques.

BACKGROUND: Lassa virus (LASV) infection causes an acute and sometimes fatal hemorrhagic disease in humans and nonhuman primates; however, little is known about the development of Lassa fever. Here, we performed a pilot study to begin to understand the progression of LASV infection in nonhuman primates. METHODS: Six cynomolgus monkeys were experimentally infected with LASV. Tissues from three animals were examined at an early- to mid-stage of disease and compared with tissues from three animals collected at terminal stages of disease. RESULTS: Dendritic cells were identified as a prominent target of LASV infection in a variety of tissues in all animals at day 7 while Kupffer cells, hepatocytes, adrenal cortical cells, and endothelial cells were more frequently infected with LASV in tissues of terminal animals (days 13.5-17). Meningoencephalitis and neuronal necrosis were noteworthy findings in terminal animals. Evidence of coagulopathy was noted; however, the degree of fibrin deposition in tissues was less prominent than has been reported in other viral hemorrhagic fevers. CONCLUSION: The sequence of pathogenic events identified in this study begins to shed light on the development of disease processes during Lassa fever and also may provide new targets for rational prophylactic and chemotherapeutic interventions.

Mixed-Methods Analysis of Evaluations From In-Person and Remote Nursing Symposium Participants.

Many healthcare organizations offer symposia for sharing nursing research, evidence-based practice, and quality improvement efforts across the system. Significant resources are spent in planning events, with added complexity when symposia are conferenced across sites. A mixed-methods study was conducted to examine the relationship between quantitative and qualitative evaluation results of onsite and remote participants at a multisite nursing symposium. Results may be used by nursing professional development practitioners to inform development of future symposia.

Effective post-exposure treatment of Ebola infection.

Ebola viruses are highly lethal human pathogens that have received considerable attention in recent years due to an increasing re-emergence in Central Africa and a potential for use as a biological weapon. There is no vaccine or treatment licensed for human use. In the past, however, important advances have been made in developing preventive vaccines that are protective in animal models. In this regard, we showed that a single injection of a live-attenuated recombinant vesicular stomatitis virus vector expressing the Ebola virus glycoprotein completely protected rodents and nonhuman primates from lethal Ebola challenge. In contrast, progress in developing therapeutic interventions against Ebola virus infections has been much slower and there is clearly an urgent need to develop effective post-exposure strategies to respond to future outbreaks and acts of bioterrorism, as well as to treat laboratory exposures. Here we tested the efficacy of the vesicular stomatitis virus-based Ebola vaccine vector in post-exposure treatment in three relevant animal models. In the guinea pig and mouse models it was possible to protect 50% and 100% of the animals, respectively, following treatment as late as 24 h after lethal challenge. More important, four out of eight rhesus macaques were protected if treated 20 to 30 min following an otherwise uniformly lethal infection. Currently, this approach provides the most effective post-exposure treatment strategy for Ebola infections and is particularly suited for use in accidentally exposed individuals and in the control of secondary transmission during naturally occurring outbreaks or deliberate release.

Postexposure protection against Marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment.

BACKGROUND: Effective countermeasures are urgently needed to prevent and treat infections caused by highly pathogenic and biological threat agents such as Marburg virus (MARV). We aimed to test the efficacy of a replication-competent vaccine based on attenuated recombinant vesicular stomatitis virus (rVSV), as a postexposure treatment for MARV haemorrhagic fever. METHODS: We used a rhesus macaque model of MARV haemorrhagic fever that produced 100% lethality. We administered rVSV vectors expressing the MARV Musoke strain glycoprotein to five macaques 20-30 min after a high-dose lethal injection of homologous MARV. Three animals were MARV-positive controls and received non-specific rVSV vectors. We tested for viraemia, undertook analyses for haematology and serum biochemistry, and measured humoral and cellular immune responses. FINDINGS: All five rhesus monkeys that were treated with the rVSV MARV vectors as a postexposure treatment survived a high-dose lethal challenge of MARV for at least 80 days. None of these five animals developed clinical symptoms consistent with MARV haemorrhagic fever. All the control animals developed fulminant disease and succumbed to the MARV challenge by day 12. MARV disease in the controls was indicated by: high titres of MARV (10(3)-10(5) plaque-forming units per mL); development of leucocytosis with concurrent neutrophilia at end-stage disease; and possible damage to the liver, kidney, and pancreas. INTERPRETATION: Postexposure protection against MARV in non-human primates provides a paradigm for the treatment of MARV haemorrhagic fever. Indeed, these data suggest that rVSV-based filoviral vaccines might not only have potential as preventive vaccines, but also could be equally useful for postexposure treatment of filoviral infections.

Cynomolgus macaque as an animal model for severe acute respiratory syndrome.

BACKGROUND: The emergence of severe acute respiratory syndrome (SARS) in 2002 and 2003 affected global health and caused major economic disruption. Adequate animal models are required to study the underlying pathogenesis of SARS-associated coronavirus (SARS-CoV) infection and to develop effective vaccines and therapeutics. We report the first findings of measurable clinical disease in nonhuman primates (NHPs) infected with SARS-CoV. METHODS AND FINDINGS: In order to characterize clinically relevant parameters of SARS-CoV infection in NHPs, we infected cynomolgus macaques with SARS-CoV in three groups: Group I was infected in the nares and bronchus, group II in the nares and conjunctiva, and group III intravenously. Nonhuman primates in groups I and II developed mild to moderate symptomatic illness. All NHPs demonstrated evidence of viral replication and developed neutralizing antibodies. Chest radiographs from several animals in groups I and II revealed unifocal or multifocal pneumonia that peaked between days 8 and 10 postinfection. Clinical laboratory tests were not significantly changed. Overall, inoculation by a mucosal route produced more prominent disease than did intravenous inoculation. Half of the group I animals were infected with a recombinant infectious clone SARS-CoV derived from the SARS-CoV Urbani strain. This infectious clone produced disease indistinguishable from wild-type Urbani strain. CONCLUSIONS: SARS-CoV infection of cynomolgus macaques did not reproduce the severe illness seen in the majority of adult human cases of SARS; however, our results suggest similarities to the milder syndrome of SARS-CoV infection characteristically seen in young children.

Chemokine gene activation in human bone marrow-derived osteoblasts following exposure to particulate wear debris.

Particulate wear debris induces the expression of pro-inflammatory cytokine and chemokine genes in various cell types of the periprosthetic region. We have previously reported that titanium particles stimulate the selective induction of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) chemokines in human osteoblast-like osteosarcoma cells. In this study, we characterize the human bone marrow-derived osteoblast chemokine response to titanium particles. We demonstrate that titanium particles result in enhanced IL-8 and MCP-1 protein secretion as well as differential chemokine gene activation. Osteoblast chemokine expression was regulated at the level of gene transcription, with a time-dependent induction of NF-kappaB activation. Inhibition studies with N-acetyl-L-cysteine (Nac) and MG-132 suggest that titanium particle activation of NF-kappaB activity and IL-8 chemokine expression involves oxidant signaling and IkappaBalpha-proteasomal degradation. Activation of the NF-kappaB transcription factor, as well as the IL-8 gene, are redox-regulated. We also demonstrate that while cytochalasin D, a potent inhibitor of phagocytosis, suppressed the titanium particle effect on IL-8 protein release in human bone marrow-derived osteoblasts, the inhibitor had no effect on IL-8 expression in MG-63 osteoblast-like cells. Collectively, these results provide insight into the potential mechanisms responsible for the particulate activation of osteoblast chemokine expression and suggest an important role for the osteoblast in the pathogenesis of periprosthetic osteolysis.

Countermeasures to the bioterrorist threat of smallpox.

Variola, the agent of smallpox, is a bioterrorist threat, as is monkeypox virus, which also occurs naturally in Africa. Development of countermeasures, in the form of improved vaccines, antiviral drugs, and other therapeutic strategies are a high priority. Recent advances in molecular biology and in animal model development have provided fresh insight into the virulence determinants for smallpox and the pathophysiology of disease. The complex replication cycle for orthopoxviruses, and the pivotal role for viral-specific immunomodulatory proteins which contribute to escape from immunologic surveillance, provide many unique targets for therapeutic intervention. The "toxemia" of smallpox has been elucidated in part by variola-infected primate studies which revealed the central role of apoptosis and the evolution of a cytokine storm leading to hemorrhagic diathesis, resembling fulminent "black" smallpox. This suggests a potential role for therapeutic strategies developed for septic shock, in treatment of smallpox. Drugs licensed for other viruses which share molecular targets with orthopoxviruses (e.g. Cidofovir) or cancer drugs (e.g. Gleevec and other tyrosine kinase inhibitors) have immediate application for treatment of smallpox and monkeypox and provide leads for second generation drugs with higher therapeutic indices. Recent advances in identification of virulence determinants and immune evasion genes facilitate the design of alternative vaccines to replace live vaccinia strains that are unsuitable for a large proportion of individuals in a mass immunization campaign.

Development of a new vaccine for the prevention of Lassa fever.

BACKGROUND: Recent importation of Lassa fever into Germany, the Netherlands, the United Kingdom, and the United States by travelers on commercial airlines from Africa underscores the public health challenge of emerging viruses. Currently, there are no licensed vaccines for Lassa fever, and no experimental vaccine has completely protected nonhuman primates against a lethal challenge. METHODS AND FINDINGS: We developed a replication-competent vaccine against Lassa virus based on attenuated recombinant vesicular stomatitis virus vectors expressing the Lassa viral glycoprotein. A single intramuscular vaccination of the Lassa vaccine elicited a protective immune response in nonhuman primates against a lethal Lassa virus challenge. Vaccine shedding was not detected in the monkeys, and none of the animals developed fever or other symptoms of illness associated with vaccination. The Lassa vaccine induced strong humoral and cellular immune responses in the four vaccinated and challenged monkeys. Despite a transient Lassa viremia in vaccinated animals 7 d after challenge, the vaccinated animals showed no evidence of clinical disease. In contrast, the two control animals developed severe symptoms including rashes, facial edema, and elevated liver enzymes, and ultimately succumbed to the Lassa infection. CONCLUSION: Our data suggest that the Lassa vaccine candidate based on recombinant vesicular stomatitis virus is safe and highly efficacious in a relevant animal model that faithfully reproduces human disease.

Chemokine IL-8 induction by particulate wear debris in osteoblasts is mediated by NF-kappaB.

Chemokines, or chemotactic cytokines, are major regulators of the inflammatory response and have been identified as pathogenic factors in the periprosthetic soft tissue. Particulate wear debris induced NF-kappaB activation, the major transcriptional regulator of IL-8 and MCP-1 pro-inflammatory genes and, indeed, both IL-8 and MCP-1 chemokine gene expressions were upregulated in titanium particulate-stimulated human osteoblasts. Here, we demonstrate that phagocytosed particles activate the IL-8 gene promoter via a NF-kappaB-mediated mechanism. Transfection of a dominant negative mutant IkappaBalpha protein that cannot be serine phosphorylated led to suppression of IL-8 promoter activity. The p65/RelA NF-kappaB subunit activity was affected in both a time- and titanium particle concentration-dependent fashion. Titanium particles led to increased ERK, JNK, and p38 activation in MG-63 osteoblast cells, and IL-8 protein release was suppressed by specific inhibitors of the ERK and p38 MAPK pathways. Together, our results suggest that wear debris particles induce chemokine expression in osteoblasts via NF-kappaB-mediated transcriptional activation, which is controlled by the MAPK signal transduction pathway.

Elimination of background in film-based applications.

Erase-It Background Eliminator is a solution used directly on processed film to remove background or improve data resolution. Traditional methods, such as optimization of the scientific protocol or better estimation of exposure time, are tedious and uncertain. Nevertheless, autoradiography continues to be a simple, effective method to visualize data. Therefore, we evaluated the ability of Erase-It Working Solution to help solve background and resolution issues. To demonstrate the efficiency of the Background Eliminator, we analyzed the product's ability to remove signal evenly, performance on several brands of film, and usefulness with various detection methods. Even reduction of signal was demonstrated by performing densitometric analysis on film generated from a dot blot with serial dilutions of analyte. In addition, overexposed films from various suppliers were effectively treated to remove background and visualize data. Autoradiographs, generated with 32P-labeled probes, and chemiluminescent substrate were also processed resulting in clearer images. Our results demonstrate that film data can be treated quickly and conveniently without fear of artificial enhancement. We show the Background Eliminator to be a universal and timesaving tool to visualize results that otherwise may be difficult to interpret.

Titanium particles induce the immediate early stress responsive chemokines IL-8 and MCP-1 in osteoblasts.

Exposure of human osteoblasts to ultrafine titanium (Ti) particles has been shown to alter osteoblast gene expression. We previously reported that Ti particles can increase IL-6 release and suppress the gene expression of procollagens alpha1[I] and alpha1[III] in human osteoblasts. In this study, we now demonstrate that Ti particles can rapidly induce the chemotactic cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), two immediate early stress responsive chemokines important for the activation and chemotaxis of neutrophils and macrophages, respectively. In MG-63 osteosarcoma cells and bone marrow derived primary osteoblasts Ti particles selectively increased the steady state levels of IL-8 and MCP-1 mRNA in a time and concentration dependent manner. The increased chemokine mRNA correlated with increased secretion of IL-8 and MCP-1 protein. Actinomycin D, a potent RNA polymerase II inhibitor, blocked the Ti particle induction of IL-8 and MCP-1 mRNA expression, whereas cycloheximide, which inhibits protein synthesis, failed to inhibit chemokine gene expression suggesting Ti particles directly target activation of chemokine gene transcription. Consistent with a transcriptional mechanism not involving new protein synthesis, we demonstrate that Ti particles induce the binding of the p65 and p50 subunits of the latent transcription factor NF-kappaB to the IL-8 gene promoter. Taken together, these data demonstrate that Ti particles can activate transcription of the stress responsive chemokine genes IL-8 and MCP-1 in human osteoblasts.

Durability of a vesicular stomatitis virus-based marburg virus vaccine in nonhuman primates.

The filoviruses, Marburg virus (MARV) and Ebola virus, causes severe hemorrhagic fever with high mortality in humans and nonhuman primates. A promising filovirus vaccine under development is based on a recombinant vesicular stomatitis virus (rVSV) that expresses individual filovirus glycoproteins (GPs) in place of the VSV glycoprotein (G). These vaccines have shown 100% efficacy against filovirus infection in nonhuman primates when challenge occurs 28-35 days after a single injection immunization. Here, we examined the ability of a rVSV MARV-GP vaccine to provide protection when challenge occurs more than a year after vaccination. Cynomolgus macaques were immunized with rVSV-MARV-GP and challenged with MARV approximately 14 months after vaccination. Immunization resulted in the vaccine cohort of six animals having anti-MARV GP IgG throughout the pre-challenge period. Following MARV challenge none of the vaccinated animals showed any signs of clinical disease or viremia and all were completely protected from MARV infection. Two unvaccinated control animals exhibited signs consistent with MARV infection and both succumbed. Importantly, these data are the first to show 100% protective efficacy against any high dose filovirus challenge beyond 8 weeks after final vaccination. These findings demonstrate the durability of VSV-based filovirus vaccines.

Cellular immune response to Marburg virus infection in cynomolgus macaques.

Marburg virus (MARV) causes a severe and usually lethal hemorrhagic disease in humans and non-human primates. Here, 16 cynomolgus macaques were experimentally infected with the Ci67 strain of MARV. Blood and spleen samples were collected at various time points after infection to study the immunological response to MARV. Beginning at day 2 and continuing throughout the course of the infection there was a rise in antigen-presenting cells in both the blood and spleen expressing MARV glycoprotein. Natural killer (NK) cells declined in the blood after infection (from 15% on day 0 to 5% on day 6), but a small increase was seen in the spleen samples. Little or no change in CD4(+) or CD8(+) T cells was observed out to day 6 post-exposure in blood, while there was a continual decline in the percentage of CD8(+) T cells in spleen samples. Circulating B cells (defined as CD20(+)) increased during the course of the infection as did CD4(+) CD8(+) (double-positive) T cells. Intracellular cytokine staining indicated that by day 6 a large population of leukocytes in the spleen were producing IFN-alpha; analysis of surface markers indicated that these cells were plasmacytoid dendritic cells based on their expression of CD123(+), but these cells had decreased expression of class II MHC. IL-6 production was detected late in the infection in CD14(+) spleen cells. These results suggest a robust innate immune response to MARV; however, this response was delayed relative to the infection.

Vesicular stomatitis virus-based vaccines protect nonhuman primates against aerosol challenge with Ebola and Marburg viruses.

Considerable progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against Ebola and Marburg viruses. A vaccine based on recombinant vesicular stomatitis virus (VSV) seems to be particularly robust as it can also confer protection when administered as a postexposure treatment. While filoviruses are not thought to be transmitted by aerosol in nature the inhalation route is among the most likely portals of entry in the setting of a bioterrorist event. At present, all candidate filoviral vaccines have been evaluated against parenteral challenges but none have been tested against an aerosol exposure. Here, we evaluated our recombinant VSV-based Zaire ebolavirus (ZEBOV) and Marburg virus (MARV) vaccines against aerosol challenge in cynomolgus macaques. All monkeys vaccinated with a VSV vector expressing the glycoprotein of ZEBOV were completely protected against an aerosol exposure of ZEBOV. Likewise, all monkeys vaccinated with a VSV vector expressing the glycoprotein of MARV were completely protected against an aerosol exposure of MARV. All control animals challenged by the aerosol route with either ZEBOV or MARV succumbed. Interestingly, disease in control animals appeared to progress slower than previously seen in macaques exposed to comparable doses by intramuscular injection.

Marburg virus Angola infection of rhesus macaques: pathogenesis and treatment with recombinant nematode anticoagulant protein c2.

BACKGROUND: The procoagulant tissue factor (TF) is thought to play a role in the coagulation disorders that characterize filoviral infections. In this study, we evaluated the pathogenesis of lethal infection with the Angola strain of Marburg virus (MARV-Ang) in rhesus macaques and tested the efficacy of recombinant nematode anticoagulant protein c2 (rNAPc2), an inhibitor of TF/factor VIIa, as a potential treatment. METHODS: Twelve rhesus macaques were challenged with a high dose (1000 pfu) of MARV-Ang. Six macaques were treated with rNAPc2, and 6 macaques served as control animals. RESULTS: All 6 control animals succumbed to MARV-Ang challenge by day 8 (mean, 7.3 days), whereas 5 of 6 rNAPc2-treated animals died on day 9 and 1 rNAPc2-treated animal survived. The disease course for MARV-Ang infection appeared to progress more rapidly in rhesus macaques than has been previously reported for other strains of MARV. In contrast to Ebola virus (EBOV) infection in macaques, up-regulation of TF was not as striking, and deposition of fibrin was a less prominent pathologic feature of disease in these animals. CONCLUSIONS: These data show that the pathogenicity of MARV-Ang infection appears to be consistent with the apparent increased human virulence attributed to this strain. The apparent reduced efficacy of rNAPc2 against MARV-Ang infection, compared with its efficacy against EBOV infection, appears to be associated with differences in TF induction and fibrin deposition.

Cross-protection against Marburg virus strains by using a live, attenuated recombinant vaccine.

Marburg virus (MARV) has been associated with sporadic episodes of hemorrhagic fever, including a recent highly publicized outbreak in Angola that produced severe disease and significant mortality in infected patients. MARV is also considered to have potential as a biological weapon. Recently, we reported the development of a promising attenuated, replication-competent vaccine against MARV based on recombinant vesicular stomatitis virus (VSV) expressing the glycoprotein of the Musoke strain of MARV (VSVDeltaG/MARVGP-Musoke). We used this vaccine to demonstrate complete protection of cynomolgus monkeys against a homologous MARV challenge. While these results are highly encouraging, an effective vaccine would need to confer protection against all relevant strains of MARV. Here, we evaluated the protective efficacy of the VSVDeltaG/MARVGP-Musoke vaccine against two heterologous MARV strains, the seemingly more pathogenic Angola strain and the more distantly related Ravn strain. In this study, seven cynomolgus monkeys were vaccinated with the VSVDeltaG/MARVGP-Musoke vector. Three of these animals were challenged with the Angola strain, three with the Ravn strain, and a single animal with the Musoke strain of MARV. Two animals served as controls and were each injected with a nonspecific VSV vector; these controls were challenged with the Angola and Ravn strains, respectively. Both controls succumbed to challenge by day 8. However, none of the specifically vaccinated animals showed any evidence of illness either from the vaccination or from the MARV challenges and all of these animals survived. These data suggest that the VSVDeltaG/MARVGP-Musoke vaccine should be sufficient to protect against all known MARV strains.

Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference.

BACKGROUND: Ebola virus (EBOV) infection causes a frequently fatal hemorrhagic fever (HF) that is refractory to treatment with currently available antiviral therapeutics. RNA interference represents a powerful, naturally occurring biological strategy for the inhibition of gene expression and has demonstrated utility in the inhibition of viral replication. Here, we describe the development of a potential therapy for EBOV infection that is based on small interfering RNAs (siRNAs). METHODS: Four siRNAs targeting the polymerase (L) gene of the Zaire species of EBOV (ZEBOV) were either complexed with polyethylenimine (PEI) or formulated in stable nucleic acid-lipid particles (SNALPs). Guinea pigs were treated with these siRNAs either before or after lethal ZEBOV challenge. RESULTS: Treatment of guinea pigs with a pool of the L gene-specific siRNAs delivered by PEI polyplexes reduced plasma viremia levels and partially protected the animals from death when administered shortly before the ZEBOV challenge. Evaluation of the same pool of siRNAs delivered using SNALPs proved that this system was more efficacious, as it completely protected guinea pigs against viremia and death when administered shortly after the ZEBOV challenge. Additional experiments showed that 1 of the 4 siRNAs alone could completely protect guinea pigs from a lethal ZEBOV challenge. CONCLUSIONS: Further development of this technology has the potential to yield effective treatments for EBOV HF as well as for diseases caused by other agents that are considered to be biological threats.