Biomedical subjects

Thomas W Geisbert

Publications and source records attributed to Thomas W Geisbert.

32 records · Page 2Linked to original sources

PubMed2004

Exotic emerging viral diseases: progress and challenges.

The agents causing viral hemorrhagic fever (VHF) are a taxonomically diverse group of viruses that may share commonalities in the process whereby they produce systemic and frequently fatal disease. Significant progress has been made in understanding the biology of the Ebola virus, one of the best known examples. This knowledge has guided our thinking about other VHF agents, including Marburg, Lassa, the South American arenaviruses, yellow fever, Crimean-Congo and Rift Valley fever viruses. Comparisons among VHFs show that a common pathogenic feature is their ability to disable the host immune response by attacking and manipulating the cells that initiate the antiviral response. Of equal importance, these comparisons highlight critical gaps in our knowledge of these pathogens.

Communicable Diseases, Emerging↗

PubMed2004

Depletion of peripheral blood T lymphocytes and NK cells during the course of ebola hemorrhagic Fever in cynomolgus macaques.

During the course of an experimentally induced Ebola virus (EBOVA) infection of cynomolgus macaques, peripheral blood mononuclear cells were isolated and characterized by multi-color flow cytometry. Both CD4+ and CD8+ lymphocyte counts decreased 60-70% during the first 4 days after infection. Among CD8+ lymphocytes, this decline was greatest among the CD8(lo) population, which was composed mostly of CD3- CD16+ NK cells. In contrast, the number of CD20+ B lymphocytes in the blood did not significantly change during the course of the infection. Phenotypic analysis of T lymphocyte subsets by flow cytometry failed to show evidence of a robust immune response to the infection. Apoptosis could be detected as early as day 2 postinfection among the CD8+ and CD16+ subsets of lymphocytes. Increased expression of CD95 (Fas) suggests that apoptosis may be induced via signaling through the Fas/Fas-L cascade. In contrast, the number of HLA-DR+ cells increased tenfold in the blood during the course of infection. These data suggest that EBOV may block dendritic cell maturation after infection, thereby inhibiting activation of lymphocytes and eliminating those subsets that are most likely to be capable of mounting an effective response to the virus.

Animals↗

PubMed2004

Interferon-beta 1a and SARS coronavirus replication.

A global outbreak of severe acute respiratory syndrome (SARS) caused by a novel coronavirus began in March 2003. The rapid emergence of SARS and the substantial illness and death it caused have made it a critical public health issue. Because no effective treatments are available, an intensive effort is under way to identify and test promising antiviral drugs. Here, we report that recombinant human interferon-beta 1a potently inhibits SARS coronavirus replication in vitro.

Animals↗

PubMed2003

Treatment of Ebola virus infection with a recombinant inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys.

BACKGROUND: Infection with the Ebola virus induces overexpression of the procoagulant tissue factor in primate monocytes and macrophages, suggesting that inhibition of the tissue-factor pathway could ameliorate the effects of Ebola haemorrhagic fever. Here, we tested the notion that blockade of fVIIa/tissue factor is beneficial after infection with Ebola virus. METHODS: We used a rhesus macaque model of Ebola haemorrhagic fever, which produces near 100% mortality. We administered recombinant nematode anticoagulant protein c2 (rNAPc2), a potent inhibitor of tissue factor-initiated blood coagulation, to the macaques either 10 min (n=6) or 24 h (n=3) after a high-dose lethal injection of Ebola virus. Three animals served as untreated Ebola virus-positive controls. Historical controls were also used in some analyses. FINDINGS: Both treatment regimens prolonged survival time, with a 33% survival rate in each treatment group. Survivors are still alive and healthy after 9 months. All but one of the 17 controls died. The mean survival for the six rNAPc2-treated macaques that died was 11.7 days compared with 8.3 days for untreated controls (p=0.0184). rNAPc2 attenuated the coagulation response as evidenced by modulation of various important coagulation factors, including plasma D dimers, which were reduced in nearly all treated animals; less prominent fibrin deposits and intravascular thromboemboli were observed in tissues of some animals that succumbed to Ebola virus. Furthermore, rNAPc2 attenuated the proinflammatory response with lower plasma concentrations of interleukin 6 and monocyte chemoattractant protein-1 (MCP-1) noted in the treated than in the untreated macaques. INTERPRETATION: Post-exposure protection with rNAPc2 against Ebola virus in primates provides a new foundation for therapeutic regimens that target the disease process rather than viral replication.

Animals↗

PubMed2003

Mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event.

Disseminated intravascular coagulation is a prominent manifestation of Ebola virus (EBOV) infection. Here, we report that tissue factor (TF) plays an important role in triggering the hemorrhagic complications that characterize EBOV infections. Analysis of samples obtained from 25 macaques showed increased levels of TF associated with lymphoid macrophages, whereas analysis of peripheral blood-cell RNA showed increased levels of TF transcripts by day 3. Plasma from macaques contained increased numbers of TF-expressing membrane microparticles. Dysregulation of the fibrinolytic system developed during the course of infection, including a rapid decrease in plasma levels of protein C. Infection of primary human monocytes/macrophages (PHMs) was used to further evaluate the role of TF in EBOV infections. Analysis of PHM RNA at 1-48 h showed increased TF transcripts, whereas levels of TF protein were dramatically increased by day 2. Thus, chemotherapeutic strategies aimed at controlling overexpression of TF may ameliorate the effects of EBOV hemorrhagic fever.

Animals↗

PubMed2003

Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus.

A previously undescribed coronavirus (CoV) is the etiologic agent responsible for severe acute respiratory syndrome (SARS). Using a panel of contiguous cDNAs that span the entire genome, we have assembled a full-length cDNA of the SARS-CoV Urbani strain, and have rescued molecularly cloned SARS viruses (infectious clone SARS-CoV) that contained the expected marker mutations inserted into the component clones. Recombinant viruses replicated as efficiently as WT virus and both were inhibited by treatment with the cysteine proteinase inhibitor (2S,3S)-transepoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester. In addition, subgenomic transcripts were initiated from the consensus sequence ACGAAC in both the WT and infectious clone SARS-CoV. Availability of a SARS-CoV full-length cDNA provides a template for manipulation of the viral genome, allowing for the rapid and rational development and testing of candidate vaccines and therapeutics against this important human pathogen.

Animals↗

PubMed2003

Accelerated vaccination for Ebola virus haemorrhagic fever in non-human primates.

Containment of highly lethal Ebola virus outbreaks poses a serious public health challenge. Although an experimental vaccine has successfully protected non-human primates against disease, more than six months was required to complete the immunizations, making it impractical to limit an acute epidemic. Here, we report the development of accelerated vaccination against Ebola virus in non-human primates. The antibody response to immunization with an adenoviral (ADV) vector encoding the Ebola glycoprotein (GP) was induced more rapidly than with DNA priming and ADV boosting, but it was of lower magnitude. To determine whether this earlier immune response could nonetheless protect against disease, cynomolgus macaques were challenged with Ebola virus after vaccination with ADV-GP and nucleoprotein (NP) vectors. Protection was highly effective and correlated with the generation of Ebola-specific CD8(+) T-cell and antibody responses. Even when animals were immunized once with ADV-GP/NP and challenged 28 days later, they remained resistant to challenge with either low or high doses of virus. This accelerated vaccine provides an intervention that may help to limit the epidemic spread of Ebola, and is applicable to other viruses.

Adenoviridae↗

PubMed2003

Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection.

Ebola virus (EBOV) infection causes a severe and fatal hemorrhagic disease that in many ways appears to be similar in humans and nonhuman primates; however, little is known about the development of EBOV hemorrhagic fever. In the present study, 21 cynomolgus monkeys were experimentally infected with EBOV and examined sequentially over a 6-day period to investigate the pathological events of EBOV infection that lead to death. Importantly, dendritic cells in lymphoid tissues were identified as early and sustained targets of EBOV, implicating their important role in the immunosuppression characteristic of EBOV infections. Bystander lymphocyte apoptosis, previously described in end-stage tissues, occurred early in the disease-course in intravascular and extravascular locations. Of note, apoptosis and loss of NK cells was a prominent finding, suggesting the importance of innate immunity in determining the fate of the host. Analysis of peripheral blood mononuclear cell gene expression showed temporal increases in tumor necrosis factor-related apoptosis-inducing ligand and Fas transcripts, revealing a possible mechanism for the observed bystander apoptosis, while up-regulation of NAIP and cIAP2 mRNA suggest that EBOV has evolved additional mechanisms to resist host defenses by inducing protective transcripts in cells that it infects. The sequence of pathogenetic events identified in this study should provide new targets for rational prophylactic and chemotherapeutic interventions.

Animals↗

PubMed2003

Pathogenesis of Ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells.

Ebola virus (EBOV) infection causes a severe and often fatal hemorrhagic disease in humans and nonhuman primates. Whether infection of endothelial cells is central to the pathogenesis of EBOV hemorrhagic fever (HF) remains unknown. To clarify the role of endothelial cells in EBOV HF, we examined tissues of 21 EBOV-infected cynomolgus monkeys throughout time, and also evaluated EBOV infection of primary human umbilical vein endothelial cells and primary human lung-derived microvascular endothelial cells in vitro. Results showed that endothelial cells were not early cellular targets of EBOV in vivo, as viral replication was not consistently observed until day 5 after infection, a full day after the onset of disseminated intravascular coagulation. Moreover, the endothelium remained relatively intact even at terminal stages of disease. Although human umbilical vein endothelial cells and human lung-derived microvascular endothelial cells were highly permissive to EBOV replication, significant cytopathic effects were not observed. Analysis of host cell gene response at 24 to 144 hours after infection showed some evidence of endothelial cell activation, but changes were unremarkable considering the extent of viral replication. Together, these data suggest that coagulation abnormalities associated with EBOV HF are not the direct result of EBOV-induced cytolysis of endothelial cells, and are likely triggered by immune-mediated mechanisms.

Animals↗

PubMed2003

Towards a vaccine against Ebola virus.

Ebola virus infection causes hemorrhagic fever with high mortality rates in humans and nonhuman primates. Currently, there are no vaccines or therapies approved for human use. Outbreaks of Ebola virus have been infrequent, largely confined to remote locations in Africa and quarantine of sick patients has been effective in controlling epidemics. In the past, this small global market has generated little commercial interest for developing an Ebola virus vaccine. However, heightened awareness of bioterrorism advanced by the events surrounding September 11, 2001, concomitant with knowledge that the former Soviet Union was evaluating Ebola virus as a weapon, has dramatically changed perspectives regarding the need for a vaccine against Ebola virus. This review takes a brief historic look at attempts to develop an efficacious vaccine, provides an overview of current vaccine candidates and highlights strategies that have the greatest potential for commercial development.

Animals↗

PubMed2002

Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses.

Spatiotemporal aspects of filovirus entry and release are poorly understood. Lipid rafts act as functional platforms for multiple cellular signaling and trafficking processes. Here, we report the compartmentalization of Ebola and Marburg viral proteins within lipid rafts during viral assembly and budding. Filoviruses released from infected cells incorporated raft-associated molecules, suggesting that viral exit occurs at the rafts. Ectopic expression of Ebola matrix protein and glycoprotein supported raft-dependent release of filamentous, virus-like particles (VLPs), strikingly similar to live virus as revealed by electron microscopy. Our findings also revealed that the entry of filoviruses requires functional rafts, identifying rafts as the site of virus attack. The identification of rafts as the gateway for the entry and exit of filoviruses and raft-dependent generation of VLPs have important implications for development of therapeutics and vaccination strategies against infections with Ebola and Marburg viruses.

Cells, Cultured↗

PubMed2002

Proinflammatory response during Ebola virus infection of primate models: possible involvement of the tumor necrosis factor receptor superfamily.

Ebola virus (EBOV) infections are characterized by dysregulation of normal host immune responses. Insight into the mechanism came from recent studies in nonhuman primates, which showed that EBOV infects cells of the mononuclear phagocyte system (MPS), resulting in apoptosis of bystander lymphocytes. In this study, we evaluated serum levels of cytokines/chemokines in EBOV-infected nonhuman primates, as possible correlates of this bystander apoptosis. Increased levels of interferon (IFN)-alpha, IFN-beta, interleukin (IL)-6, IL-18, MIP-1alpha, and MIP-1beta were observed in all EBOV-infected monkeys, indicating the occurrence of a strong proinflammatory response. To investigate the mechanism(s) involved in lymphoid apoptosis, soluble Fas (sFas) and nitrate accumulation were measured. sFas was detected in 4/9 animals, while, elevations of nitrate accumulation occurred in 3/3 animals. To further evaluate the potential role of these factors in the observed bystander apoptosis and intact animals, in vitro cultures were prepared of adherent human monocytes/macrophages (PHM), and monocytes differentiated into immature dendritic cells (DC). These cultures were infected with EBOV and analyzed for cytokine/chemokine induction and expression of apoptosis-related genes. In addition, the in vitro EBOV infection of peripheral blood mononuclear cells (PBMC) resulted in strong cytokine/chemokine induction, a marked increase in lactate dehydrogenase (LDH) activity, and an increase in the number of apoptotic lymphocytes examined by electron microscopy. Increased levels of sFAS were detected in PHM cultures, although, <10% of the cells were positive by immunohistochemistry. In contrast, >90% of EBOV-infected PHM were positive for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by immunohistochemistry, RNA analysis, and flow cytometry. Inactivated EBOV also effected increased TRAIL expression in PHM, suggesting that the TNF receptor superfamily may be involved in apoptosis of the host lymphoid cells, and that induction may occur independent of viral replication. In further studies with infected PHM, expression of MHC II was remarkably suppressed after 6 days, an additional correlate of immunological dysregulation. In conclusion, our findings suggest that infection of mononuclear phagocytes is critical, triggering a cascade of events involving cytokines/chemokines and oxygen free radicals. It is the consequence of these events rather than direct viral infection that results in much of the observed pathology. Identification of cytokine/chemokine, nitric oxide, and reactive oxygen species involvement in the observed filoviral pathogenesis may lend insight into the rational design of therapeutic countermeasures of filoviral pathogenesis.

Animals↗

PubMed2002

Evidence against an important role for infectivity-enhancing antibodies in Ebola virus infections.

The neutralizing and enhancing activities of Ebola virus (EBOV)-specific antibodies were tested among four murine antibodies specific to the surface glycoprotein (GP), a recombinant human monoclonal antibody specific to GP, a polyclonal equine IgG, and serum obtained from a convalescent monkey. All but one of these antibodies neutralized EBOV infectivity of primary human monocytes/macrophages or Vero cells. None of the antibodies enhanced EBOV infectivity in these cells. Taken together with in vivo observations that early deaths were not observed in animals immunized with various viral vectors expressing EBOV GP, it is unlikely that any EBOV-enhancing antibodies profoundly affected EBOV pathogenesis.

Animals↗

PubMed2002

Evaluation in nonhuman primates of vaccines against Ebola virus.

Ebola virus (EBOV) causes acute hemorrhagic fever that is fatal in up to 90% of cases in both humans and nonhuman primates. No vaccines or treatments are available for human use. We evaluated the effects in nonhuman primates of vaccine strategies that had protected mice or guinea pigs from lethal EBOV infection. The following immunogens were used: RNA replicon particles derived from an attenuated strain of Venezuelan equine encephalitis virus (VEEV) expressing EBOV glycoprotein and nucleoprotein; recombinant Vaccinia virus expressing EBOV glycoprotein; liposomes containing lipid A and inactivated EBOV; and a concentrated, inactivated whole-virion preparation. None of these strategies successfully protected nonhuman primates from robust challenge with EBOV. The disease observed in primates differed from that in rodents, suggesting that rodent models of EBOV may not predict the efficacy of candidate vaccines in primates and that protection of primates may require different mechanisms.

Animals↗