From the Cover: Vaccinating captive chimpanzees to save wild chimpanzees

Infectious disease has only recently been recognized as a major threat to the survival of Endangered chimpanzees and Critically Endangered gorillas in the wild. One potentially powerful tool, vaccination, has not been deployed in fighting this disease threat, in good part because of fears about vaccine safety. Here we report on what is, to our knowledge, the first trial in which captive chimpanzees were used to test a vaccine intended for use on wild apes rather than humans. We tested a virus-like particle vaccine against Ebola virus, a leading source of death in wild gorillas and chimpanzees. The vaccine was safe and immunogenic. Captive trials of other vaccines and of methods for vaccine delivery hold great potential as weapons in the fight against wild ape extinction.There is growing recognition that infectious diseases pose a threat to the survival of African apes: a threat on par with poaching and habitat loss. Heightened awareness is due both to better data on rates of disease mortality in gorillas and chimpanzees and to new molecular diagnostic assays that pinpoint the cause of death. These assays tell us that wild apes are regularly infected by a variety of virulent pathogens, including simian immunodeficiency virus (SIV) (1), anthrax (2), and malaria (3). The ethical finger has been pointed squarely and quantitatively at researchers and conservationists with the discovery that “spillover” of human respiratory viruses cause about half of deaths among chimpanzees (4, 5) and gorillas (6) habituated to human approach for research or tourism. Even more widely recognized have been massive Ebola virus (EBOV) outbreaks in gorillas and chimpanzees (7, 8), which have killed roughly one-third of the world gorilla population and led to the 2007 upgrading of western gorillas to Critically Endangered status on the World Conservation Union’s Red List of Threatened Species (9).The ability to accurately diagnose diseases that afflict wild apes has opened the door to an active management response: vaccination (10). The door has been pushed further open by recent advances in vaccinology, including experimental vaccines against several previously unpreventable disease threats to wild apes, new vaccine platforms that reduce or eliminate the risk of infection or vaccine spillover into nontarget species, and new adjuvants that enhance vaccine efficacy (11). Although recognition of the magnitude of the disease threat has made a historically noninterventionist ape conservation community increasingly receptive to vaccination, park managers are still adamant that any experimental vaccine be tested for safety and immunogenicity in captive apes before being used on apes in the wild.Both to address a salient disease threat and to evaluate whether captive testing of vaccines is feasible with the meager budgets typically available to ape conservationists, we decided to test an experimental vaccine against EBOV. During a consultative process lasting several years, vaccine experts, veterinarians, and park managers persistently expressed concerns about the safety of using live (replicating) vaccines on immunologically stressed wild animals. Some cited the case of SIV, which is typically benign in well-cared-for captive chimpanzees but virulent in environmentally challenged wild chimpanzees (1). Therefore, we chose to test a new virus-like particle (VLP) that does not contain an entire replicating virus but only a fragment of viral coat protein. Because they do not cause infection, VLP-based vaccines are particularly safe and several have been recently approved for human use (12). We tested a VLP vaccine based on the virulent Zaire species of EBOV that previously has been given to more than 80 captive macaques without serious health complications (13). In the present study, we did not challenge vaccinated chimpanzees with EBOV. Rather, we simply evaluated whether the vaccine caused health complications that had not been observed in macaques and whether the vaccine induced immune responses comparable to those observed in macaques who survived Ebola challenge. We also tested whether antibodies harvested from vaccinated chimpanzees could protect mice against EBOV challenge.     

Cell adhesion-dependent membrane trafficking of a binding partner for the ebolavirus glycoprotein is a determinant of viral entry

Ebolavirus is a hemorrhagic fever virus associated with high mortality. Although much has been learned about the viral lifecycle and pathogenesis, many questions remain about virus entry. We recently showed that binding of the receptor binding region (RBR) of the ebolavirus glycoprotein (GP) and infection by GP pseudovirions increase on cell adhesion independently of mRNA or protein synthesis. One model to explain these observations is that, on cell adhesion, an RBR binding partner translocates from an intracellular vesicle to the cell surface. Here, we provide evidence for this model by showing that suspension 293F cells contain an RBR binding site within a membrane-bound compartment associated with the trans-Golgi network and microtubule-organizing center. Consistently, trafficking of the RBR binding partner to the cell surface depends on microtubules, and the RBR binding partner is internalized when adherent cells are placed in suspension. Based on these observations, we reexamined the claim that lymphocytes, which are critical for ebolavirus pathogenesis, are refractory to infection because they lack an RBR binding partner. We found that both cultured and primary human lymphocytes (in suspension) contain an intracellular pool of an RBR binding partner. Moreover, we identified two adherent primate lymphocytic cell lines that bind RBR at their surface and strikingly, support GP-mediated entry and infection. In summary, our results reveal a mode of determining viral entry by a membrane-trafficking event that translocates an RBR binding partner to the cell surface, and they suggest that this process may be operative in cells important for ebolavirus pathogenesis (e.g., lymphocytes and macrophages).     

Ubiquitin Ligase SMURF2 Interacts with Filovirus VP40 and Promotes Egress of VP40 VLPs

Filoviruses Ebola (EBOV) and Marburg (MARV) are devastating high-priority pathogens capable of causing explosive outbreaks with high human mortality rates. The matrix proteins of EBOV and MARV, as well as eVP40 and mVP40, respectively, are the key viral proteins that drive virus assembly and egress and can bud independently from cells in the form of virus-like particles (VLPs). The matrix proteins utilize proline-rich Late (L) domain motifs (e.g., PPxY) to hijack specific host proteins that contain WW domains, such as the HECT family E3 ligases, to facilitate the last step of virus–cell separation. We identified E3 ubiquitin ligase Smad Ubiquitin Regulatory Factor 2 (SMURF2) as a novel interactor with VP40 that positively regulates VP40 VLP release. Our results show that eVP40 and mVP40 interact with the three WW domains of SMURF2 via their PPxY motifs. We provide evidence that the eVP40–SMURF2 interaction is functional as the expression of SMURF2 positively regulates VLP egress, while siRNA knockdown of endogenous SMURF2 decreases VLP budding compared to controls. In sum, our identification of novel interactor SMURF2 adds to the growing list of identified host proteins that can regulate PPxY-mediated egress of VP40 VLPs. A more comprehensive understanding of the modular interplay between filovirus VP40 and host proteins may lead to the development of new therapies to combat these deadly infections.     

Development of treatment strategies to combat Ebola and Marburg viruses

Ebola and Marburg viruses are emerging/re-emerging pathogens that pose a significant threat to human health. These naturally occurring viral infections frequently cause a lethal hemorrhagic fever in humans and nonhuman primates. The disastrous consequences of infection with these viruses have been pursued as potential biological weapons. To date, there are no therapeutic options available for the prophylaxis or treatment of infected individuals. The recognition that Ebola and Marburg viruses may be exploited as biological weapons has resulted in major efforts to develop modalities to counter infection. In this review, select technologies and approaches will be highlighted as part of the critical path for the development of therapeutics to ameliorate the invariably devastating outcomes of human filoviral infections.     

An Interagency Collaboration to Facilitate Development of Filovirus Medical Countermeasures

The Filovirus Animal Non-Clinical Group (FANG) is a US interdepartmental and interagency group established to support and facilitate the advanced development of filovirus Medical Countermeasures (MCM), both vaccines and therapeutics. It is co-led by one representative from the Department of Defense (DoD), the first author, and one from the Department of Health and Human Services (HHS), the second author. The FANG membership includes operational level program staff and Subject Matter Experts (SME) from performing organizations as well as scientific staff and program managers from DoD and HHS funding and regulatory agencies. Focus areas include animal models, assays, reagents, product manufacture and characterization, and other interagency product development issues that will support Food and Drug Administration (FDA) licensure of safe and effective filovirus MCMs. The FANG continues to develop strategies to address broadly applicable and interagency product development challenges relevant to filovirus MCM development. This paper summarizes FANG structure and accomplishments and is meant to heighten community awareness of this government-led collaborative effort.     

Filovirus entry: a novelty in the viral fusion world

Ebolavirus (EBOV) and Marburgvirus (MARV) that compose the filovirus family of negative strand RNA viruses infect a broad range of mammalian cells. Recent studies indicate that cellular entry of this family of viruses requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses and others are commonly used by all viral glycoproteins. Details of this entry pathway are highlighted here. Virus entry into cells is initiated by the interaction of the viral glycoprotein(1) subunit (GP(1)) with both adherence factors and one or more receptors on the surface of host cells. On epithelial cells, we recently demonstrated that TIM-1 serves as a receptor for this family of viruses, but the cell surface receptors in other cell types remain unidentified. Upon receptor binding, the virus is internalized into endosomes primarily via macropinocytosis, but perhaps by other mechanisms as well. Within the acidified endosome, the heavily glycosylated GP(1) is cleaved to a smaller form by the low pH-dependent cellular proteases Cathepsin L and B, exposing residues in the receptor binding site (RBS). Details of the molecular events following cathepsin-dependent trimming of GP(1) are currently incomplete; however, the processed GP(1) specifically interacts with endosomal/lysosomal membranes that contain the Niemann Pick C1 (NPC1) protein and expression of NPC1 is required for productive infection, suggesting that GP/NPC1 interactions may be an important late step in the entry process. Additional events such as further GP(1) processing and/or reducing events may also be required to generate a fusion-ready form of the glycoprotein. Once this has been achieved, sequences in the filovirus GP(2) subunit mediate viral/cellular membrane fusion via mechanisms similar to those previously described for other enveloped viruses. This multi-step entry pathway highlights the complex and highly orchestrated path of internalization and fusion that appears unique for filoviruses.     

Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies

The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines.