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1.  A Recombinant Hendra Virus G Glycoprotein Subunit Vaccine Protects Nonhuman Primates against Hendra Virus Challenge 
Journal of Virology  2014;88(9):4624-4631.
ABSTRACT
Hendra virus (HeV) is a zoonotic emerging virus belonging to the family Paramyxoviridae. HeV causes severe and often fatal respiratory and/or neurologic disease in both animals and humans. Currently, there are no licensed vaccines or antiviral drugs approved for human use. A number of animal models have been developed for studying HeV infection, with the African green monkey (AGM) appearing to most faithfully reproduce the human disease. Here, we assessed the utility of a newly developed recombinant subunit vaccine based on the HeV attachment (G) glycoprotein in the AGM model. Four AGMs were vaccinated with two doses of the HeV vaccine (sGHeV) containing Alhydrogel, four AGMs received the sGHeV with Alhydrogel and CpG, and four control animals did not receive the sGHeV vaccine. Animals were challenged with a high dose of infectious HeV 21 days after the boost vaccination. None of the eight specifically vaccinated animals showed any evidence of clinical illness and survived the challenge. All four controls became severely ill with symptoms consistent with HeV infection, and three of the four animals succumbed 8 days after exposure. Success of the recombinant subunit vaccine in AGMs provides pivotal data in supporting its further preclinical development for potential human use.
IMPORTANCE A Hendra virus attachment (G) glycoprotein subunit vaccine was tested in nonhuman primates to assess its ability to protect them from a lethal infection with Hendra virus. It was found that all vaccinated African green monkeys were completely protected against subsequent Hendra virus infection and disease. The success of this new subunit vaccine in nonhuman primates provides critical data in support of its further development for future human use.
doi:10.1128/JVI.00005-14
PMCID: PMC3993805  PMID: 24522928
2.  Durability of a Vesicular Stomatitis Virus-Based Marburg Virus Vaccine in Nonhuman Primates 
PLoS ONE  2014;9(4):e94355.
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.
doi:10.1371/journal.pone.0094355
PMCID: PMC3997383  PMID: 24759889
3.  A Recently Isolated Lassa Virus From Mali Demonstrates Atypical Clinical Disease Manifestations and Decreased Virulence in Cynomolgus Macaques 
The Journal of Infectious Diseases  2013;207(8):1316-1327.
The virulence of Soromba-R, a Lassa virus strain recently isolated from southern Mali, was assessed in 2 animal models of Lassa fever: inbred strain 13 guinea pigs and cynomolgus macaques. In both models, the Malian isolate demonstrated tissue tropism and viral titers similar to those of historical Lassa virus isolates from Sierra Leone (Josiah) and Liberia (Z-132); however, the Soromba-R isolate was found to be less pathogenic, as determined by decreased mortality and prolonged time to euthanasia in macaques. Interestingly, in addition to the classic indicators of Lassa fever, Soromba-R infection presented with moderate to severe pulmonary manifestations in the macaque model. Analysis of host responses demonstrated increased immune activation in Soromba-R–infected macaques, particularly in neutrophil-activating or -potentiating proinflammatory cytokines or growth factors, including tumor necrosis factor α, macrophage inflammatory protein 1α, interleukin 1β, and granulocyte colony-stimulating factor, as well as interleukin 5, which may be responsible for the decreased lethality and uncharacteristic clinical presentation. These results suggest that the strain of Lassa virus circulating in Mali might be less pathogenic than strains circulating in the historical region of endemicity and may result in an atypical presentation for Lassa fever, which could complicate clinical diagnosis.
doi:10.1093/infdis/jit004
PMCID: PMC3603532  PMID: 23303805
Lassa fever; pathogenesis; disease modeling; guinea pigs; non-human primates; West Africa
4.  Animal models for highly pathogenic emerging viruses 
Current opinion in virology  2013;3(2):205-209.
Exotic and emerging viral pathogens associated with high morbidity and mortality in humans are being identified annually with recent examples including Lujo virus in southern Africa, Severe fever with Thrombocytopenia virus in China and a SARS-like coronavirus in the Middle East. The sporadic nature of these infections hampers our understanding of these diseases and limits the opportunities to design appropriate medical countermeasures against them. Due to this, animal models are utilized to gain insight into the pathogenesis of disease with the overall goal of identifying potential targets for intervention and evaluating specific therapeutics and vaccines. For these reasons it is imperative that animal models of disease recapitulate the human condition as closely as possible in order to provide the best predictive data with respect to the potential efficacy in humans. In this article we review the current status of disease models for highly pathogenic and emerging viral pathogens.
doi:10.1016/j.coviro.2013.01.001
PMCID: PMC3644300  PMID: 23403208
5.  Molecular Epidemiology of Crimean-Congo Hemorrhagic Fever Virus in Kosovo 
Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic agent that causes severe, life-threatening disease, with a case fatality rate of 10–50%. It is the most widespread tick-borne virus in the world, with cases reported in Africa, Asia and Eastern Europe. CCHFV is a genetically diverse virus. Its genetic diversity is often correlated to its geographical origin. Genetic variability of CCHFV was determined within few endemic areas, however limited data is available for Kosovo. Furthermore, there is little information about the spatiotemporal genetic changes of CCHFV in endemic areas. Kosovo is an important endemic area for CCHFV. Cases were reported each year and the case-fatality rate is significantly higher compared to nearby regions. In this study, we wanted to examine the genetic variability of CCHFV obtained directly from CCHF-confirmed patients, hospitalized in Kosovo from 1991 to 2013. We sequenced partial S segment CCHFV nucleotide sequences from 89 patients. Our results show that several viral variants are present in Kosovo and that the genetic diversity is high in relation to the studied area. We also show that variants are mostly uniformly distributed throughout Kosovo and that limited evolutionary changes have occurred in 22 years. Our results also suggest the presence of a new distinct lineage within the European CCHF phylogenetic clade. Our study provide the largest number of CCHFV nucleotide sequences from patients in 22 year span in one endemic area.
Author Summary
Crimean-Congo hemorrhagic fever (CCHF) is an acute, tick-borne disease with a case fatality rate of 10–30%. It is geographically the most widespread tick-borne disease in the world. In recent years there has been an increase of the disease incidence in several countries, mainly in the countries of the Balkan. The disease is also endemic in Kosovo. Since CCHF virus is very genetically diverse we aimed to determine the genetic variability of the virus in Kosovo in the span of 22 years. We obtained the largest number of patient derived nucleotide sequences and found great genetic variability which has been more or less stable during the 22 year period. Our results also suggest that significant changes in viral population occur in different years. We show that ecological factors such as temperature could play a role in the composition of the viral population.
doi:10.1371/journal.pntd.0002647
PMCID: PMC3886908  PMID: 24416468
6.  Production of CXC and CC Chemokines by Human Antigen-Presenting Cells in Response to Lassa Virus or Closely Related Immunogenic Viruses, and in Cynomolgus Monkeys with Lassa Fever 
The pathogenesis of Lassa fever (LF), a hemorrhagic fever endemic to West Africa, remains unclear. We previously compared Lassa virus (LASV) with its genetically close, but nonpathogenic homolog Mopeia virus (MOPV) and demonstrated that the strong activation of antigen-presenting cells (APC), including type I IFN production, observed in response to MOPV probably plays a crucial role in controlling infection. We show here that human macrophages (MP) produce large amounts of CC and CXC chemokines in response to MOPV infection, whereas dendritic cells (DC) release only moderate amounts of CXC chemokines. However, in the presence of autologous T cells, DCs produced CC and CXC chemokines. Chemokines were produced in response to type I IFN synthesis, as the levels of both mediators were strongly correlated and the neutralization of type I IFN resulted in an inhibition of chemokine production. By contrast, LASV induced only low levels of CXCL-10 and CXCL-11 production. These differences in chemokine production may profoundly affect the generation of virus-specific T-cell responses and may therefore contribute to the difference of pathogenicity between these two viruses. In addition, a recombinant LASV (rLASV) harboring the NP-D389A/G392A mutations, which abolish the inhibition of type I IFN response by nucleoprotein (NP), induced the massive synthesis of CC and CXC chemokines in both DC and MP, confirming the crucial role of arenavirus NP in immunosuppression and pathogenicity. Finally, we confirmed, using PBMC samples and lymph nodes obtained from LASV-infected cynomolgus monkeys, that LF was associated with high levels of CXC chemokine mRNA synthesis, suggesting that the very early synthesis of these mediators may be correlated with a favourable outcome.
Author Summary
Lassa virus (LASV) causes a viral hemorrhagic fever that affects about 300,000 people and leads to 5,000 deaths annually. Lassa fever (LF) is a public health problem in West Africa, where it is endemic, because of the number of cases, deaths and disabling effects. There is no vaccine against LASV and the only treatment, ribavirin, is not useful in the field. Little is known about the pathogenesis and immune responses associated with LF. Chemokines are involved in the induction of immunity and attraction of immune cells to inflamed sites. We compared the ability of antigen-presenting cells to produce chemokines in response to infection with LASV, the closely related but nonpathogenic Mopeia virus (MOPV) and a LASV unable to inhibit the type I IFN response due to mutations in its nucleoprotein gene. We found that MOPV and the mutant LASV, but not wild-type LASV, strongly induced CC and CXC chemokine production by dendritic cells and macrophages, in a type I IFN-dependent manner. We confirmed in cynomolgus monkeys that these mediators probably play a role during LF. These results highlight the role of innate immunity in LF control and provide insight into the mechanisms leading to survival or death after infection.
doi:10.1371/journal.pntd.0002637
PMCID: PMC3888467  PMID: 24421914
7.  Vesicular Stomatitis Virus-Based Vaccines Protect Nonhuman Primates against Bundibugyo ebolavirus 
Ebola virus (EBOV) causes severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Currently, there are no licensed vaccines or therapeutics for human use. Recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode an EBOV glycoprotein in place of the VSV glycoprotein, have shown 100% efficacy against homologous Sudan ebolavirus (SEBOV) or Zaire ebolavirus (ZEBOV) challenge in NHPs. In addition, a single injection of a blend of three rVSV vectors completely protected NHPs against challenge with SEBOV, ZEBOV, the former Côte d'Ivoire ebolavirus, and Marburg virus. However, recent studies suggest that complete protection against the newly discovered Bundibugyo ebolavirus (BEBOV) using several different heterologous filovirus vaccines is more difficult and presents a new challenge. As BEBOV caused nearly 50% mortality in a recent outbreak any filovirus vaccine advanced for human use must be able to protect against this new species. Here, we evaluated several different strategies against BEBOV using rVSV-based vaccines. Groups of cynomolgus macaques were vaccinated with a single injection of a homologous BEBOV vaccine, a single injection of a blended heterologous vaccine (SEBOV/ZEBOV), or a prime-boost using heterologous SEBOV and ZEBOV vectors. Animals were challenged with BEBOV 29–36 days after initial vaccination. Macaques vaccinated with the homologous BEBOV vaccine or the prime-boost showed no overt signs of illness and survived challenge. In contrast, animals vaccinated with the heterologous blended vaccine and unvaccinated control animals developed severe clinical symptoms consistent with BEBOV infection with 2 of 3 animals in each group succumbing. These data show that complete protection against BEBOV will likely require incorporation of BEBOV glycoprotein into the vaccine or employment of a prime-boost regimen. Fortunately, our results demonstrate that heterologous rVSV-based filovirus vaccine vectors employed in the prime-boost approach can provide protection against BEBOV using an abbreviated regimen, which may have utility in outbreak settings.
Author Summary
Ebola viruses (EBOV), of which there are five species, are categorized as Category A Priority Pathogens and Tier 1 Select Agents by several US Government agencies as a result of their high mortality rates and potential for use as agents of bioterrorism. Currently, there are no vaccines or therapeutics approved for human use. Replication-competent, recombinant vesicular stomatitis virus (rVSV) vectors expressing filovirus glycoproteins (GP), in place of the VSV glycoprotein have shown promise in lethal nonhuman primate (NHP) models of filovirus infection as both single injection preventive vaccines and as post-exposure treatments. The recent outbreak of the fifth recognized EBOV species, Bundibugyo ebolavirus (BEBOV), demonstrates the need for vaccines that can be rapidly deployed to combat an outbreak of a new filovirus species. To date, rVSV-filovirus GP-based vaccines have only been able to protect against challenge with a homologous species of EBOV. Here, we show that the two heterologous rVSV-based filovirus vaccines available at the time of the original BEBOV outbreak can protect NHPs against BEBOV challenge using a short prime-boost vaccination strategy. While the prime-boost strategy was successful, a single injection blended vaccination strategy with the same vaccine vectors failed to provide protection. These data suggest that an abbreviated prime-boost regimen of 36 days may have utility for quickly responding to outbreaks caused by new species of EBOV.
doi:10.1371/journal.pntd.0002600
PMCID: PMC3868506  PMID: 24367715
8.  Single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal Nipah virus disease 
Virology Journal  2013;10:353.
Background
Nipah virus (NiV) is a highly pathogenic zoonotic agent in the family Paramyxoviridae that is maintained in nature by bats. Outbreaks have occurred in Malaysia, Singapore, India, and Bangladesh and have been associated with 40 to 75% case fatality rates. There are currently no vaccines or postexposure treatments licensed for combating human NiV infection.
Methods and results
Four groups of ferrets received a single vaccination with different recombinant vesicular stomatitis virus vectors expressing: Group 1, control with no glycoprotein; Group 2, the NiV fusion protein (F); Group 3, the NiV attachment protein (G); and Group 4, a combination of the NiV F and G proteins. Animals were challenged intranasally with NiV 28 days after vaccination. Control ferrets in Group 1 showed characteristic clinical signs of NiV disease including respiratory distress, neurological disorders, viral load in blood and tissues, and gross lesions and antigen in target tissues; all animals in this group succumbed to infection by day 8. Importantly, all specifically vaccinated ferrets in Groups 2-4 showed no evidence of clinical illness and survived challenged. All animals in these groups developed anti-NiV F and/or G IgG and neutralizing antibody titers. While NiV RNA was detected in blood at day 6 post challenge in animals from Groups 2-4, the levels were orders of magnitude lower than animals from control Group 1.
Conclusions
These data show protective efficacy against NiV in a relevant model of human infection. Further development of this technology has the potential to yield effective single injection vaccines for NiV infection.
doi:10.1186/1743-422X-10-353
PMCID: PMC3878732  PMID: 24330654
Nipah virus; Henipavirus; Vaccine; Vesicular stomatitis virus; Ferret; Fusion protein; Attachment protein; Glycoprotein; Single-injection; Immunity
9.  Transcriptome Analysis of Human Peripheral Blood Mononuclear Cells Exposed to Lassa Virus and to the Attenuated Mopeia/Lassa Reassortant 29 (ML29), a Vaccine Candidate 
Lassa virus (LASV) is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV) are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC) exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. Results were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG), as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease.
Author Summary
The virulent Lassa fever virus (LASV) and the non-pathogenic Mopeia virus (MOPV) infect rodents and, incidentally, people in West Africa. The mechanism of LASV damage in human beings is unclear. There is no licensed Lassa fever vaccine and therapeutic intervention is usually too late. The ML29 vaccine candidate derived from Lassa and Mopeia viruses protects rodents and primates from Lassa fever disease. Peripheral blood mononuclear cells from healthy human subjects were exposed to either LASV or ML29 in order to identify early cellular responses that could be attributed to the difference in virulence between the two viruses. Differential expression of interferon-stimulated genes as well as coagulation-related genes could lead to an explanation for Lassa fever pathogenesis and indicate protective treatments for Lassa fever disease.
doi:10.1371/journal.pntd.0002406
PMCID: PMC3772037  PMID: 24069471
10.  Protection Against Lethal Marburg Virus Infection Mediated by Lipid Encapsulated Small Interfering RNA 
The Journal of Infectious Diseases  2013;209(4):562-570.
Background. Marburg virus (MARV) infection causes severe morbidity and mortality in humans and nonhuman primates. Currently, there are no licensed therapeutics available for treating MARV infection. Here, we present the in vitro development and in vivo evaluation of lipid-encapsulated small interfering RNA (siRNA) as a potential therapeutic for the treatment of MARV infection.
Methods. The activity of anti-MARV siRNAs was assessed using dual luciferase reporter assays followed by in vitro testing against live virus. Lead candidates were tested in lethal guinea pig models of 3 different MARV strains (Angola, Ci67, Ravn).
Results. Treatment resulted in 60%–100% survival of guinea pigs infected with MARV. Although treatment with siRNA targeting other MARV messenger RNA (mRNA) had a beneficial effect, targeting the MARV NP mRNA resulted in the highest survival rates. NP-718m siRNA in lipid nanoparticles provided 100% protection against MARV strains Angola and Ci67, and 60% against Ravn. A cocktail containing NP-718m and NP-143m provided 100% protection against MARV Ravn.
Conclusions. These data show protective efficacy against the most pathogenic Angola strain of MARV. Further development of the lipid nanoparticle technology has the potential to yield effective treatments for MARV infection.
doi:10.1093/infdis/jit465
PMCID: PMC3903369  PMID: 23990568
Marburg virus; filovirus; LNP; RNAi; siRNA; treatment; therapeutics; guinea pig
11.  Chikungunya Virus Infection Results in Higher and Persistent Viral Replication in Aged Rhesus Macaques Due to Defects in Anti-Viral Immunity 
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1–5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys.
Author Summary
Chikungunya virus (CHIKV) is a re-emerging Alphavirus that has caused recent massive outbreaks in the Indian Ocean region. In addition, outbreaks have been documented in Europe and elsewhere in the world, initiated by infected travelers returning to their homelands. The recent outbreak strains possess extended vector range and as such, raise the potential of CHIKV outbreaks in the Southeastern parts of the United States. In this study, we examined CHIKV immunity in adult and aged Rhesus macaques following infection with two different CHIKV strains (recent outbreak strain CHIKV-LR and a West African Strain CHIKV-37997). CHIKV-LR causes persistent infection in the aged animals and replicates, on average, to higher levels than CHIKV-37997. Irrespective of the viral strain used, aged animals had delayed and/or reduced immunity compared to adult animals. Our data support the clinical findings of CHIKV susceptibility in vulnerable populations including the aged and provide mechanistic evidence that an effective immune response directed against the virus is required for preventing persistent CHIKV infection.
doi:10.1371/journal.pntd.0002343
PMCID: PMC3723534  PMID: 23936572
12.  Transcriptional Profiling of the Circulating Immune Response to Lassa Virus in an Aerosol Model of Exposure 
Lassa virus (LASV) is a significant human pathogen that is endemic to several countries in West Africa. Infection with LASV leads to the development of hemorrhagic fever in a significant number of cases, and it is estimated that thousands die each year from the disease. Little is known about the complex immune mechanisms governing the response to LASV or the genetic determinants of susceptibility and resistance to infection. In the study presented here, we have used a whole-genome, microarray-based approach to determine the temporal host response in the peripheral blood mononuclear cells (PBMCs) of non-human primates (NHP) following aerosol exposure to LASV. Sequential sampling over the entire disease course showed that there are strong transcriptional changes of the immune response to LASV exposure, including the early induction of interferon-responsive genes and Toll-like receptor signaling pathways. However, this increase in early innate responses was coupled with a lack of pro-inflammatory cytokine response in LASV exposed NHPs. There was a distinct lack of cytokines such as IL1β and IL23α, while immunosuppressive cytokines such as IL27 and IL6 were upregulated. Comparison of IRF/STAT1-stimulated gene expression with the viral load in LASV exposed NHPs suggests that mRNA expression significantly precedes viremia, and thus might be used for early diagnostics of the disease. Our results provide a transcriptomic survey of the circulating immune response to hemorrhagic LASV exposure and provide a foundation for biomarker identification to allow clinical diagnosis of LASV infection through analysis of the host response.
Author Summary
Lassa virus (LASV), a member of the Arenaviridae family, is a viral hemorrhagic fever causing virus endemic to several countries in West Africa with a history of sporadic importation into the United States. It has been characterized as a Category A agent, and despite the significant public health issues posed by LASV and the potential biodefense risks, little is known about the immune response to the virus. In the study presented here, we have taken an unbiased genomics approach to map the temporal host response in the peripheral blood mononuclear cells (PBMCs) of non-human primates (NHP) exposed to LASV. Gene expression patterns over the entire disease course showed that there are strong transcriptional changes of the immune response to LASV exposure, including the upregulation of Toll-like receptor signaling pathways and innate antiviral transcription factors. However, there was a lack of pro-inflammatory cytokine response in LASV exposed NHPs similar to what is seen in human disease. Our data suggests that LASV induces negative regulation of immunological events, leading to an inefficient adaptive immune response as observed in LASV-infected human patients. Our results provide a picture of the host's circulating immune response to hemorrhagic LASV exposure and demonstrate that gene expression patterns correlate with specific stages of disease progression.
doi:10.1371/journal.pntd.0002171
PMCID: PMC3636129  PMID: 23638192
13.  A Hendra Virus G Glycoprotein Subunit Vaccine Protects African Green Monkeys from Nipah Virus Challenge 
Science translational medicine  2012;4(146):146ra107.
In the 1990s, Hendra virus and Nipah virus (NiV), two closely related and previously unrecognized paramyxoviruses that cause severe disease and death in humans and a variety of animals, were discovered in Australia and Malaysia, respectively. Outbreaks of disease have occurred nearly every year since NiV was first discovered, with case fatality ranging from 10 to 100%. In the African green monkey (AGM), NiV causes a severe lethal respiratory and/or neurological disease that essentially mirrors fatal human disease. Thus, the AGM represents a reliable disease model for vaccine and therapeutic efficacy testing. We show that vaccination of AGMs with a recombinant subunit vaccine based on the henipavirus attachment G glycoprotein affords complete protection against subsequent NiV infection with no evidence of clinical disease, virus replication, or pathology observed in any challenged subjects. Success of the recombinant subunit vaccine in nonhuman primates provides crucial data in supporting its further preclinical development for potential human use.
doi:10.1126/scitranslmed.3004241
PMCID: PMC3516289  PMID: 22875827
14.  Comparison of the Pathogenicity of Nipah Virus Isolates from Bangladesh and Malaysia in the Syrian Hamster 
Nipah virus is a zoonotic pathogen that causes severe disease in humans. The mechanisms of pathogenesis are not well described. The first Nipah virus outbreak occurred in Malaysia, where human disease had a strong neurological component. Subsequent outbreaks have occurred in Bangladesh and India and transmission and disease processes in these outbreaks appear to be different from those of the Malaysian outbreak. Until this point, virtually all Nipah virus studies in vitro and in vivo, including vaccine and pathogenesis studies, have utilized a virus isolate from the original Malaysian outbreak (NiV-M). To investigate potential differences between NiV-M and a Nipah virus isolate from Bangladesh (NiV-B), we compared NiV-M and NiV-B infection in vitro and in vivo. In hamster kidney cells, NiV-M-infection resulted in extensive syncytia formation and cytopathic effects, whereas NiV-B-infection resulted in little to no morphological changes. In vivo, NiV-M-infected Syrian hamsters had accelerated virus replication, pathology and death when compared to NiV-B-infected animals. NiV-M infection also resulted in the activation of host immune response genes at an earlier time point. Pathogenicity was not only a result of direct effects of virus replication, but likely also had an immunopathogenic component. The differences observed between NiV-M and NiV-B pathogeneis in hamsters may relate to differences observed in human cases. Characterization of the hamster model for NiV-B infection allows for further research of the strain of Nipah virus responsible for the more recent outbreaks in humans. This model can be used to study NiV-B pathogenesis, transmission, and countermeasures that could be used to control outbreaks.
Author Summary
Nipah virus causes severe disease in humans and outbreaks have occurred in two geographic regions, Malaysia and Bangladesh, and viruses have been isolated during outbreaks from both of these regions (NiV-M and NiV-B, respectively). The original outbreak of Nipah virus occurred in Malaysia and caused severe encephalitis in humans. All subsequent outbreaks of Nipah virus have occurred in Bangladesh or India and disease has been characterized as having a strong respiratory component. Nipah virus is a public health concern that can cause up to 100% lethality in humans and there is no approved treatment or vaccine. Current research should focus on understanding disease progression and pathogenicity. We compared NiV-M and NiV-B infection and disease progression using the Syrian hamster model. We found that NiV-M is more destructive in cultured hamster cells and has faster onset of cytopathogenicity compared to NiV-B. This is also true in hamsters, where although both viruses are pathogenic and cause a similar disease, pathology caused by NiV-M infection is accelerated. These data show that there is a difference in disease progression between the two strains of Nipah virus and will allow for a more detailed understanding of the events leading to disease caused by these viruses.
doi:10.1371/journal.pntd.0002024
PMCID: PMC3547834  PMID: 23342177
15.  Cathepsin B & L Are Not Required for Ebola Virus Replication 
Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB−/− and catL−/− mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.
Author Summary
It is currently believed that Ebola virus (EBOV) enters cells via macropinocytosis following which, the cysteine proteases cathepsin B and L (CatB, CatL) cleave the viral glycoprotein (GP) allowing exposure of its core receptor-binding and fusion domain thus facilitating subsequent infection. We studied the effect of CatB and CatL on in vitro and in vivo EBOV replication. Our results demonstrate a reduction of Zaire ebolavirus (ZEBOV) entry upon selective inhibition of CatB, but not CatL in cell culture. Interestingly, all other EBOV species enter the cells efficiently when CatB and/or CatL activity is blocked. Moreover, when wild-type (control), catB−/− and catL−/− mice were infected with a lethal dose of mouse-adapted ZEBOV, all animals were equally susceptible to lethal challenge with no difference in virus replication and time to death. Therefore, we conclude that EBOV replication is dispensable of CatB and CatL, and proteolytic processing of GP can also be mediated by other endosomal proteases.
doi:10.1371/journal.pntd.0001923
PMCID: PMC3516577  PMID: 23236527
16.  Vesicular Stomatitis Virus–Based Ebola Vaccines With Improved Cross-Protective Efficacy 
The Journal of Infectious Diseases  2011;204(Suppl 3):S1066-S1074.
For Ebola virus (EBOV), 4 different species are known: Zaire, Sudan, Côte d’Ivoire, and Reston ebolavirus. The newly discovered Bundibugyo ebolavirus has been proposed as a 5th species. So far, no cross-neutralization among EBOV species has been described, aggravating progress toward cross-species protective vaccines. With the use of recombinant vesicular stomatitis virus (rVSV)–based vaccines, guinea pigs could be protected against Zaire ebolavirus (ZEBOV) infection only when immunized with a vector expressing the homologous, but not a heterologous, EBOV glycoprotein (GP). However, infection of guinea pigs with nonadapted wild-type strains of the different species resulted in full protection of all animals against subsequent challenge with guinea pig–adapted ZEBOV, showing that cross-species protection is possible. New vectors were generated that contain EBOV viral protein 40 (VP40) or EBOV nucleoprotein (NP) as a second antigen expressed by the same rVSV vector that encodes the heterologous GP. After applying a 2-dose immunization approach, we observed an improved cross-protection rate, with 5 of 6 guinea pigs surviving the lethal ZEBOV challenge if vaccinated with rVSV-expressing SEBOV-GP and -VP40. Our data demonstrate that cross-protection between the EBOV species can be achieved, although EBOV-GP alone cannot induce the required immune response.
doi:10.1093/infdis/jir348
PMCID: PMC3203393  PMID: 21987743
17.  Recombinant Vesicular Stomatitis Virus–Based Vaccines Against Ebola and Marburg Virus Infections 
The Journal of Infectious Diseases  2011;204(Suppl 3):S1075-S1081.
The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with a high mortality rate in humans and nonhuman primates. Among the most-promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Importantly, a single injection of blended rVSV-based filovirus vaccines was shown to completely protect nonhuman primates against Marburg virus and 3 different species of Ebola virus. These rVSV-based vaccines have also shown utility when administered as a postexposure treatment against filovirus infections, and a rVSV-based Ebola virus vaccine was recently used to treat a potential laboratory exposure. Here, we review the history of rVSV-based vaccines and pivotal animal studies showing their utility in combating Ebola and Marburg virus infections.
doi:10.1093/infdis/jir349
PMCID: PMC3218670  PMID: 21987744
18.  Single Immunization With a Monovalent Vesicular Stomatitis Virus–Based Vaccine Protects Nonhuman Primates Against Heterologous Challenge With Bundibugyo ebolavirus 
The Journal of Infectious Diseases  2011;204(Suppl 3):S1082-S1089.
The recombinant vesicular stomatitis virus (rVSV) vector-based monovalent vaccine platform expressing a filovirus glycoprotein has been demonstrated to provide protection from lethal challenge with Ebola (EBOV) and Marburg (MARV) viruses both prophylactically and after exposure. This platform provides protection between heterologous strains within a species; however, protection from lethal challenge between species has been largely unsuccessful. To determine whether the rVSV-EBOV vaccines have the potential to provide protection against a newly emerging, phylogenetically related species, cynomolgus macaques were vaccinated with an rVSV vaccine expressing either the glycoprotein of Zaire ebolavirus (ZEBOV) or Côte d’Ivoire ebolavirus (CIEBOV) and then challenged with Bundibugyo ebolavirus (BEBOV), which was recently proposed as a new EBOV species following an outbreak in Uganda in 2007. A single vaccination with the ZEBOV–specific vaccine provided cross-protection (75% survival) against subsequent BEBOV challenge, whereas vaccination with the CIEBOV–specific vaccine resulted in an outcome similar to mock-immunized animals (33% and 25% survival, respectively). This demonstrates that monovalent rVSV-based vaccines may be useful against a newly emerging species; however, heterologous protection across species remains challenging and may depend on enhancing the immune responses either through booster immunizations or through the inclusion of multiple immunogens.
doi:10.1093/infdis/jir350
PMCID: PMC3189995  PMID: 21987745
19.  Severe Hemorrhagic Fever in Strain 13/N Guinea Pigs Infected with Lujo Virus 
Lujo virus (LUJV) is a novel member of the Arenaviridae family that was first identified in 2008 after an outbreak of severe hemorrhagic fever (HF). In what was a small but rapidly progressing outbreak, this previously unknown virus was transmitted from the critically ill index patient to 4 attending healthcare workers. Four persons died during this outbreak, for a total case fatality of 80% (4/5). The suspected rodent source of the initial exposure to LUJV remains a mystery. Because of the ease of transmission, high case fatality, and novel nature of LUJV, we sought to establish an animal model of LUJV HF. Initial attempts in mice failed, but infection of inbred strain 13/N guinea pigs resulted in lethal disease. A total of 41 adult strain 13/N guinea pigs were infected with either wild-type LUJV or a full-length recombinant LUJV. Results demonstrated that strain 13/N guinea pigs provide an excellent model of severe and lethal LUJV HF that closely resembles what is known of the human disease. All infected animals experienced consistent weight loss (3–5% per day) and clinical illness characterized by ocular discharge, ruffled fur, hunched posture, and lethargy. Uniform lethality occurred by 11–16 days post-infection. All animals developed disseminated LUJV infection in various organs (liver, spleen, lung, and kidney), and leukopenia, lymphopenia, thrombocytopenia, coagulopathy, and elevated transaminase levels. Serial euthanasia studies revealed a temporal pattern of virus dissemination and increasing severity of disease, primarily targeting the liver, spleen, lungs, and lower gastrointestinal tract. Establishing an animal LUJV model is an important first step towards understanding the high pathogenicity of LUJV and developing vaccines and antiviral therapeutic drugs for this highly transmissible and lethal emerging pathogen.
Author Summary
The pathogenic arenaviruses are a diverse group of human pathogens capable of causing a wide range of human illness ranging from encephalitis to severe hemorrhagic fever throughout the New and Old World. In 2008, a previously unknown virus (now named Lujo virus) caused a high case fatality outbreak (80%) in southern Africa. Limited data available from these patients indicated that LUJV HF was characterized by thrombocytopenia, elevated liver transaminases, coagulopathy, viral antigen in multiple tissues, neurological symptoms in some cases, and eventual death. The source of exposure of the index patient remains unknown. Due to the unusually high lethality and rapid human to human spread, we sought to develop an animal model of Lujo hemorrhagic fever. We report here that after infection with Lujo virus, Strain 13/N guinea pigs develop a hemorrhagic fever syndrome similar to the disease observed in human patients. This animal model of severe Lujo hemorrhagic fever is a critical first step to increase our understanding of this highly pathogenic virus, and to develop anti-viral therapeutics or experimental vaccines for this new and unique threat to human health.
doi:10.1371/journal.pntd.0001801
PMCID: PMC3429401  PMID: 22953019
20.  Vesicular stomatitis virus-based vaccines protect nonhuman primates against aerosol challenge with Ebola and Marburg viruses 
Vaccine  2008;26(52):6894-6900.
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.
doi:10.1016/j.vaccine.2008.09.082
PMCID: PMC3398796  PMID: 18930776
Ebola virus; Marburg virus; Filovirus; Nonhuman primates; Aerosol; Vaccines
21.  Progress in filovirus vaccine development: evaluating the potential for clinical use 
Expert Review of Vaccines  2011;10(1):63-77.
Marburg and Ebola viruses cause severe hemorrhagic fever in humans and nonhuman primates. Currently, there are no effective treatments and no licensed vaccines; although a number of vaccine platforms have proven successful in animal models. The ideal filovirus vaccine candidate should be able to provide rapid protection following a single immunization, have the potential to work postexposure and be cross-reactive or multivalent against all Marburg virus strains and all relevant Ebola virus species and strains. Currently, there are multiple platforms that have provided prophylactic protection in nonhuman primates, including DNA, recombinant adenovirus serotype 5, recombinant human parainfluenza virus 3 and virus-like particles. In addition, a single platform, recombinant vesicular stomatitis virus, has demonstrated both prophylactic and postexposure protection in nonhuman primates. These results demonstrate that achieving a vaccine that is protective against filoviruses is possible; the challenge now is to prove its safety and efficacy in order to obtain a vaccine that is ready for human use.
doi:10.1586/erv.10.152
PMCID: PMC3398800  PMID: 21162622
Ebola virus; filovirus; Marburg virus; postexposure; prophylactic; vaccine
22.  Prospects for immunisation against Marburg and Ebola viruses 
Reviews in Medical Virology  2010;20(6):344-357.
SUMMARY
For more than 30 years the filoviruses, Marburg virus and Ebola virus, have been associated with periodic outbreaks of hemorrhagic fever that produce severe and often fatal disease. The filoviruses are endemic primarily in resource-poor regions in Central Africa and are also potential agents of bioterrorism. Although no vaccines or antiviral drugs for Marburg or Ebola are currently available, remarkable progress has been made over the last decade in developing candidate preventive vaccines against filoviruses in nonhuman primate models. Due to the generally remote locations of filovirus outbreaks, a single-injection vaccine is desirable. Among the prospective vaccines that have shown efficacy in nonhuman primate models of filoviral hemorrhagic fever, two candidates, one based on a replication-defective adenovirus serotype 5 and the other on a recombinant VSV (rVSV), were shown to provide complete protection to nonhuman primates when administered as a single injection. The rVSV-based vaccine has also shown utility when administered for postexposure prophylaxis against filovirus infections. A VSV-based Ebola vaccine was recently used to manage a potential laboratory exposure.
doi:10.1002/rmv.661
PMCID: PMC3394174  PMID: 20658513
23.  Ebola and Marburg Hemorrhagic Fevers: Neglected Tropical Diseases? 
Ebola hemorrhagic fever (EHF) and Marburg hemorrhagic fever (MHF) are rare viral diseases, endemic to central Africa. The overall burden of EHF and MHF is small in comparison to the more common protozoan, helminth, and bacterial diseases typically referred to as neglected tropical diseases (NTDs). However, EHF and MHF outbreaks typically occur in resource-limited settings, and many aspects of these outbreaks are a direct consequence of impoverished conditions. We will discuss aspects of EHF and MHF disease, in comparison to the “classic” NTDs, and examine potential ways forward in the prevention and control of EHF and MHF in sub-Saharan Africa, as well as examine the potential for application of novel vaccines or antiviral drugs for prevention or control of EHF and MHF among populations at highest risk for disease.
doi:10.1371/journal.pntd.0001546
PMCID: PMC3385614  PMID: 22761967
24.  Junín Virus Infection Activates the Type I Interferon Pathway in a RIG-I-Dependent Manner 
Junín virus (JUNV), an arenavirus, is the causative agent of Argentine hemorrhagic fever, an infectious human disease with 15–30% case fatality. The pathogenesis of AHF is still not well understood. Elevated levels of interferon and cytokines are reported in AHF patients, which might be correlated to the severity of the disease. However the innate immune response to JUNV infection has not been well evaluated. Previous studies have suggested that the virulent strain of JUNV does not induce IFN in human macrophages and monocytes, whereas the attenuated strain of JUNV was found to induce IFN response in murine macrophages via the TLR-2 signaling pathway. In this study, we investigated the interaction between JUNV and IFN pathway in human epithelial cells highly permissive to JUNV infection. We have determined the expression pattern of interferon-stimulated genes (ISGs) and IFN-β at both mRNA and protein levels during JUNV infection. Our results clearly indicate that JUNV infection activates the type I IFN response. STAT1 phosphorylation, a downstream marker of activation of IFN signaling pathway, was readily detected in JUNV infected IFN-competent cells. Our studies also demonstrated for the first time that RIG-I was required for IFN production during JUNV infection. IFN activation was detected during infection by either the virulent or attenuated vaccine strain of JUNV. Curiously, both virus strains were relatively insensitive to human IFN treatment. Our studies collectively indicated that JUNV infection could induce host type I IFN response and provided new insights into the interaction between JUNV and host innate immune system, which might be important in future studies on vaccine development and antiviral treatment.
Author Summary
Junín virus (JUNV), which is endemic to the Argentinean Pampas region, is the causative agent of Argentine hemorrhagic fever (AHF), a severe illness with hemorrhagic and neurological manifestations and with a case fatality of 15–30%. Clinical studies demonstrate that elevated levels of interferon and cytokines are produced in AHF patients, which might be correlated to the severity of disease. However it remains unclear, especially during virus infection, how human cells can sense virus infection and respond by activation of IFN pathway. Our studies clearly demonstrated that JUNV infection could activate type I IFN response in human cells. IFN pathway activation occurred in cells infected with either virulent strain or attenuated vaccine strain of JUNV. Our data also revealed for the first time that RIG-I was required for type I IFN production during virus infection in human cells. Interestingly, both strains of JUNV were relatively insensitive to human IFN treatment, which might have implications for the role of the IFN on virus infection in vivo. Overall, these results indicate that JUNV infection could induce host IFN response and provide new insights into JUNV and host interaction as well as the mechanism underlying AHF.
doi:10.1371/journal.pntd.0001659
PMCID: PMC3358329  PMID: 22629479
25.  High Diversity of Rabies Viruses Associated with Insectivorous Bats in Argentina: Presence of Several Independent Enzootics 
Background
Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites. Rabies virus (RABV) circulates through two different epidemiological cycles: terrestrial and aerial, where dogs, foxes or skunks and bats, respectively, act as the most relevant reservoirs and/or vectors. It is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory.
Methods
We studied the positivity rate of RABV detection in different areas of the country, and the antigenic and genetic diversity of 99 rabies virus (RABV) strains obtained from 14 species of insectivorous bats collected in Argentina between 1991 and 2008.
Results
Based on the analysis of bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats ranged from 3.1 to 5.4%, depending on the geographic location. The findings were distributed among an extensive area of the Argentinean territory. The 99 strains of insectivorous bat-related sequences were divided into six distinct lineages associated with Tadarida brasiliensis, Myotis spp, Eptesicus spp, Histiotus montanus, Lasiurus blosseviilli and Lasiurus cinereus. Comparison with RABV sequences obtained from insectivorous bats of the Americas revealed co-circulation of similar genetic variants in several countries. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples.
Conclusions
This study demonstrates the presence of several independent enzootics of rabies in insectivorous bats of Argentina. This information is relevant to identify potential areas at risk for human and animal infection.
Author Summary
In Argentina, successful vaccination and control of terrestrial rabies in the 1980s revealed the importance of the aerial route in RABV transmission. Current distribution of cases shows a predominance of rabies by hematophagous bats in the Northern regions where rabies is a major public health concern; in contrast, in Central and Southern regions where rabies is not a major public health concern, little surveillance is performed. Based on the analysis of insectivorous bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats in these regions ranged from 3.1 to 5.4%. This rate is comparable to other nations such as the United States (9–10%) where insectivorous bats are an important cause of concern for RABV surveillance systems. Antigenic and genetic analysis of a wide collection of rabies strains shows the presence of multiple endemic cycles associated with six bat insectivorous species distributed among an extensive area of the Argentinean territory and several countries of the Americas. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples. Increased public education about the relationship between insectivorous bats and rabies are essential to avoid human cases and potential spread to terrestrial mammals.
doi:10.1371/journal.pntd.0001635
PMCID: PMC3348165  PMID: 22590657

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