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

Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication

Ebolavirus (EBOV) is an RNA virus that is known to cause severe hemorrhagic fever in humans and other primates. EBOV successfully enters and replicates in many cell types. This replication is dependent on the virus successfully coopting a number of cellular factors. Many of these factors are currently unidentified but represent potential targets for antiviral therapeutics. Here we show that cellular polyamines are critical for EBOV replication. We found that small-molecule inhibitors of polyamine synthesis block gene expression driven by the viral RNA-dependent RNA polymerase. Short hairpin RNA (shRNA) knockdown of the polyamine pathway enzyme spermidine synthase also resulted in reduced EBOV replication. These findings led us to further investigate spermidine, a polyamine that is essential for the hypusination of eukaryotic initiation factor 5A (eIF5A). Blocking the hypusination of eIF5A (and thereby inhibiting its function) inhibited both EBOV gene expression and viral replication. The mechanism appears to be due to the importance of hypusinated eIF5A for the accumulation of VP30, an essential component of the viral polymerase. The same reduction in hypusinated eIF5A did not alter the accumulation of other viral polymerase components. This action makes eIF5A function an important gate for proper EBOV polymerase assembly and function through the control of a single virus protein

Transcriptome Analysis of Circulating Immune Cell Subsets Highlight the Role of Monocytes in Zaire Ebola Virus Makona Pathogenesis

Existing models of Ebola virus disease (EVD) suggest antigen-presenting cells are initial targets of Zaire ebolavirus (ZEBOV). In vitro studies have shown that ZEBOV infection of monocytes and macrophages results in the production of inflammatory mediators, which may cause lymphocyte apoptosis. However, these findings have not been corroborated by in vivo studies. In this study, we report the first longitudinal analysis of transcriptional changes in purified monocytes, T-cells, and B-cells isolated from cynomolgus macaques following infection with ZEBOV-Makona. Our data reveal monocytes as one of the major immune cell subsets that supports ZEBOV replication in vivo. In addition, we report a marked increase in the transcription of genes involved in inflammation, coagulation, and vascular disease within monocytes, suggesting that monocytes contribute to EVD manifestations. Further, genes important for antigen presentation and regulation of immunity were downregulated, potentially subverting development of adaptive immunity. In contrast, lymphocytes, which do not support ZEBOV replication, showed transcriptional changes limited to a small number of interferon-stimulated genes (ISGs) and a failure to upregulate genes associated with an antiviral effector immune response. Collectively, these data suggest that ZEBOV-infected monocytes play a significant role in ZEBOV-Makona pathogenesis and strategies to suppress virus replication or modify innate responses to infection in these cells should be a priority for therapeutic intervention

Crimean-Congo hemorrhagic fever virus strains Hoti and Afghanistan cause viremia and mild clinical disease in cynomolgus monkeys.

BackgroundDevelopment of vaccines and therapies against Crimean-Congo hemorrhagic fever virus (CCHFV) have been hindered by the lack of immunocompetent animal models. Recently, a lethal nonhuman primate model based on the CCHFV Hoti strain was reported. CCHFV Hoti caused severe disease in cynomolgus monkeys with 75% lethality when given by the intravenous (i.v.) route.Methodology/principal findingsIn a series of experiments, eleven cynomologus monkeys were exposed i.v. to CCHFV Hoti and four macaques were exposed i.v. to CCHFV Afghanistan. Despite transient viremia and changes in clinical pathology such as leukopenia and thrombocytopenia developing in all 15 animals, all macaques survived to the study endpoint without developing severe disease.Conclusions/significanceWe were unable to attribute differences in the results of our study versus the previous report to differences in the CCHFV Hoti stock, challenge dose, origin, or age of the macaques. The observed differences are most likely the result of the outbred nature of macaques and low animal numbers often used by necessity and for ethical considerations in BSL-4 studies. Nonetheless, while we were unable to achieve severe disease or lethality, the CCHFV Hoti and Afghanistan macaque models are useful for screening medical countermeasures using biomarkers including viremia and clinical pathology to assess efficacy

IgG antibody response to rVSV-filovirus-GP vaccination.

<p>Reciprocal endpoint dilution titers for IgG against SEBOV GP (A), ZEBOV GP (B), and BEBOV GP (C) were determined from serum samples in each Group at 29, 8, and 0 days before challenge. Group 1 (PBS only control, black), Group 2 (rVSV-BEBOV-GP only, red), Group 3 (rVSV-SEBOV-GP plus rVSV-ZEBOV-GP, green), and Group 4 (rVSV-SEBOV-GP only, then rVSV-ZEBOV-GP 14 days post, yellow/blue). Red **, p<0.01 (Group 2 vs Group 4), Red ***, p<0.001 (Group 2 vs Group 4), Blue **, p<0.01 (Group 3 vs. Group 4), and Blue >, p>0.05 (Group 3 vs Group 4).</p

Clinical findings and viremia for NHPs challenged with BEBOV.

<p> 98C007 expired before sampling at day 10 could be achieved.</p><p>^a rVSV blend; rVSV-SEBOV-GP plus rVSV-ZEBOV-GP, rVSV Prime-boost; rVSV-SEBOV-GP first then rVSV-ZEBOV-GP 14 days after.</p><p>°F over baseline or at least 1.5°F over baseline and ≥103.5°F. Moderate rash refers to petechiae coverage of more than 20% of the skin. Lymphopenia and thrombocytopenia are defined by a ≥35% drop in numbers of lymphocytes and platelets, respectively. (ALP) alkaline phosphatase, (AST) aspartate aminotransferase, (BUN) blood urea nitrogen, (GGT) gamma glutamyltransferase: 2- to 3-fold increase,→; 4- to 5-fold increase, →→; >5 fold increase, →→→.^b Days after BEBOV challenge are in parentheses. Fever is defined as a temperature more than 2.5</p><p>^c No symptoms observed.</p><p>₁₀ PFU/ml<b>/</b>qRT-PCR positive (+) or negative (−). +, ≤5 log₁₀; ++, ≥6 log₁₀; +++, ≥7 log₁₀.^d Days after BEBOV challenge are in parentheses. Viral load for each day is depicted as: log</p

Use of Single-Injection Recombinant Vesicular Stomatitis Virus Vaccine to Protect Nonhuman Primates Against Lethal Nipah Virus Disease

Nipah virus (NiV) is a zoonotic pathogen that causes high case-fatality rates (CFRs) in humans. Two NiV strains have caused outbreaks: the Malaysia strain (NiVM), discovered in 1998–1999 in Malaysia and Singapore (≈40% CFR); and the Bangladesh strain (NiVB), discovered in Bangladesh and India in 2001 (≈80% CFR). Recently, NiVB in African green monkeys resulted in a more severe and lethal disease than NiVM. No NiV vaccines or treatments are licensed for human use. We assessed replication-restricted single-injection recombinant vesicular stomatitis vaccine NiV vaccine vectors expressing the NiV glycoproteins against NiVB challenge in African green monkeys. All vaccinated animals survived to the study endpoint without signs of NiV disease; all showed development of NiV F Ig, NiV G IgG, or both, as well as neutralizing antibody titers. These data show protective efficacy against a stringent and relevant NiVB model of human infection

Group outcomes of BEBOV challenge.

<p>(A) Kapplan-Meier survival curve for each Group post BEBOV challenge. (B) Clinical scores for each individual within each Group after BEBOV challenge. Group 1 (PBS only control, black), Group 2 (rVSV-BEBOV-GP only, red), Group 3 (rVSV-SEBOV-GP plus rVSV-ZEBOV-GP, green), and Group 4 (rVSV-SEBOV-GP only, then rVSV-ZEBOV-GP 14 days post, yellow/blue). The x-axis represents clinical scores from Day 0 to Day 14 post challenge for each individual animal to show disease progression.</p