Gene Transfer of ZMapp Antibodies Mediated by Recombinant Adeno-Associated Virus Protects Against Ebola Infections.

Vectored delivery of the ZMapp antibody cocktail (c2G4, c4G7, and c13C6) by using recombinant adeno-associated viruses (rAAVs) could be useful for preventive immunization against Ebola virus infections because rAAVs can generate long-term antibody expression. Three rAAVs (serotype 9) encoding chimeric ZMapp antibodies were produced by triple-plasmid transfection up to 10 L-scale in WAVE bioreactors using HEK293 cells grown in suspension/serum-free conditions. Efficacy of AAV-c2G4 via intravenous (i.v.), intramuscular (i.m.), and intranasal (i.n.) routes of administration was evaluated in mice with two different doses of 2.7 × 1010 and 13.0 × 1010 vector genomes (vg). The best protective efficacies after Ebola challenge were obtained with the i.v. and i.m. routes. Serum concentrations of ZMapp antibodies positively correlated with survivability. Efficacy of the rAAV-ZMapp cocktail was then evaluated at a higher dose of 30.0 × 1010 vg. It conferred a more robust protection (90% i.v. and 60% i.m.) than rAAV-c4G7 (30%) and rAAV-c13C6 (70%), both administered separately at the same dose. Delivery of rAAV-c2G4 alone achieved up to 100% protection (100% i.v. and 90% i.m.) at the same dose. In conclusion, the preventive treatment was effective in mice. However, no advantage was observed for using the rAAV-ZMapp cocktail in comparison to the utilization of the single rAAV-c2G4.

Development of an HIV vaccine using a vesicular stomatitis virus vector expressing designer HIV-1 envelope glycoproteins to enhance humoral responses.

Vesicular stomatitis virus (VSV), like many other Rhabdoviruses, have become the focus of intense research over the past couple of decades based on their suitability as vaccine vectors, transient gene delivery systems, and as oncolytic viruses for cancer therapy. VSV as a vaccine vector platform has multiple advantages over more traditional viral vectors including low level, non-pathogenic replication in diverse cell types, ability to induce both humoral and cell-mediate immune responses, and the remarkable expression of foreign proteins cloned into multiple intergenic sites in the VSV genome. The utility and safety of VSV as a vaccine vector was recently demonstrated near the end of the recent Ebola outbreak in West Africa where VSV pseudotyped with the Ebola virus (EBOV) glycoprotein was proven safe in humans and provided protective efficacy against EBOV in a human phase III clinical trial. A team of Canadian scientists, led by Dr. Gary Kobinger, is now working with International AIDS Vaccine Initiative (IAVI) in developing a VSV-based HIV vaccine that will combine unique Canadian research on the HIV-1 Env glycoprotein and on the VSV vaccine vector. The goal of this collaboration is to develop a vaccine with a robust and potent anti-HIV immune response with an emphasis on generating quality antibodies to protect against HIV challenges.

Assessing the safety and immunogenicity of recombinant vesicular stomatitis virus Ebola vaccine in healthy adults: a randomized clinical trial.

BACKGROUND: The 2013-2016 Ebola virus outbreak in West Africa was the most widespread in history. In response, alive attenuated recombinant vesicular stomatitis virus (rVSV) vaccine expressing Zaire Ebolavirus glycoprotein (rVSVΔG-ZEBOV-GP) was evaluated in humans. METHODS: In a phase 1, randomized, dose-ranging, observer-blind, placebo-controlled trial, healthy adults aged 18-65 years were randomized into 4 groups of 10 to receive one of 3 vaccine doses or placebo. Follow-up visits spanned 180 days postvaccination for safety monitoring, immunogenicity testing and any rVSV virus shedding. RESULTS: Forty participants were injected with rVSVΔG-ZEBOV-GP vaccine (n = 30) or saline placebo (n = 10). No serious adverse events related to the vaccine or participant withdrawals were reported. Solicited adverse events during the 14-day follow-up period were mild to moderate and self-limited, with the exception of injection-site pain and headache. Viremia following vaccination was transient and no longer detectable after study day 3, with no virus shedding in saliva or urine. All vaccinated participants developed serum immunoglobulin G (IgG), as measured by Ebola virus envelope glycoprotein-based enzyme-linked immunosorbent assay (ELISA). Immunogenicity was comparable across all dose groups, and sustained IgG titers were detectable through to the last visit, at study day 180. INTERPRETATION: In this phase 1 study, there were no safety concerns after a single dose of rVSVΔG-ZEBOV-GP vaccine. IgG ELISA showed persistent high titers at 180 days postimmunization. There was a period of reactogenicity, but in general, the vaccine was well tolerated. This study provides evidence of the safety and immunogenicity of rVSVΔG-ZEBOV-GP vaccine and importance of its further investigation. Trial registration: Clinical-Trials.gov no., NCT02374385.

DNA vaccination protects mice against Zika virus-induced damage to the testes.

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract.

Adeno-Associated Virus Serotype 9-Expressed ZMapp in Mice Confers Protection Against Systemic and Airway-Acquired Ebola Virus Infection.

Adeno-associated viral vectors can be used as a platform for delivering biological countermeasures against pandemic and biological threats. We show that vector delivery of two antibody components of the ZMapp product is effective in mice against systemic and airway challenge with a mouse-adapted strain of Ebola virus. This platform provides a generic manufacturing solution and overcomes some of the delivery challenges associated with repeated administration of the protective protein.

A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogs as Effective Inhibitors of Ebola Virus.

To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy in vitro and/or in non-human primate studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles (trVLPs), requiring only level 2 biosafety containment, we compared the activities of the type I interferons (IFNs) IFN-α and IFN-ß, a panel of viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF)), and the estrogen receptor modulator, toremifene (TOR), in inhibiting viral replication in dose-response and time course studies. We also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-ß inhibited viral replication 24 hours post-infection (IC50 0.038µM and 0.016µM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50-62%) or in combination (87%). They exhibited lower IC50 (0.98-6.2µM) compared with FPV (36.8µM), when administered 24 hours post-infection. Unexpectedly, CDF had a narrow therapeutic window (6.25-25µM). When dosed >50µM, CDF treatment enhanced viral infection. IFN-ß exhibited strong synergy with 3TC (97.3% inhibition) or in triple combination with 3TC and AZT (95.8% inhibition). This study demonstrates that IFNs and viral polymerase inhibitors may have utility in EVD. We identified several 2 and 3 drug combinations with strong anti-Ebola activity, confirmed in studies using fully infectious ZEBOV, providing a rationale for testing combination therapies in animal models of lethal Ebola challenge. These studies open up new possibilities for novel therapeutic options, in particular combination therapies, which could prevent and treat Ebola infection and potentially reduce drug resistance.

Adenovirus-Vectored Vaccine Provides Postexposure Protection to Ebola Virus-Infected Nonhuman Primates.

Ebola virus (EBOV) causes lethal disease in up to 90% of EBOV-infected humans. Among vaccines, only the vesicular stomatitis virus platform has been successful in providing postexposure protection in nonhuman primates. Here, we show that an adjuvanted human adenovirus serotype 5 (Ad5)-vectored vaccine (Ad5-Zaire EBOV glycoprotein) protected 67% (6 of 9) and 25% (1 of 4) of cynomolgus macaques when administered 30 minutes and 24 hours following EBOV challenge, respectively. The treatment also protected 33% of rhesus macaques (1 of 3) when given at 24 hours. The results highlight the utility of adjuvanted Ad5 vaccines for rapid immunization against EBOV.

Enhanced fusion pore expansion mediated by the trans-acting Endodomain of the reovirus FAST proteins.

The reovirus fusion-associated small transmembrane (FAST) proteins are virus-encoded membrane fusion proteins that function as dedicated cell-cell fusogens. The topology of these small, single-pass membrane proteins orients the majority of the protein on the distal side of the membrane (i.e., inside the cell). We now show that ectopic expression of the endodomains of the p10, p14, and p15 FAST proteins enhances syncytiogenesis induced by the full-length FAST proteins, both homotypically and heterotypically. Results further indicate that the 68-residue cytoplasmic endodomain of the p14 FAST protein (1) is endogenously generated from full-length p14 protein expressed in virus-infected or transfected cells; (2) enhances syncytiogenesis subsequent to stable pore formation; (3) increases the syncytiogenic activity of heterologous fusion proteins, including the differentiation-dependent fusion of murine myoblasts; (4) exerts its enhancing activity from the cytosol, independent of direct interactions with either the fusogen or the membranes being fused; and (5) contains several regions with protein-protein interaction motifs that influence enhancing activity. We propose that the unique evolution of the FAST proteins as virus-encoded cellular fusogens has allowed them to generate a trans-acting, soluble endodomain peptide to harness a cellular pathway or process involved in the poorly understood process that facilitates the transition from microfusion pores to macrofusion and syncytiogenesis.