Role of Antibodies in Protection Against Ebola Virus in Nonhuman Primates Immunized With Three Vaccine Platforms.

Background: Several vaccine platforms have been successfully evaluated for prevention of Ebola virus (EBOV) disease (EVD) in nonhuman primates and humans. Despite remarkable efficacy by multiple vaccines, the immunological correlates of protection against EVD are incompletely understood. Methods: We systematically evaluated the antibody response to various EBOV proteins in 79 nonhuman primates vaccinated with various EBOV vaccine platforms. We evaluated the serum immunoglobulin (Ig)G titers against EBOV glycoprotein (GP), the ability of the vaccine-induced antibodies to bind GP at acidic pH or to displace ZMapp, and virus neutralization titers. The correlation of these outcomes with survival from EVD was evaluated by appropriate statistical methods. Results: Irrespective of the vaccine platform, protection from EVD strongly correlated with anti-GP IgG titers. The GP-directed antibody levels required for protection in animals vaccinated with virus-like particles (VLPs) lacking nucleoprotein (NP) was significantly higher than animals immunized with NP-containing VLPs or adenovirus-expressed GP, platforms that induce strong T-cell responses. Furthermore, protective immune responses correlated with anti-GP antibody binding strength at acidic pH, neutralization of GP-expressing pseudovirions, and the ability to displace ZMapp components from GP. Conclusions: These findings suggest key quantitative and qualitative attributes of antibody response to EVD vaccines as potential correlates of protection.

AVI-7288 for Marburg Virus in Nonhuman Primates and Humans.

BACKGROUND: AVI-7288 is a phosphorodiamidate morpholino oligomer with positive charges that targets the viral messenger RNA that encodes Marburg virus (MARV) nucleoprotein. Its safety in humans is undetermined. METHODS: We assessed the efficacy of AVI-7288 in a series of studies involving a lethal challenge with MARV in nonhuman primates. The safety of AVI-7288 was evaluated in a randomized, multiple-ascending-dose study in which 40 healthy humans (8 humans per dose group) received 14 once-daily infusions of AVI-7288 (1 mg, 4 mg, 8 mg, 12 mg, or 16 mg per kilogram of body weight) or placebo, in a 3:1 ratio. We estimated the protective dose in humans by comparing pharmacokinetic variables in infected nonhuman primates, uninfected nonhuman primates, and uninfected humans. RESULTS: Survival in infected nonhuman primates was dose-dependent, with survival rates of 0%, 30%, 59%, 87%, 100%, and 100% among monkeys treated with 0 mg, 3.75 mg, 7.5 mg, 15 mg, 20 mg, and 30 mg of AVI-7288 per kilogram, respectively (P<0.001 with the use of the log-rank test for the comparison of survival across groups). No safety concern was identified at doses up to 16 mg per kilogram per day in humans. No serious adverse events were reported. Drug exposure (the area under the curve) was dose-dependent in both nonhuman primates and humans; drug clearance was independent of dose but was higher in nonhuman primates than in humans. The protective dose in humans was initially estimated, on the basis of exposure, to be 9.6 mg per kilogram per day (95% confidence interval, 6.6 to 12.5) for 14 days. Monte Carlo simulations supported a dose of 11 mg per kilogram per day to match the geometric mean protective exposure in nonhuman primates. CONCLUSIONS: This study shows that, on the basis of efficacy in nonhuman primates and pharmacokinetic data in humans, AVI-7288 has potential as postexposure prophylaxis for MARV infection in humans. (Funded by the Department of Defense; ClinicalTrials.gov number, NCT01566877.).

Role of natural killer cells in innate protection against lethal ebola virus infection.

Ebola virus is a highly lethal human pathogen and is rapidly driving many wild primate populations toward extinction. Several lines of evidence suggest that innate, nonspecific host factors are potentially critical for survival after Ebola virus infection. Here, we show that nonreplicating Ebola virus-like particles (VLPs), containing the glycoprotein (GP) and matrix protein virus protein (VP)40, administered 1-3 d before Ebola virus infection rapidly induced protective immunity. VLP injection enhanced the numbers of natural killer (NK) cells in lymphoid tissues. In contrast to live Ebola virus, VLP treatment of NK cells enhanced cytokine secretion and cytolytic activity against NK-sensitive targets. Unlike wild-type mice, treatment of NK-deficient or -depleted mice with VLPs had no protective effect against Ebola virus infection and NK cells treated with VLPs protected against Ebola virus infection when adoptively transferred to naive mice. The mechanism of NK cell-mediated protection clearly depended on perforin, but not interferon-gamma secretion. Particles containing only VP40 were sufficient to induce NK cell responses and provide protection from infection in the absence of the viral GP. These findings revealed a decisive role for NK cells during lethal Ebola virus infection. This work should open new doors for better understanding of Ebola virus pathogenesis and direct the development of immunotherapeutics, which target the innate immune system, for treatment of Ebola virus infection.

Chemical modifications of antisense morpholino oligomers enhance their efficacy against Ebola virus infection.

Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5' or the 3' terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.

Analysis of Ebola virus and VLP release using an immunocapture assay.

Ebola virus (EBOV), an emerging pathogen, is the causative agent of a rapidly progressive hemorrhagic fever with high mortality rates. There are currently no approved vaccines or treatments available for Ebola hemorrhagic fever. Standard plaque assays are currently the only reliable techniques for enumerating the virus. Effective drug-discovery screening as well as target identification and validation require simple and more rapid detection methods. This report describes the development of a rapid ELISA that measures virus release with high sensitivity. This assay detects both Ebola virus and EBOV-like particles (VLPs) directly from cell-culture supernatants with the VP40 matrix protein serving as antigen. Using this assay, the contribution of the EBOV nucleocapsid (NC) proteins in VLP release was determined. These findings indicate that a combination of NC proteins together with the envelope components is optimal for VLP formation and release, a finding that is important for vaccination with Ebola VLPs. Furthermore, this assay can be used in surrogate models in non-biocontainment environment, facilitating both basic research on the mechanism of EBOV assembly and budding as well as drug-discovery research.

Homologous and heterologous protection of nonhuman primates by Ebola and Sudan virus-like particles.

Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components.

Generation of Marburg virus-like particles by co-expression of glycoprotein and matrix protein.

Marburg virus (MARV), the causative agent of a severe hemorrhagic fever, has a characteristic filamentous morphology. Here we report that co-expression of MARV glycoprotein and matrix protein (VP40) in mammalian cells leads to spontaneous budding of filamentous particles strikingly similar to wild-type MARV. In addition, these particles elicit an immune response in BALB/c mice. The generation of non-replicating Marburg virus-like particles (VLPs) should significantly facilitate the research on molecular mechanisms of MARV assembly and release. Furthermore, VLPs may be an excellent vaccine candidate against Marburg infection.

Advanced antisense therapies for postexposure protection against lethal filovirus infections.

Currently, no vaccines or therapeutics are licensed to counter Ebola or Marburg viruses, highly pathogenic filoviruses that are causative agents of viral hemorrhagic fever. Here we show that administration of positively charged phosphorodiamidate morpholino oligomers (PMOplus), delivered by various dosing strategies initiated 30-60 min after infection, protects>60% of rhesus monkeys against lethal Zaire Ebola virus (ZEBOV) and 100% of cynomolgus monkeys against Lake Victoria Marburg virus (MARV) infection. PMOplus may be useful for treating these and other highly pathogenic viruses in humans.

Monovalent virus-like particle vaccine protects guinea pigs and nonhuman primates against infection with multiple Marburg viruses.

BACKGROUND: Virus-like particle (VLP)-based vaccines have the advantage of being morphologically and antigenically similar to the live virus from which they are derived. Expression of the glycoprotein and VP40 matrix protein from Lake Victoria marburgvirus (MARV) results in spontaneous production of VLPs in mammalian cells. Guinea pigs vaccinated with Marburg virus VLPs (mVLPs) or inactivated MARV (iMARV) develop homologous humoral and T-cell responses and are completely protected from a lethal homologous MARV challenge. AIMS & METHODS: To determine whether mVLPs based on the Musoke (aka Lake Victoria) isolate of MARV could broadly protect against diverse isolates of MARV, guinea pigs were vaccinated with mVLPs or iMARV-Musoke and challenged with MARV-Musoke, -Ravn or -Ci67. RESULTS: Prior to challenge, the mVLP- and iMARV-vaccinated guinea pigs had high levels of homologous MARV-Musoke and heterologous MARV-Ravn and -Ci67 antibodies. The Musoke-based mVLPs and iMARV vaccines provided complete protection in guinea pigs against viremia, viral replication and pathological changes in tissues, and lethal disease following challenge with MARV-Musoke, -Ravn or -Ci67. Guinea pigs vaccinated with RIBI adjuvant alone and infected with guinea pig-adapted MARV-Musoke, -Ravn or -Ci67 had histopathologic findings similar to those seen in the nonhuman primate model for MARV infection. Based on the strong protection observed in guinea pigs, we next vaccinated cynomolgus macaques with Musoke-based mVLPs and showed the VLP-vaccinated monkeys were broadly protected against three isolates of MARV (Musoke, Ravn and Ci67). CONCLUSION: Musoke mVLPs are effective at inducing broad heterologous immunity and protection against multiple MARV isolates.

Vaccine to confer to nonhuman primates complete protection against multistrain Ebola and Marburg virus infections.

Filoviruses (Ebola and Marburg viruses) are among the deadliest viruses known to mankind, with mortality rates nearing 90%. These pathogens are highly infectious through contact with infected body fluids and can be easily aerosolized. Additionally, there are currently no licensed vaccines available to prevent filovirus outbreaks. Their high mortality rates and infectious capabilities when aerosolized and the lack of licensed vaccines available to prevent such infectious make Ebola and Marburg viruses serious bioterrorism threats, placing them both on the category A list of bioterrorism agents. Here we describe a panfilovirus vaccine based on a complex adenovirus (CAdVax) technology that expresses multiple antigens from five different filoviruses de novo. Vaccination of nonhuman primates demonstrated 100% protection against infection by two species of Ebola virus and three Marburg virus subtypes, each administered at 1,000 times the lethal dose. This study indicates the feasibility of vaccination against all current filovirus threats in the event of natural hemorrhagic fever outbreak or biological attack.

Ebola virus-like particle-based vaccine protects nonhuman primates against lethal Ebola virus challenge.

BACKGROUND: Currently, there are no licensed vaccines or therapeutics for the prevention or treatment of infection by the highly lethal filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), in humans. We previously had demonstrated the protective efficacy of virus-like particle (VLP)-based vaccines against EBOV and MARV infection in rodents. METHODS: To determine the efficacy of vaccination with Ebola VLPs (eVLPs) in nonhuman primates, we vaccinated cynomolgus macaques with eVLPs containing EBOV glycoprotein (GP), nucleoprotein (NP), and VP40 matrix protein and challenged the macaques with 1000 pfu of EBOV. RESULTS: Serum samples from the eVLP-vaccinated nonhuman primates demonstrated EBOV-specific antibody titers, as measured by enzyme-linked immunosorbent assay, complement-mediated lysis assay, and antibody-dependent cell-mediated cytotoxicity assay. CD44+ T cells from eVLP-vaccinated macaques but not from a naive macaque responded with vigorous production of tumor necrosis factor- alpha after EBOV-peptide stimulation. All 5 eVLP-vaccinated monkeys survived challenge without clinical or laboratory signs of EBOV infection, whereas the control animal died of infection. CONCLUSION: On the basis of safety and efficacy, eVLPs represent a promising filovirus vaccine for use in humans.

NKp30-dependent cytolysis of filovirus-infected human dendritic cells.

Understanding how protective innate immune responses are generated is crucial to defeating highly lethal emerging pathogens. Accumulating evidence suggests that potent innate immune responses are tightly linked to control of Ebola and Marburg filoviral infections. Here, we report that unlike authentic or inactivated Ebola and Marburg, filovirus-derived virus-like particles directly activated human natural killer (NK) cells in vitro, evidenced by pro-inflammatory cytokine production and enhanced cytolysis of permissive target cells. Further, we observed perforin- and CD95L-mediated cytolysis of filovirus-infected human dendritic cells (DCs), primary targets of filovirus infection, by autologous NK cells. Gene expression knock-down studies directly linked NK cell lysis of infected DCs to upregulation of the natural cytotoxicity receptor, NKp30. These results are the first to propose a role for NK cells in the clearance of infected DCs and the potential involvement of NKp30-mediated cytolysis in control of viral infection in vivo. Further elucidation of the biology of NK cell activation, specifically natural cytotoxicity receptors like NKp30 and NKp46, promises to aid our understanding of microbial pathology.

Involvement of vacuolar protein sorting pathway in Ebola virus release independent of TSG101 interaction.

Budding of Ebola virus (EBOV) particles from the plasma membrane of infected cells requires viral and host proteins. EBOV virus matrix protein VP40 recruits TSG101, an ESCRT-1 (host cell endosomal sorting complex required for transport-1) complex protein in the vacuolar protein sorting (vps) pathway, to the plasma membrane during budding. Involvement of other vps proteins in EBOV budding has not been established. Therefore, we used VP40 deletion analysis, virus-like particle-release assays, and confocal microscopy to investigate the potential role of ESCRT-1 proteins VPS4, VPS28, and VPS37B in EBOV budding. We found that VP40 could redirect each protein from endosomes to the cell surface independently of TSG101 interaction. A lack of VPS4 adenosine triphosphatase activity reduced budding by up to 80%. Inhibition of VPS4 gene expression by use of phosphorodiamidite morpholino antisense oligonucleotides protected mice from lethal EBOV infection. These data show that EBOV can use vps proteins independently of TSG101 for budding and reveal VPS4 as a potential target for filovirus therapeutics.

Ebola virus inactivation with preservation of antigenic and structural integrity by a photoinducible alkylating agent.

Current methods for inactivating filoviruses are limited to high doses of irradiation or formalin treatment, which may cause structural perturbations that are reflected by poor immunogenicity. In this report, we describe a novel inactivation technique for Zaire Ebola virus (ZEBOV) that uses the photoinduced alkylating probe 1,5-iodonaphthylazide (INA). INA is incorporated into lipid bilayers and, when activated by ultraviolet irradiation, alkylates the proteins therein. INA treatment of ZEBOV resulted in the complete loss of infectivity in cells. Results of electron microscopy and virus-capture assays suggested the preservation of conformational surface epitopes. Challenge with 50,000 pfu of INA-inactivated, mouse-adapted ZEBOV did not cause disease or death in mice. A single vaccination with INA-inactivated ZEBOV (equivalent to 5 x 10(4) pfu) protected mice against lethal challenge with 1000 pfu of ZEBOV. INA-inactivated virus induced a protective response in 100% of mice when administered 3 days before challenge. Thus, INA may have significant potential for the development of vaccines and immunotherapeutics for filoviruses and other enveloped viruses.

Filovirus-like particles produced in insect cells: immunogenicity and protection in rodents.

BACKGROUND: Virus-like particles (VLPs) of Ebola virus (EBOV) and Marburg virus (MARV) produced in human 293T embryonic kidney cells have been shown to be effective vaccines against filoviral infection. In this study, we explored alternative strategies for production of filovirus-like particle-based vaccines, to accelerate the development process. The goal of this work was to increase the yield of VLPs, while retaining their immunogenic properties. METHODS: Ebola and Marburg VLPs (eVLPs and mVLPs, respectively) were generated by use of recombinant baculovirus constructs expressing glycoprotein, VP40 matrix protein, and nucleoprotein from coinfected insect cells. The baculovirus-derived eVLPs and mVLPs were characterized biochemically, and then the immune responses produced by the eVLPs in insect cells were studied further. RESULTS: The baculovirus-derived eVLPs elicited maturation of human myeloid dendritic cells (DCs), indicating their immunogenic properties. Mice vaccinated with insect cell-derived eVLPs generated antibody and cellular responses equivalent to those vaccinated with mammalian 293T cell-derived eVLPs and were protected from EBOV challenge in a dose-dependent manner. CONCLUSION: Together, these data suggest that filovirus-like particles produced by baculovirus expression systems, which are amenable to large-scale production, are highly immunogenic and are suitable as safe and effective vaccines for the prevention of filoviral infection.

Gene-specific countermeasures against Ebola virus based on antisense phosphorodiamidate morpholino oligomers.

The filoviruses Marburg virus and Ebola virus (EBOV) quickly outpace host immune responses and cause hemorrhagic fever, resulting in case fatality rates as high as 90% in humans and nearly 100% in nonhuman primates. The development of an effective therapeutic for EBOV is a daunting public health challenge and is hampered by a paucity of knowledge regarding filovirus pathogenesis. This report describes a successful strategy for interfering with EBOV infection using antisense phosphorodiamidate morpholino oligomers (PMOs). A combination of EBOV-specific PMOs targeting sequences of viral mRNAs for the viral proteins (VPs) VP24, VP35, and RNA polymerase L protected rodents in both pre- and post-exposure therapeutic regimens. In a prophylactic proof-of-principal trial, the PMOs also protected 75% of rhesus macaques from lethal EBOV infection. The work described here may contribute to development of designer, "druggable" countermeasures for filoviruses and other microbial pathogens.

Activation of triggering receptor expressed on myeloid cells-1 on human neutrophils by marburg and ebola viruses.

Marburg virus (MARV) and Ebola virus (EBOV), members of the viral family Filoviridae, cause fatal hemorrhagic fevers in humans and nonhuman primates. High viral burden is coincident with inadequate adaptive immune responses and robust inflammatory responses, and virus-mediated dysregulation of early host defenses has been proposed. Recently, a novel class of innate receptors called the triggering receptors expressed in myeloid cells (TREM) has been discovered and shown to play an important role in innate inflammatory responses and sepsis. Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. A peptide specific to TREM-1 diminished the release of tumor necrosis factor alpha by filovirus-activated human neutrophils in vitro, and a soluble recombinant TREM-1 competitively inhibited the loss of cell surface TREM-1 that otherwise occurred on neutrophils exposed to filoviruses. These data imply direct activation of TREM-1 by filoviruses and also indicate that neutrophils may play a prominent role in the immune and inflammatory responses to filovirus infections.

VP35 knockdown inhibits Ebola virus amplification and protects against lethal infection in mice.

Phosphorodiamidate morpholino oligomers (PMO) are a class of uncharged single-stranded DNA analogs modified such that each subunit includes a phosphorodiamidate linkage and morpholine ring. PMO antisense agents have been reported to effectively interfere with the replication of several positive-strand RNA viruses in cell culture. The filoviruses, Marburg virus and Ebola virus (EBOV), are negative-strand RNA viruses that cause up to 90% lethality in human outbreaks. There is currently no commercially available vaccine or efficacious therapeutic for any filovirus. In this study, PMO conjugated to arginine-rich cell-penetrating peptide (P-PMO) and nonconjugated PMO were assayed for the ability to inhibit EBOV infection in cell culture and in a mouse model of lethal EBOV infection. A 22-mer P-PMO designed to base pair with the translation start site region of EBOV VP35 positive-sense RNA generated sequence-specific and time- and dose-dependent inhibition of EBOV amplification in cell culture. The same oligomer provided complete protection to mice when administered before or after an otherwise lethal infection of EBOV. A corresponding nonconjugated PMO, as well as nonconjugated truncated versions of 16 and 19 base residues, provided length-dependent protection to mice when administered prophylactically. Together, these data suggest that antisense PMO and P-PMO have the potential to control EBOV infection and are promising therapeutic candidates.

Filovirus-like particles as vaccines and discovery tools.

Ebola and Marburg viruses are members of the family Filoviridae, which cause severe hemorrhagic fevers in humans. Filovirus outbreaks have been sporadic, with mortality rates currently ranging from 30 to 90%. Unfortunately, there is no efficacious human therapy or vaccine available to treat disease caused by either Ebola or Marburg virus infection. Expression of the filovirus matrix protein, VP40, is sufficient to drive spontaneous production and release of virus-like particles (VLPs) that resemble the distinctively filamentous infectious virions. The addition of other filovirus proteins, including virion proteins (VP)24, 30 and 35 and glycoprotein, increases the efficiency of VLP production and results in particles containing multiple filovirus antigens. Vaccination with Ebola or Marburg VLPs containing glycoprotein and VP40 completely protects rodents from lethal challenge with the homologous virus. These candidate vaccines are currently being tested for immunogenicity and efficacy in nonhuman primates. Furthermore, the Ebola and Marburg VLPs are being used as a surrogate model to further understand the filovirus life cycle, with the goal of developing rationally designed vaccines and therapeutics. Thus, in addition to their use as a vaccine, VLPs are currently being used as tools to learn lessons about filovirus pathogenesis, immunology, replication and assembly requirements.

Virus-like particles exhibit potential as a pan-filovirus vaccine for both Ebola and Marburg viral infections.

A safe and effective pan-filovirus vaccine is highly desirable since the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) cause highly lethal disease typified by unimpeded viral replication and severe hemorrhagic fever. Previously, we showed that expression of the homologous glycoprotein (GP) and matrix protein VP40 from a single filovirus, either EBOV or MARV, resulted in formation of wild-type virus-like particles (VLPs) in mammalian cells. When used as a vaccine, the wild-type VLPs protected from homologous filovirus challenge. The aim of this work was to generate a multi-agent vaccine that would simultaneously protect against multiple and diverse members of the Filoviridae family. Our initial approach was to construct hybrid VLPs containing heterologous viral proteins, of EBOV and MARV, and test the efficacy of the hybrid VLPs in a guinea pig model. Our data indicate that vaccination with GP was required and sufficient to protect against a homologous filovirus challenge, as heterologous wild-type VLPs or hybrid VLPs that did not contain the homologous GP failed to protect. Alternately, we vaccinated guinea pigs with a mixture of wild-type Ebola and Marburg VLPs. Vaccination with a single dose of the multivalent VLP vaccine elicited strong immune responses to both viruses and protected animals against EBOV and MARV challenge. This work provides a critical foundation towards the development of a pan-filovirus vaccine that is safe and effective for use in primates and humans.