Dana L. Swenson

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.

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.

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.

Induction of Humoral and CD8+ T Cell Responses Are Required for Protection against Lethal Ebola Virus Infection

Ebola virus (EBOV)-like particles (eVLP), composed of the EBOV glycoprotein and matrix viral protein (VP)40 with a lipid membrane, are a highly efficacious method of immunization against EBOV infection. The exact requirements for immunity against EBOV infection are poorly defined at this time. The goal of this work was to determine the requirements for EBOV immunity following eVLP vaccination. Vaccination of BALB/c or C57BL/6 mice with eVLPs in conjunction with QS-21 adjuvant resulted in mixed IgG subclass responses, a Th1-like memory cytokine response, and protection from lethal EBOV challenge. Further, this vaccination schedule led to the generation of both CD4+ and CD8+ IFN-γ+ T cells recognizing specific peptides within glycoprotein and VP40. The transfer of both serum and splenocytes, but not serum or splenocytes alone, from eVLP-vaccinated mice conferred protection against lethal EBOV infection in these studies. B cells were required for eVLP-mediated immunity to EBOV because B cell-deficient mice vaccinated with eVLPs were not protected from lethal EBOV challenge. We also found that CD8+, but not CD4+, T cells are absolutely required for eVLP-mediated protection against EBOV infection. Further, eVLP-induced protective mechanisms were perforin-independent, but IFN-γ-dependent. Taken together, both EBOV-specific humoral and cytotoxic CD8+ T cell responses are critical to mediate protection against filoviruses following eVLP vaccination.

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-γ 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.

VP35 Knockdown Inhibits Ebola Virus Amplification and Protects against Lethal Infection in Mice

ABSTRACT 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.

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.

Vaccine to Confer to Nonhuman Primates Complete Protection Against Multistrain Ebola and Marburg Virus Infections

ABSTRACT 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.

Activation of Triggering Receptor Expressed on Myeloid Cells-1 on Human Neutrophils by Marburg and Ebola Viruses

ABSTRACT 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.

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.