Kelly L. Warfield

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.

Ebola virus-like particles protect from lethal Ebola virus infection

The filovirus Ebola causes hemorrhagic fever with 70–80% human mortality. High case-fatality rates, as well as known aerosol infectivity, make Ebola virus a potential global health threat and possible biological warfare agent. Development of an effective vaccine for use in natural outbreaks, response to biological attack, and protection of laboratory workers is a higher national priority than ever before. Coexpression of the Ebola virus glycoprotein (GP) and matrix protein (VP40) in mammalian cells results in spontaneous production and release of virus-like particles (VLPs) that resemble the distinctively filamentous infectious virions. VLPs have been tested and found efficacious as vaccines for several viruses, including papillomavirus, HIV, parvovirus, and rotavirus. Herein, we report that Ebola VLPs (eVLPs) were immunogenic in vitro as eVLPs matured and activated mouse bone marrow-derived dendritic cells, assessed by increases in cell-surface markers CD40, CD80, CD86, and MHC class I and II and secretion of IL-6, IL-10, macrophage inflammatory protein (MIP)-1α, and tumor necrosis factor α by the dendritic cells. Further, vaccinating mice with eVLPs activated CD4+ and CD8+ T cells, as well as CD19+ B cells. After vaccination with eVLPs, mice developed high titers of Ebola virus-specific antibodies, including neutralizing antibodies. Importantly, mice vaccinated with eVLPs were 100% protected from an otherwise lethal Ebola virus inoculation. Together, our data suggest that eVLPs represent a promising vaccine candidate for protection against Ebola virus infections and a much needed tool to examine the genesis and nature of immune responses to Ebola virus.

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.

Seasonal Pulses of Marburg Virus Circulation in Juvenile Rousettus aegyptiacus Bats Coincide with Periods of Increased Risk of Human Infection

Marburg virus (family Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ∼2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (∼six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide a basis for developing Marburg hemorrhagic fever risk reduction strategies.

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.

Conserved Receptor-binding Domains of Lake Victoria Marburgvirus and Zaire Ebolavirus Bind a Common Receptor

The GP1,2 envelope glycoproteins (GP) of filoviruses (marburg- and ebolaviruses) mediate cell-surface attachment, membrane fusion, and entry into permissive cells. Here we show that a 151-amino acid fragment of the Lake Victoria marburgvirus GP1 subunit bound filovirus-permissive cell lines more efficiently than full-length GP1. An homologous 148-amino acid fragment of the Zaire ebolavirus GP1 subunit similarly bound the same cell lines more efficiently than a series of longer GP1 truncation variants. Neither the marburgvirus GP1 fragment nor that of ebolavirus bound a nonpermissive lymphocyte cell line. Both fragments specifically inhibited replication of infectious Zaire ebolavirus, as well as entry of retroviruses pseudotyped with either Lake Victoria marburgvirus or Zaire ebolavirus GP1,2. These studies identify the receptor-binding domains of both viruses, indicate that these viruses utilize a common receptor, and suggest that a single small molecule or vaccine can be developed to inhibit infection of all filoviruses.

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.