M. Javad Aman

Mutation of Jak3 in a patient with SCID : essential role of Jak3 in lymphoid development

Males with X-linked severe combined immunodeficiency (XSCID) have defects in the common cytokine receptor γ chain (γc) gene that encodes a shared, essential component of the receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. The Janus family tyrosine kinase Jak3 is the only signaling molecule known to be associated with γc, so it was hypothesized that defects in Jak3 might cause an XSCID-like phenotype. A girl with immunological features indistinguishable from those of XSCID was therefore selected for analysis. An Epstein-Barr virus (EBV)-transformed cell line derived from her lymphocytes had normal γc expression but lacked Jak3 protein and had greatly diminished Jak3 messenger RNA. Sequencing revealed a different mutation on each allele: a single nucleotide insertion resulting in a frame shift and premature termination in the Jak3 JH4 domain and a nonsense mutation in the Jak3 JH2 domain. The lack of Jak3 expression correlated with impaired B cell signaling, as demonstrated by the inability of IL-4 to activate Stat6 in the EBV-transformed cell line from the patient. These observations indicate that the functions of γc are dependent on Jak3 and that Jak3 is essential for lymphoid development and signaling.

Lipid Raft Microdomains A Gateway for Compartmentalized Trafficking of Ebola and Marburg Viruses

Spatiotemporal aspects of filovirus entry and release are poorly understood. Lipid rafts act as functional platforms for multiple cellular signaling and trafficking processes. Here, we report the compartmentalization of Ebola and Marburg viral proteins within lipid rafts during viral assembly and budding. Filoviruses released from infected cells incorporated raft-associated molecules, suggesting that viral exit occurs at the rafts. Ectopic expression of Ebola matrix protein and glycoprotein supported raft-dependent release of filamentous, virus-like particles (VLPs), strikingly similar to live virus as revealed by electron microscopy. Our findings also revealed that the entry of filoviruses requires functional rafts, identifying rafts as the site of virus attack. The identification of rafts as the gateway for the entry and exit of filoviruses and raft-dependent generation of VLPs have important implications for development of therapeutics and vaccination strategies against infections with Ebola and Marburg viruses.

cDNA cloning and characterization of the human interleukin 13 receptor alpha chain.

We have cloned cDNAs corresponding to the human interleukin 13 receptor α chain (IL-13Rα). The protein has 76% homology to murine IL-13Rα, with 95% amino acid identity in the cytoplasmic domain. Only weak IL-13 binding activity was found in cells transfected with only IL-13Rα; however, the combination of both IL-13Rα and IL-4Rα resulted in substantial binding activity, with a Kd of approximately 400 pM, indicating that both chains are essential components of the IL-13 receptor. Whereas IL-13Rα serves as an alternative accessory protein to the common cytokine receptor γ chain (γc) for IL-4 signaling, it could not replace the function of γc in allowing enhanced IL-2 binding activity. Nevertheless, the overall size and length of the cytoplasmic domain of IL-13Rα and γc are similar, and like γc, IL-13Rα is located on chromosome X.

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 and Marburg Viruses Replicate in Monocyte-Derived Dendritic Cells without Inducing the Production of Cytokines and Full Maturation

Ebola virus (EBOV) and Marburg virus (MARV) cause rapidly progressive hemorrhagic fever with high mortality and may possess specialized mechanisms to evade immune destruction. We postulated that immune evasion could be due to the ability of EBOV and MARV to interfere with dendritic cells (DCs), which link innate and adaptive immune responses. We demonstrate that EBOV and MARV infected and replicated in primary human DCs without inducing cytokine secretion. Infected DC cultures supported exponential viral growth without releasing interferon (IFN)-alpha and were impaired in IFN-alpha production if treated with double-stranded RNA. Moreover, EBOV and MARV impaired the ability of DCs to support T cell proliferation, and infected, immature DCs underwent an anomalous maturation. These findings may explain the profound virulence of EBOV and MARV--DCs are disabled, and an effective early host response is delayed by the necessary reliance on less-efficient secondary mechanisms.

In vivo oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding

The matrix protein VP40 plays a critical role in Ebola virus assembly and budding, a process that utilizes specialized membrane domains known as lipid rafts. Previous studies with purified protein suggest a role for oligomerization of VP40 in this process. Here, we demonstrate VP40 oligomers in lipid rafts of mammalian cells, virus-like particles, and in the authentic Ebola virus. By mutagenesis, we identify several critical C-terminal sequences that regulate oligomerization at the plasma membrane, association with detergent-resistant membranes, and vesicular release of VP40, directly linking these phenomena. Furthermore, we demonstrate the active recruitment of TSG101 into lipid rafts by VP40. We also report the successful application of the biarsenic fluorophore, FlAsH, combined with a tetracysteine tag for imaging of Ebola VP40 in live cells.

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.

Identification of small molecule inhibitors of anthrax lethal factor

The virulent spore-forming bacterium Bacillus anthracis secretes anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease that inactivates key signaling molecules, such as mitogen-activated protein kinase kinases (MAPKK), to ultimately cause cell death. We report here the identification of small molecule (nonpeptidic) inhibitors of LF. Using a two-stage screening assay, we determined the LF inhibitory properties of 19 compounds. Here, we describe six inhibitors on the basis of a pharmacophoric relationship determined using X-ray crystallographic data, molecular docking studies and three-dimensional (3D) database mining from the US National Cancer Institute (NCI) chemical repository. Three of these compounds have Ki values in the 0.5–5 μM range and show competitive inhibition. These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.

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.