High-Dose Mannose-Binding Lectin Therapy for Ebola Virus Infection

Mannose-binding lectin (MBL) targets diverse microorganisms for phagocytosis and complement-mediated lysis by binding specific surface glycans. Although recombinant human MBL (rhMBL) trials have focused on reconstitution therapy, safety studies have identified no barriers to its use at higher levels. Ebola viruses cause fatal hemorrhagic fevers for which no treatment exists and that are feared as potential biothreat agents. We found that mice whose rhMBL serum concentrations were increased ≥7-fold above average human levels survived otherwise fatal Ebola virus infections and became immune to virus rechallenge. Because Ebola glycoproteins potentially model other glycosylated viruses, rhMBL may offer a novel broad-spectrum antiviral approach

Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes

Multiple Cationic Amphiphiles Induce a Niemann-Pick C Phenotype and Inhibit Ebola Virus Entry and Infection

<div><p>Ebola virus (EBOV) is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC₅₀ 1.6 to 8.0 µM) at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann–Pick C1 protein (NPC1), a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents) inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.</p> </div

Models for how CADs May Block EBOV Entry.

<p>(A) In the first model, the CADs (yellow star) interact directly with NPC1, but at a site distinct from the C loop of NPC1 (which binds GP_{19 kDa}). Binding to NPC1 inhibits a second function of NPC1 (i.e. in addition to its role in binding GP_{19 kDa}) that is critical for EBOV entry. (B) In the second model, the CADs intercalate into the LE/Lys membrane, indirectly inhibiting a second function of NPC1 that promotes EBOV entry. (C) In a final model, the CADs disrupt a target distinct from NPC1 that is critical for EBOV fusion with LE/Lys) and is regulated by NPC1. The target may be another LE/Lys protein (e.g. ASM) or a lipid of the LE/Lys membrane system. (See text for details.) Alternatively, the CADs may interfere with NPC1-dependent membrane trafficking <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone.0056265-Ko1" target="_blank">[47]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone.0056265-Sobo1" target="_blank">[50]</a> such that the virus is never found in an NPC1-containing compartment. In all of the models, the yellow star denotes a CAD and red in each middle image denotes the target molecule.</p

CADs that strongly inhibit EBOV entry and infection cause cholesterol accumulation in LE/Lys.

<p>SNB19 cells were treated for 21 hr with either DMSO or inhibitor (concentrations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone-0056265-g002" target="_blank">Fig. 2</a>). Cells were then fixed, stained with filipin, and imaged on a fluorescence microscope. Images were inverted and uniformly adjusted for contrast and brightness. Representative images are shown. Arrows indicate sites of cholesterol accumulation. Each compound was tested at least 3 times, and scored (+/−) by a blind observer (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone-0056265-t001" target="_blank">Table 1</a>).</p

U18666A inhibits EBOV entry at a post internalization step and does not inhibit endosome acidification or cathepsin levels.

<p>Effects of U18666A (indicated concentration in A, 5 µM in B-E) on: (A) VLP-GPΔ and VLP-G entry; one representative of two experiments (done in triplicate). (B) VLP-GPΔ and VLP-LCMV entry; one representative of three experiments (done in duplicate). (C) VLP-GPΔ internalization and entry (controls as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone-0056265-g003" target="_blank">Fig. 3C</a>); one representative of two experiments (done in triplicate). (D) Endosomal pH detected by Lysotracker Red; 10 mM NH₄Cl was used as the control for pH neutralization; representative images from multiple coverslips from a single experiment (E) Cathepsin B, L, and combined B/L activity; E64d was used as the positive control as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone-0056265-g003" target="_blank">Fig. 3E</a>; results from a single experiment performed in duplicate. In all assays, SNB19 cells were pre-treated with the indicated concentration of inhibitor for 1 hr at 37°C, and inhibitors were maintained throughout the assays. Error bars represent standard deviation from the mean of mock-treated samples: * (P<.01), ** (P<.001), or *** (P<.0001).</p

Ro 48-8071 inhibits EBOV entry at a post internalization step and does not inhibit endosome acidification or cathepsin levels.

<p>Effects of Ro 48-8071 (indicated concentration in A, 5 µM in B-E) on: (A) VLP-GPΔ and VLP-G entry; one representative of two experiments (done in triplicate). (B) VLP-GPΔ and VLP-LCMV entry; one representative of three experiments (done in duplicate). (C) VLP-GPΔ internalization and entry; 50 µM EIPA was used as the positive control for an inhibitor of EBOV internalization <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone.0056265-Saeed1" target="_blank">[8]</a>; 10 µM EIPA was used as the control for the entry assay (50 µM EIPA caused high background fluorescence in the entry assay); one representative of two experiments (done in triplicate). (D) Low endosomal pH was detected by incubating cells with Lysotracker Red; 10 mM NH₄Cl was used as the control for pH neutralization; representative images from multiple coverslips from a single experiment. (E) Cathepsin B, L, and combined B/L activity; 10 µM E64d was used as the positive control for inhibition of cysteine protease activity; results from a single experiment performed in duplicate. In all assays, SNB19 cells were pre-treated with the indicated concentration of inhibitor for 1 hr at 37°C, and inhibitors were maintained throughout the assays. Error bars represent standard deviation from the mean of mock-treated samples: * (P<.01), ** (P<.001), or *** (P<.0001).</p