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

FGI-104: a broad-spectrum small molecule inhibitor of viral infection

The treatment of viral diseases remains an intractable problem facing the medical community. Conventional antivirals focus upon selective targeting of virus-encoded targets. However, the plasticity of viral nucleic acid mutation, coupled with the large number of progeny that can emerge from a single infected cells, often conspire to render conventional antivirals ineffective as resistant variants emerge. Compounding this, new viral pathogens are increasingly recognized and it is highly improbable that conventional approaches could address emerging pathogens in a timely manner. Our laboratories have adopted an orthogonal approach to combat viral disease: Target the host to deny the pathogen the ability to cause disease. The advantages of this novel approach are many-fold, including the potential to identify host pathways that are applicable to a broad-spectrum of pathogens. The acquisition of drug resistance might also be minimized since selective pressure is not directly placed upon the viral pathogen. Herein, we utilized this strategy of host-oriented therapeutics to screen small molecules for their abilities to block infection by multiple, unrelated virus types and identified FGI-104. FGI-104 demonstrates broad-spectrum inhibition of multiple blood-borne pathogens (HCV, HBV, HIV) as well as emerging biothreats (Ebola, VEE, Cowpox, PRRSV infection). We also demonstrate that FGI-104 displays an ability to prevent lethality from Ebola in vivo. Altogether, these findings reinforce the concept of host-oriented therapeutics and present a much-needed opportunity to identify antiviral drugs that are broad-spectrum and durable in their application

Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection

Outbreaks of emerging infections present health professionals with the unique challenge of trying to select appropriate pharmacologic treatments in the clinic with little time available for drug testing and development. Typically, clinicians are left with general supportive care and often untested convalescent-phase plasma as available treatment options. Repurposing of approved pharmaceutical drugs for new indications presents an attractive alternative to clinicians, researchers, public health agencies, drug developers, and funding agencies. Given the development times and manufacturing requirements for new products, repurposing of existing drugs is likely the only solution for outbreaks due to emerging viruses. In the studies described here, a library of 290 compounds was screened for antiviral activity against Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Selection of compounds for inclusion in the library was dependent on current or previous FDA approval or advanced clinical development. Some drugs that had a well-defined cellular pathway as target were included. In total, 27 compounds with activity against both MERS-CoV and SARS-CoV were identified. The compounds belong to 13 different classes of pharmaceuticals, including inhibitors of estrogen receptors used for cancer treatment and inhibitors of dopamine receptor used as antipsychotics. The drugs identified in these screens provide new targets for in vivo studies as well as incorporation into ongoing clinical studies. Copyright © 2014, American Society for Microbiology. All Rights Reserved

MBL mediates HIV-EBOV GP infection via the canonical macropinocytosis pathway for EBOV but with less dependence on actin.

<p>We preincubated HEK293F cells with (A) EIPA (5-(<i>N</i>-Ethyl-<i>N</i>-isopropyl)amiloride, a potent and specific inhibitor of Na⁺/H⁺ exchanger activity), (B) methyl-β-cyclodextrin (extracts or sequesters cholesterol from the plasma membrane), (C) latrunculin B (blocks actin polymerization), (D) cytochalasin D (inhibits actin microfilament function), (E) nocodazole (disrupts microtubules), or (F) jasplakinolide (disrupts microtubules) in 5% MBL-deficient serum in the absence or presence of rhMBL at 37°C for 1 hour. We then infected cells with HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl). Percentages of infected cells are relative to DMSO controls. Luciferase values were adjusted for cell viability. Experiments were performed twice in quadruplicate. Significant differences are shown. (G) Absorbance values of an ELISA assay are shown indicating the difference in amount of rhMBL within the physiological range that binds to immobilized mannan or FITC-dextran (1 µg/100 µl). (H) We preincubated FITC-dextran with various concentrations of rhMBL at 37°C for 30 minutes and then added the products to PMA-stimulated (10 ng/ml), IL-4-supplemented (100 ng/ml) THP-1 cells at 37°C for 1 hour. We measured FITC-dextran uptake by flow cytometry and reported the results as mean fluorescence intensity (geometric mean fluorescence × percentage of cells). Experiments were performed twice in triplicate.</p

Proposed model of MBL-mediated macropinocytosis of EBOV.

<p>MBL carbohydrate recognition domains (CRD) bind to highly glycosylated mucin-rich regions of EBOV GP and the MBL-virion complex is presented to the cell surface. Then MBL binds to cognate cellular receptors, such as C1QBP or calreticulin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060838#pone.0060838-Michelow1" target="_blank">[6]</a> via MBL collagenous stalks. In this manner, MBL concentrates virus at the cell surface and may facilitate cross-linking of EBOV to its cognate receptor or to other attachment factors such as dectin-2, which may facilitate subsequent membrane fusion and internalization. The relatively large size of EBOV particles (800 to 1,400 nm) is amenable to bulk fluid-phase uptake pathways such as macropinocytosis which is the canonical pathway for EBOV entry. Our data indicate that MBL also mediates internalization of virus via macropinocytosis but suggests that MBL-mediated uptake preferentially utilizes microtubules compared with the canonical EBOV pathway which is dependent on both microtubules and actin.</p

MBL enhances infections by wild type-like EBOV and other glycosylated virions.

<p>(A) We preincubated wild type-like EBOV-eGFP (1976 Mayinga variant) with media alone or 5% MBL-deficient serum with or without rhMBL at 37°C for 1 hour and then infected 4×10⁴ HEK293T cells (multiplicity of infection, 0.1) at 37°C for 1 hour. We measured cellular fluorescence after 72 hours of incubation in fresh media. Comparisons are with baseline values, * p = 0.028; ** p = 0.037. (B) We preincubated native Hendra and Nipah viruses (10,000 TCID₅₀/ml) with 10% heat-inactivated MBL-deficient serum with or without rhMBL and then infected Vero E6 cells at 37°C for 1 hour. After 24 hours, infection was detected by chemiluminescence-based viral protein immunoassays. Comparisons are with baseline values, *p = 0.001; ** p = 0.029. (C) We preincubated 250 µl West Nile virion-like particle-GFP with media alone or 2% MBL-deficient human serum, with or without rhMBL at 37°C for 1 hour and then transduced 1×10⁴ HEK293T cells. Cells were detached using TrypLE and washed three times with PBS at 4°C. Rates of transduction were assayed by flow cytometry. Comparisons are with baseline values, * p = 0.002; ** p = 0.001. WT refers to wild type; mutant refers to glycosylation mutant of WNV E protein; hMBL refers to human MBL.</p

MBL targets <i>N</i>-linked glycans on viral and cellular surfaces.

<p>The cleavage sites of two endoglycosidases are shown (A,B). N-glycosidase F (PNGase F) is an amidase that cleaves the linkages between the innermost GlcNAc and asparagine residues within high-mannose, hybrid and complex oligosaccharides of <i>N</i>-linked glycoproteins, thereby producing carbohydrate-free peptides without any potential ligands for MBL. Endoglycosidase H (endo H) cleaves linkages within the diacetylchitobiose stem of high-mannose of <i>N</i>-linked glycoproteins, thereby generating a truncated sugar molecule with one <i>N</i>-acetylglucosamine residue (a potential target for MBL) remaining on the asparagine. Man, mannose; GlcNAc, <i>N</i>-acetylglucosamine; asn, asparagine; × and y, various oligosaccharides; n = 2–150 residues. We preincubated HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl) at 37°C for 1 hour with (C) PNGase F or endo H (0–10,000 U/ml), or (D) the same concentrations of heat inactivated enzymes. Then we incubated the viruses with 5% MBL-deficient serum in the presence or absence of rhMBL at 37°C for 1 hour before infecting HEK293F cells. Significant differences are shown. (E) We preincubated HEK293F cells at 37°C for 1 hour with chemicals (tunicamycin, swainsonine or deoxynojirimycin) that inhibit various stages of <i>N</i>-linked glycosylation. Then we infected cells with HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl) that had been preincubated with 5% MBL-deficient serum and supplemented with various concentrations of rhMBL. Significant differences are shown. * and **, p<0.001 (all pairwise comparisons at 1 and 10 µg/ml rhMBL, respectively). (F) We cultivated HEK293F and HEK293S (deficient in <i>N</i>-acetylglucosaminyltransferase I) cells in 5% MBL-deficient serum which was supplemented with various concentrations of rhMBL. We infected cells with HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl) in the absence or presence of 1 µg/ml tunicamycin. Statistical differences among inhibitors at various rhMBL concentrations are shown. *, ** and †, p<0.005 (all pairwise comparisons at 0.1, 1 and 10 µg/ml rhMBL, respectively). Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay). All experiments were performed twice in quadruplicate.</p

MBL interacts with HIV-EBOV GP via MBL carbohydrate recognition domains.

<p>We preincubated 5% serum containing native human MBL (3,621 ng/ml) with (A) 0, 1 and 10 mM of hexose monosaccharides or EDTA diluted in media, or (B) 0–100 µg/ml of mannan or polydisperse polyethylene glycol (PEG)(D) at room temperature for 30 minutes. Then we incubated the serum with HIV-EBOV GP (1200 pg p24/100 µl) at 37°C for 1 hour before infecting adherent HEK293F cells. Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay). We observed relatively more toxicity associated with 10 mM EDTA but this did not invalidate our results because of our adjustment for cell viability. (C) We repeated the previous experiments with 3F8, an anti-human MBL monoclonal antibody or an IgG1 isotype control (preincubation at 37°C for 30 minutes). Significant differences are shown. (D) We preincubated HIV-EBOV GP virion-like particles with cyanovirin (0–600 nM) at 37°C for 1 hour before incubating the particles with 5% serum in the presence or absence of rhMBL. Luciferase values were adjusted for cell viability. Experiments were performed twice in quadruplicate.</p