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

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

Assessment of Ebola Specific Antibody Responses in Vaccinated Macaques (128.20)

Abstract Ebola virus (EBOV) infection causes severe disease accompanied by high mortality rates. Several vaccine platforms have shown promise in protecting against Ebola exposure; however, there are no licensed vaccines or therapies approved and available for human use. A vaccine approach utilizing Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) expressing filovirus protein(s) was the first vaccine approach that provided complete protection against Marburg infection in non-human primates (NHP). We evaluated the ability of VEE replicons expressing the glycoprotein (GP), nucleoprotein (NP), VP24, VP30, VP35, and VP40 to protect against Ebola Zaire (ZEBOV) exposure in NHP by various routes of administration, intramuscular (IM) versus subcutaneous (SC). To further evaluate protective efficacy, NHP were vaccinated IM or SC with VEE replicon expressing the viral glycoprotein or expressing all six proteins. Protective efficacy was demonstrated in the both cynomolgus macaques and rhesus macaques. Enzyme-Linked ImmunoSorbent Assays (ELISA) were developed using the whole antigen or recombinant Ebola GP to assess vaccine induced immunity against EBOV. The ELISA based assay assessing humoral responses did not accurately predict protection. The protection generated by the VEE replicon expression of Ebola proteins demonstrates the efficacy of the vaccines platform and warrants further evaluation as a human use vaccine. Further studies are needed to develop accurate correlate of immunity assays.</jats:p

A Novel L-Ficolin-Mannose Binding Lectin Chimeric Molecule With Enhanced Activity Against Lethal Viral Infections (39.33)

Abstract Ebola viruses are potentially lethal enveloped RNA viruses for which no effective therapy exists. Human mannose-binding lectin (MBL) and L-ficolin (FCN) bind to conserved epitopes on pathogens and eradicate microbes by opsonophagocytosis, complement activation, and/or regulation of adaptive immune responses. Three FCN-MBL chimeras containing the carbohydrate-recognition domains of hMBL and varying lengths of the collagen stalks of FCN were stably expressed in HEK293F cells. rhMBL (Enzon, NJ) formed octadecameric and larger oligomers vs. smaller chimera multimers. Compared with rhMBL, chimeras bound a similar range of carbohydrate ligands but FCN-MBL76 had significantly improved function as shown by: 1) greater affinity to mannan (KD, 47 vs 73 pM, p&amp;lt;0.004); 2) greater capacity to activate complement in the range of 7-30 ng/mL, p&amp;lt;0.001; and 3) greater binding to human placental calreticulin, a putative cellular receptor for MBL (p&amp;lt;0.001). All chimeras and rhMBL inhibited Ebola glycoprotein-pseudotyped lentivirus and native Ebola virus infection of HepG2 cells although FCN-MBL76 was the most effective protein against native virus. Tapping-force atomic force microscopy revealed that FCN-MBL76 was less tall than the other proteins suggesting enhanced flexibility. Compared with rhMBL, a novel FCN-MBL chimeric protein exhibited superior binding to select carbohydrates, activation of complement, binding to calreticulin, and greater inhibition of Ebola virus infection in vitro.</jats:p

The cyanobacterial lectin scytovirin displays potent in vitro and in vivo activity against Zaire Ebola virus

The cyanobacterial lectin scytovirin (SVN) binds with high affinity to mannose-rich oligosaccharides on the envelope glycoprotein (GP) of a number of viruses, blocking entry into target cells. In this study, we assessed the ability of SVN to bind to the envelope GP of Zaire Ebola virus (ZEBOV) and inhibit its replication. SVN interacted specifically with the protein’s mucin-rich domain. In cell culture, it inhibited ZEBOV replication with a 50% virus-inhibitory concentration (EC(50)) of 50 nM, and was also active against the Angola strain of the related Marburg virus (MARV), with a similar EC(50). Injected subcutaneously in mice, SVN reached a peak plasma level of 100 nm in 45 minutes, but was cleared within 4 hours. When ZEBOV-infected mice were given 30 mg/kg/day of SVN by subcutaneous injection every 6 hours, beginning the day before virus challenge, 9 of 10 animals survived the infection, while all infected, untreated mice died. When treatment was begun one hour or one day after challenge, 70-90% of mice survived. Quantitation of infectious virus and viral RNA in samples of serum, liver and spleen collected on days 2 and 5 postinfection showed a trend toward lower titers in treated than control mice, with a significant decrease in liver titers on day 2. Our findings provide further evidence of the potential of natural lectins as therapeutic agents for viral infections

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

A screen of approved drugs and molecular probes identifies therapeutics with anti–Ebola virus activity

Several FDA-approved drugs, including bepridil and sertraline, may be therapeutics against filovirus infections.</jats:p

Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses

ABSTRACT The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. IMPORTANCE Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective immunotherapeutics are urgently needed. Here, we describe monoclonal antibodies with cross-reactivity to several filoviruses, including the first report of a cross-neutralizing antibody that exhibits protection against Ebola virus and Sudan virus in mice. Our results further describe a novel combination of antibodies with enhanced protective efficacy. These results form a basis for further development of effective immunotherapeutics against filoviruses for human use. Understanding the cross-protective epitopes are also important for rational design of pan-ebolavirus and pan-filovirus vaccines. </jats:p