FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection.

Ebola viruses remain a substantial threat to both civilian and military populations as bioweapons, during sporadic outbreaks, and from the possibility of accidental importation from endemic regions by infected individuals. Currently, no approved therapeutics exist to treat or prevent infection by Ebola viruses. Therefore, we performed an in vitro screen of Food and Drug Administration (FDA)- and ex-US-approved drugs and selected molecular probes to identify drugs with antiviral activity against the type species Zaire ebolavirus (EBOV). From this screen, we identified a set of selective estrogen receptor modulators (SERMs), including clomiphene and toremifene, which act as potent inhibitors of EBOV infection. Anti-EBOV activity was confirmed for both of these SERMs in an in vivo mouse infection model. This anti-EBOV activity occurred even in the absence of detectable estrogen receptor expression, and both SERMs inhibited virus entry after internalization, suggesting that clomiphene and toremifene are not working through classical pathways associated with the estrogen receptor. Instead, the response appeared to be an off-target effect where the compounds interfere with a step late in viral entry and likely affect the triggering of fusion. These data support the screening of readily available approved drugs to identify therapeutics for the Ebola viruses and other infectious diseases. The SERM compounds described in this report are an immediately actionable class of approved drugs that can be repurposed for treatment of filovirus infections.

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.

Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques.

Filovirus infections can cause a severe and often fatal disease in humans and nonhuman primates, including great apes. Here, three anti-Ebola virus mouse/human chimeric mAbs (c13C6, h-13F6, and c6D8) were produced in Chinese hamster ovary and in whole plant (Nicotiana benthamiana) cells. In pilot experiments testing a mixture of the three mAbs (MB-003), we found that MB-003 produced in both manufacturing systems protected rhesus macaques from lethal challenge when administered 1 h postinfection. In a pivotal follow-up experiment, we found significant protection (P < 0.05) when MB-003 treatment began 24 or 48 h postinfection (four of six survived vs. zero of two controls). In all experiments, surviving animals that received MB-003 experienced little to no viremia and had few, if any, of the clinical symptoms observed in the controls. The results represent successful postexposure in vivo efficacy by a mAb mixture and suggest that this immunoprotectant should be further pursued as a postexposure and potential therapeutic for Ebola virus exposure.

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.

A novel L-ficolin/mannose-binding lectin chimeric molecule with enhanced activity against Ebola virus.

Ebola viruses constitute a newly emerging public threat because they cause rapidly fatal hemorrhagic fevers for which no treatment exists, and they can be manipulated as bioweapons. We targeted conserved N-glycosylated carbohydrate ligands on viral envelope surfaces using novel immune therapies. Mannose-binding lectin (MBL) and L-ficolin (L-FCN) were selected because they function as opsonins and activate complement. Given that MBL has a complex quaternary structure unsuitable for large scale cost-effective production, we sought to develop a less complex chimeric fusion protein with similar ligand recognition and enhanced effector functions. We tested recombinant human MBL and three L-FCN/MBL variants that contained the MBL carbohydrate recognition domain and varying lengths of the L-FCN collagenous domain. Non-reduced chimeric proteins formed predominantly nona- and dodecameric oligomers, whereas recombinant human MBL formed octadecameric and larger oligomers. Surface plasmon resonance revealed that L-FCN/MBL76 had the highest binding affinities for N-acetylglucosamine-bovine serum albumin and mannan. The same chimeric protein displayed superior complement C4 cleavage and binding to calreticulin (cC1qR), a putative receptor for MBL. L-FCN/MBL76 reduced infection by wild type Ebola virus Zaire significantly greater than the other molecules. Tapping mode atomic force microscopy revealed that L-FCN/MBL76 was significantly less tall than the other molecules despite similar polypeptide lengths. We propose that alterations in the quaternary structure of L-FCN/MBL76 resulted in greater flexibility in the collagenous or neck region. Similarly, a more pliable molecule might enhance cooperativity between the carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics. L-FCN/MBL chimeric proteins should be considered as potential novel therapeutics.

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.