The characterisation of the viral entry mechanism of any virus allows to identify targets for antiviral drugs which can be used immediately following the (suspected or proven) exposure to the virus as well as preventing further spread of the infection within an infected person. This is especially important if treating viral diseases to which no vaccine is available or in situations where the goal is to contain a local epidemic or in the case of emerging viruses who only cause sporadic outbreaks but are not persistently present in the population.
Currently we are seeing and following the outbreak of one such a disease, Ebola associated hemorrhagic fever (Ebola-HF), in a number of west African countries. While this is not the first outbreak of Ebola in Africa, by all accounts it is the most devastating recorded so far. Treatment consists mostly of treating the symptoms, and although experimental drugs are available in countries of the northern hemisphere, these are mostly not clinical tested and even if so, then the effectiveness during an actual outbreak (“in the field” one might say) might very well differ from the controlled conditions of laboratory based animal testing.
Nevertheless, it is worth taking a look at some of these drugs or compounds available and to examine which point of the viral life cycle they actually inhibit with a focus on the viral GP.
As mentioned above, one of the most obvious targets of any antiviral compound is the entry of viruses. In the case of Ebola Virus (EBOV), targeting the entry of the virus is complicated by the fact that -although the entry process is relatively well understood- the cellular receptor for the virus is not known, although it emerges that entry is mediated by multiple receptors in a clathrin independent and dependent process. What is known however, is the localisation of the putative receptor-binding domain (RBD) within the EBOV-GP protein as well as the structure of the full length as well as cleaved GP. Therefore it should be possible to design molecules, which bind to the RBD of EBOV-GP, thus preventing the viral particle from entering the cell.
In practice however, the epitope is masked by a dense clustering of O- and N-linked oligosaccharides within the Mucin-like domain (MLD), glycans which prevent efficient binding of otherwise neutralising antibodies or compounds particularly to the RBD.
Although the EBOV-GP MLD is highly immunogenic, this domain is also dispensable for viral entry and antibodies directed against this region do not interfere with viral entry. It is only when mice infected with EBOV Δ MLD produce antibodies targeting the conserved core structure of EBOV. Highly specific antibodies however are produced against a small epitope close to the viral membrane but given the proximity of this epitope to the viral membrane and the GP1 subunit it is not clear how accessible this epitope is. So does this mean that the application of antibodies does not prevent Ebola-HF? In October 2019, a company from California published a study using a mixture of three monoclonal antibodies to treat mice and non-human primates infected with a lethal dose of EBOV. Following infection, the monkeys were treated with three doses of the mixture beginning either at 24 hours at 48 hours p.i.; compared with the controls, 100% and 50% of the infected monkeys respectively survived. While this study is certainly encouraging, one should cautious to extrapolate these findings to the current outbreak. The symptoms of Ebola-HF during the early stages can be easily confused with other diseases and thus patients only be recognised as being infected with EBOV later in the disease. Furthermore, patients might simply not reach healthcare facilities in time to start effective treatment due to travel. The study also showed that treating animals prior infection does not protect from the disease. Also, the application of the mAb cocktail does not prevent disease but alleviates the symptoms by leading a transient control of the infection thus allowing the immune system to mount an antiviral defense. Finally, the application of highly specific monoclonal antibodies presumes that escape mutants do not exist or that the mutation rate is low, which is the case for EBOV (so far). In general however the results are encouraging and certainly warrant further investigation and testing.
In practice however, the epitope is masked by a dense clustering of O- and N-linked oligosaccharides within the Mucin-like domain (MLD), glycans which prevent efficient binding of otherwise neutralising antibodies or compounds particularly to the RBD.
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| Formation of the vision attached and secreted GP trimers. See next figure for legend. Note the presence of glycosylated residues and localisation of the RBD |
Although the EBOV-GP MLD is highly immunogenic, this domain is also dispensable for viral entry and antibodies directed against this region do not interfere with viral entry. It is only when mice infected with EBOV Δ MLD produce antibodies targeting the conserved core structure of EBOV. Highly specific antibodies however are produced against a small epitope close to the viral membrane but given the proximity of this epitope to the viral membrane and the GP1 subunit it is not clear how accessible this epitope is. So does this mean that the application of antibodies does not prevent Ebola-HF? In October 2019, a company from California published a study using a mixture of three monoclonal antibodies to treat mice and non-human primates infected with a lethal dose of EBOV. Following infection, the monkeys were treated with three doses of the mixture beginning either at 24 hours at 48 hours p.i.; compared with the controls, 100% and 50% of the infected monkeys respectively survived. While this study is certainly encouraging, one should cautious to extrapolate these findings to the current outbreak. The symptoms of Ebola-HF during the early stages can be easily confused with other diseases and thus patients only be recognised as being infected with EBOV later in the disease. Furthermore, patients might simply not reach healthcare facilities in time to start effective treatment due to travel. The study also showed that treating animals prior infection does not protect from the disease. Also, the application of the mAb cocktail does not prevent disease but alleviates the symptoms by leading a transient control of the infection thus allowing the immune system to mount an antiviral defense. Finally, the application of highly specific monoclonal antibodies presumes that escape mutants do not exist or that the mutation rate is low, which is the case for EBOV (so far). In general however the results are encouraging and certainly warrant further investigation and testing.
Using EBOV-GP as a target for antiviral drugs is further complicated by production of secreted -soluble- GP, a feature shared with other viruses including HIV, Lassa, RSV, and Herpes Simplex Virus. These proteins can act as a decoy for antibodies and indeed EBOV encodes for a GP variant of 110 kDa in size which either is being shed as trimeric protein (sGP) via cleavage at the membrane-proximal external region by the tumor necrosis factor-αconverting enzyme (TACE), or alternatively small secreted (ss) GP is expressed. In EBOV patients as well as in the sera of infected macaques, most antibodies are indeed targeted against the sGP rather the virion attached form of GP; even neutralising antibodies cross reactive for both forms may be simply absorbed by the more abundant form sGP. This might explain why antibody treatment is more efficient in early times post exposure.
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| Formation of sGP and ssGP |
As mentioned in a previous post, the EBOV-GP is cleaved into the GP1 and GP2 subunit prior to the fusion of the viral membrane with the endosomal membrane in a Cathepsin-B and -L dependent manner. Cathepsin inhibitors indeed have been shown to prevent replication of a number of viruses such as SARS-CoV), Hendra and Nipah viruses which depend on Cathepsin-L for fusion with the endosome and subsequent release of the viral nucleocapsid and one of these small molecule inhibitors out of a library of 5000, 5705213, has recently identified as being effective not only in inhibiting pseudotyped VSV-GP, but also SARS-CoV S as well as the NiV-F0 and HeV-F0 mediated fusion. It remains to be seen if 5705213 is effective in animal models as well, but the current results suggests that cytotoxicity is low. In a similar way, binding of an inhibitory peptide to the GP2 subunit prior entry inhibits the fusion of the GP2 subunit to the endosome following cleavage of EBOV-GP.
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| Cathepsin-B/-L cleaves EBOV-GP and facilitates fusion with the endosomal membrane |
Finally, where are we going from here? Personally, I think the future belongs to a multi pronged approach, intertwining antibody-based treatments with small molecule inhibitors and “classic” treatment with Interferon and Ribavirin once an outbreak has started. To prevent an outbreak or limit the spread of the disease, local customs need to be changed - slowly, without condemning the local population. Also, the healthcare system in the affected countries needs to be improved, not only to prevent outbreaks of Ebola, but also to cope with everyday disease. We need also to improve nutrition - a healthy, well-nourished body will always improve chances of recovery. In the case of Ebola, viral shedding by survivors might prolong an epidemic, so we need to take care of this as well. In the developed countries we need to continue the research on the viral life cycle as well as searching for antivirals, not only against Ebola but also other zoonotic diseases as well. Some circles blame the pharmaceutical industry for not investing enough resources in designing antivirals against rare diseases. I would argue however, that this could be done also in academia, and we should not always expect industry to solve problems that take time and manpower.
Further reading
Lee JE, & Saphire EO (2009). Ebolavirus glycoprotein structure and mechanism of entry. Future virology, 4 (6), 621-635 PMID: 20198110
Cook JD, & Lee JE (2019). The secret life of viral entry glycoproteins: moonlighting in immune evasion. PLoS pathogens, 9 (5) PMID: 23696729
Qiu X, Audet J, Wong G, Pillet S, Bello A, Cabral T, Strong JE, Plummer F, Corbett CR, Alimonti JB, & Kobinger GP (2012). Successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies. Science translational medicine, 4 (138) PMID: 22700957
Suzuki, Y., & Gojobori, T. (1997). The origin and evolution of Ebola and Marburg viruses Molecular Biology and Evolution, 14 (8), 800-806 DOI: 10.1093/oxfordjournals.molbev.a025820
Elshabrawy HA, Fan J, Haddad CS, Ratia K, Broder CC, Caffrey M, & Prabhakar BS (2019). Identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and Ebola, Hendra, and Nipah viruses by using a novel high-throughput screening assay. Journal of virology, 88 (8), 4353-65 PMID: 24501399
Koehler JW, Smith JM, Ripoll DR, Spik KW, Taylor SL, Badger CV, Grant RJ, Ogg MM, Wallqvist A, Guttieri MC, Garry RF, & Schmaljohn CS (2019). A fusion-inhibiting peptide against Rift Valley fever virus inhibits multiple, diverse viruses. PLoS neglected tropical diseases, 7 (9) PMID: 24069485
Qiu X, Wong G, Fernando L, Audet J, Bello A, Strong J, Alimonti JB, & Kobinger GP (2019). mAbs and Ad-vectored IFN-α therapy rescue Ebola-infected nonhuman primates when administered after the detection of viremia and symptoms. Science translational medicine, 5 (207) PMID: 24132638
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