Wednesday, September 21, 2016

Building the "Trojan Horse" to Combat Ebolaviruses

    From it's discovery a short four decades ago, Ebola viruses have directly affected the lives of more than 30 000 people (1).  Despite the many historical outbreaks, the recent outbreak in Western Africa in 2014 marks the largest and most complex yet officially bringing the infection across seas to the United States.  As a result, a "Public Health Emergency of International Concern" was declared and an international response to combat Ebola virus has commenced.
     Ebola virus is a severe and often fatal disease in humans with an average case fatality rate of about 50% (2).  The disease can be contracted through various forms of direct contact with anything that has been contaminated or infected with the virus.  Evidence suggests that bats are the "reservoir hosts", transmitting the disease to other animals or humans (3).  Affected individuals experience a range of symptoms including severe headache and weakness as well as fever and muscle pain.  The illness is caused by infection with a member of the Filoviridae family of viruses, genus Ebolavirus.  To date, there are five identified species of Ebola virus with four of which causing disease in humans (2).  Nonetheless, the recent outbreak in 2014 caused specifically by the Zaire ebolavirus has driven scientific research to find protection against any or all of the Ebola viruses to promote the well-being and health of the world's population.  
     In order to understand the scientific foundation for the development of therapeutics, one must become familiar with the viral entry process of the Ebola viruses.  In terms of "viral entry", this is the point in the infection cycle in which the Ebola virus genetic material is released from a vesicle (lysosome) located inside the target cell.  The released viral genetic material can then undergo replication and transcription/translation to form more viral components and ultimately assemble more virions that can be released to commence the infection process in other target cells.  In other words, "viral entry" in the case of Ebola viruses does not refer directly to it's physical entry into the target cell, but rather the release of the viral genetic material from within an internal compartment of the target cell.  

     Generally, the sequence of events for viruses similar to Ebola the  are as follows: a viral adhesin (in Ebola's case, it's glycoprotein) attaches the virion to the target cell membrane through receptor interactions, the virion is engulfed via endocytosis now encapsulating the virion in an endosome, the virion is delivered to another vesicle known as a lysosome, then the environment within the lysosome triggers a conformational change of the protein (Ebola's glycoprotein) that will ultimately undergo a series of conformational changes to fuse the virion to the lysosomal membrane by interacting with a membrane receptor, allowing the virion to release its genetic material outside of the lysosome.  Very simply put, an Ebola virus finds a way into the cell via endocytosis and then finds a way out of the lysosome for it's genetic material via fusion with the lysosomal membrane to reach the cytoplasm. 

     Filoviruses, the family that Ebola viruses are members of, display a unique characteristic in terms of this entry process.  Once the endosomal materials, including the virion, are within the lysosome, a protease cleaves the glycoprotein (GP) of the virion resulting in a GP that now reveals affinities for molecules that it could not interact with prior.  Specifically, the cleaved GP on the virus in the lysosome now has a receptor-binding site that can interact with the Niemann-Pick C1 (NPC1) intracellular receptor (the uncleaved version of GP cannot interact with NPC1).  This fusion event located in the lysosomal membrane is what ultimately leads to the release of the viral genetic material from the lysosome, and thus this cleavage event of the GP is essential for filovirus infection.  An excellent visual representation for this process can be found from the Albert Einstein College of Medicine at "How Ebola Virus Infects a Cell".  
     Now that there is an understanding of viral entry, a discussion can be had regarding a developed therapy for Ebola viruses.  Researchers have developed what's known as "ZMapp", which consists of three antibodies that target the Zaire Ebola virus glycoprotein.  These antibodies block the ability for the glycoprotein to interact with the receptors it needs to for successful infection (ie. reaching the cytoplasm).  The research team found that the ZMapp concoction of antibodies can rescue nonhuman primates already infected with the Ebola virus (4).  As this brings light to the potential of antiviral therapies, there still exist a great concern for another viral epidemic to occur from another species of Ebola virus.  In other words, ZMapp targets the Zaire Ebola virus responsible for the 2014 epidemic but has yet to prove successful with others.  This is because ZMapp is designed to recognize a "binding site" (epitope) specific to the glycoprotein of the Zaire Ebola virus, and thus cannot target the other Ebola viruses' glycoproteins.  As a result, there is a need to be prepared for any epidemic by another filovirus by constructing broad protection immunotherapies (A.Z. Wec et al., 2016). 

     A team of researchers have recently brought a "Trojan horse" antibody strategy to life that preliminary results have shown potential for combating a whole scope of filoviruses.  The idea in this therapeutic method is to ultimately attack the cleaved glycoprotein in the lysosome and/or the NPC1 receptor in the lysosomal membrane to block the entry of viral genetic material into the cell, by bypassing the obvious obstacle that the lysosome is a closed off compartment within the cell.  The "Trojan horse" title to this therapy describes how the researchers are utilizing the virus itself to deliver the aforementioned antibodies to the lysosome.  This is done by coupling the cleaved GP or NPC1 targeting antibody to an antibody targeting a "binding site" (epitope) broadly conserved across filoviruses in the uncleaved version of the glycoprotein.  Using a well-known dual variable domain strategy, DVD-Ig, this coupling of antibodies can be achieved (A.Z. Wec et al., 2016).  In a simple sense, this "Trojan horse" therapy is a very hopeful strategy for combating filovirus infections because it attacks the unique characteristic (cleavage of the GP in the lysosome) of filoviruses in their mechanism for successful viral entry.  Blocking the interaction of the cleaved GP with the NPC1 receptor by either blocking the cleaved GP or the NPC1 receptor disables the ability to release the genetic material into the cytoplasm.  This therefore disables the ability to replicate and create more viral components to survive and assemble more virions, combating infection. 

     Preliminary results have shown, in short, that the DVD-Igs are localizing successfully within the lysosome, and that the DVD-Ig targeting the cleaved glycoprotein is fully protective against the human Sudan Ebola virus in a model system.  Although more work needs to be carried out before such a therapy is "perfected" and could be used on the human population, the recent work has demonstrated that broad protection against ebolaviruses is a strong possibility.  If this is a possibility, then this also demonstrates that broad protection against other infectious families of viruses is a possibility... and that building a shield against epidemics is a likelihood. 

Primary Article: 
A. Z. Wec et al. (2016).  A "Trojan horse" bispecific antibody strategy for broad protection against 
     ebolaviruses.  Science 10.1126/science.aag3267.  

Works Cited:
1.  http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html 
2.  http://www.who.int/mediacentre/factsheets/fs103/en/
3.  http://www.cdc.gov/vhf/ebola/about.html
4.  Qui et al., (2014).  Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.  Nature 514: 47-53.  doi:10.1038/nature13777.
5.  http://www.newswise.com/articles/researchers-find-key-used-by-ebola-virus-to-unlock-cells-and-spread-deadly-infection

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