"Trojan Horse" Strategy Uses Bispecific Antibodies to Block Ebola from Invading Cells

In a collaborative effort, researchers from Albert Einstein College of Medicine, US Army Medical Research Institute of Infectious Diseases (USAMRIID), Integrated Biotherapeutics, Vanderbilt University Medical Center, and The Scripps Research Institute have discovered what they refer to as a “Trojan Horse” strategy that uses two developed bispecific antibodies that have proved active against all five strains of the Ebolavirus.

Previously known as Ebola hemorrhagic fever, Ebola is a rare, deadly disease that is caused by infection with one of the Ebola virus strains, according to the Centers for Disease Control and Prevention (CDC). First discovered in 1976 near what is now the Democratic Republic of the Congo, Ebolavirus can be found in several African countries. The largest outbreak in Ebola history started in March 2014 and resulted in over 11,000 deaths.

Although monoclonal antibodies are currently the best treatment in the fight against Ebola, there exists at least one major setback: monoclonal antibodies only target one of the Ebolavirus species out of the five known to exist. ZMapp, composed of three different monoclonal antibodies, targets Zaire ebolavirus, one of the most dangerous Ebola virus species, but it fails to deliver protection against the Bundibugyo ebolavirus and Sudan ebolavirus, according to the press release.

This knowledge prompted researchers to discover a different way to combat Ebola, one that could offer protection to a number of the viruses instead of just a single specific one. When developing the monoclonal antibodies, researchers set out to target viruses after they entered the host’s lysosomes, according to the press release.

Filoviruses, such as Ebola, are able to enter cells through the use of glycoproteins that allow the filovirus to attach to the outer membrane of the host cell. Lysosomes are soon created, when part of the cell membrane encompasses the virus and then “pinches off.” By utilizing the resources of the host cell, filoviruses are then able to break out of these lysosomes in an attempt to enter the cell’s cytoplasm. Once there, the filovirus is free to replicate, according to the press release.

However, “Enzymes in the lysosome slice a ‘cap’ from the virus’s glycoproteins, unveiling a site that binds to the NPC1 embedded in the lysosome membrane,” according to the press release. Attaching to the protein, Niemann-Pick C1 (NPC1) is the only opportunity that the Ebola virus has to break out of the lysosome and replicate.

Due to the fact that the virus can typically be found in lysosomes that are so far within the cell that they tend to remain undetected by the host’s immune system, the researchers found that targeting the viral protein that binds to NCP1 or neutralizing NCP1 itself would prove difficult, so Kartik Chandran, PhD, professor of microbiology and immunology at the Albert Einstein School of Medicine, John M. Dye, PhD, chief of viral immunology at USAMRIID, and author Jonathan R. Lai, PhD, associate professor of biochemistry at Einstein developed what they refer to as a “Trojan Horse” strategy, where the filoviruses would carry virus-destructive bispecific antibodies with them into the host cell.

Dr. Dye said, “Our team of scientists took the ‘Trojan Horse’ concept from the chalkboard to a product that has the capacity to provide a viable treatment for all filoviruses, both known and emerging. This work highlights the power of governmental, academic and industrial researchers collaborating together to solve a complex and important public health concern.”

Dr. Lai, experienced in engineering antibodies, assisted in the development of the two different bispecific antibodies: one would target NCP1 and the other would target the viral protein that binds to it, according to the press release.

Through the use of an antibody called FVM09, developed by M. Javad Aman, PhD, both bispecific antibodies were able to travel with the virus into the lysosome. When the “glycoprotein caps” are severed from the lysosome, the bispecific antibodies are then freed from the virus and are able to carry out their respective duties. A combination of the FVM09 with an antibody called MR72 (which is an isolate taken from an individual who had survived the Marburg virus by James E. Crowe Jr., MD, director of the Vanderbilt Vaccine Center), the MR72 antibody targets the viral protein that binds to NPC1. The other bispecific antibody, created at the Albert Einstein College of Medicine, combines FVM09 with an antibody called mAb-548, which specifically targets NPC1, according to the press release. Both of the bispecific antibodies could potentially prevent the filovirus from breaking free from the lysosome, and thus, could prevent the replication of the virus throughout the host cell.

In order to test their hypothesis, researchers created a “harmless” virus that would “display glycoproteins from all five ebolaviruses on its surface,” according to the press release. They found that both of the bispecific antibodies were successful in their intended purpose, in all five of the ebolaviruses. The USAMRIID found that in addition, the antibodies were also successful in blocking infection by three of the real Ebola virus strains (specifically Sudan, Zaire, and Bundibugyo), according to the press release.

Lastly, researchers at USAMRIID took the research a step further by administering the bispecific antibodies to mice, two days after being given with a deadly dose of Zaire and Sudan ebolaviruses to measure the amount of protection, if any, that the antibodies would offer. They found that the MR72 antibody offered protection from both viruses but the mAb-548 one did not. “It was designed to bind specifically to human NPC1, which differs slightly in structure from the NPC1 found in mice,” according to the press release.

Dr. Lai, said, “It’s impossible to predict where the next ebolavirus outbreak will occur or which virus will cause it. So the best therapy would be a monoclonal antibody that is active against the glycoproteins of all five ebolaviruses—and until our study, no such antibody had demonstrated the ability to do that. We hope that further testing in nonhuman primates will establish our antibodies are safe and effective for treating those exposed to any ebolavirus.”