This research was conducted at the University of Minnesota.
This post was reviewed by Dr. Sebastian Lequime, one of our subject matter experts.
(A little disclaimer, this paper and review does not talk about domain antibodies, but the opportunity to make a pun with NAbs and DAbs was right there, and I had to do it).
The paper we’ll be demystifying today can be found here, if you’d like to follow along.
This team of researchers hail from Minnesota (University of Minnesota), New York (Kimball Research Institute), Beijing (Beijing Institute of Microbiology and Epidemiology), and Wuhan (Wuhan Institute of Virology). They investigated a phenomenon known as antibody-dependent enhancement (ADE for short) of viral entry. They looked at ADE for the coronaviruses SARS-CoV (responsible for the 2003 SARS outbreak) and MERS-CoV. This paper was first submitted for publication before the first SARS-CoV-2 infection was identified in China, so they do not talk about the implications of ADE viral entry for SARS-CoV-2. Nonetheless, these researchers shine a light on a legitimate concern about an enhanced viral entry mechanism for coronaviruses. This enhanced viral entry has implications for vaccine development and viral epidemiology. This means that there is no specific focus on the SARS-CoV-2 virus in this paper. However, it does give us clues and suggests that there are likely research groups investigating this phenomenon for this particular virus right now.
Before we dive into the paper, I also want to spend some time explaining what ADE of viral entry is, and why we care so much about it in virology and immunology. The researchers also talk about it in their introduction, so I’ll be simplifying what they say as a quick lesson on ADE for all of us to understand their discoveries.
So, antibody-dependent enhancement (ADE) of viral entry is an occurrence that takes place after you get infected with a virus (say SARS-CoV). When you first get infected with a novel virus that you haven’t been infected with before, your adaptive immune system kicks into gear. One of the things your immune system does is make something known as neutralizing antibodies (NAbs). Neutralizing antibodies are like what they sound like, if you get reinfected with the exact same SARS-CoV virus (that means you got infected with the same serotype, not a close relative), these antibodies will swarm the virus and prevent it from entering your cells.
Think of neutralizing antibodies as a bunch of cute puppies swarming you with lots of licks and cuddles. This leaves you helpless, because you can’t do anything but play with the cute puppies (if only that were a reality)! So, neutralizing antibodies basically do that to an existing virus you already encountered. They help prevent the virus from infecting a new set of cells.
But the problem arises when you get infected with a closely related SARS-CoV virus. The neutralizing antibodies were meant for a specific type of virus and can’t fully swarm and neutralize a different type. What happens instead is that the neutralizing antibody binds the related SARS-CoV and then it binds an immunoglobulin G fragment crystallizable region (known as IgG Fc) receptor on your immune cells. What ends up happening when a neutralizing antibody binds to a related SARS-CoV-2 virus, is that it forces the antibody-virus combo to bind to our immune cells. This means that these viruses now have entry to our immune cells. Neutralizing antibodies that would have stopped SARS-CoV could thus potentially lead to a worsen infection in the case of a close relative virus.
But hold-up, what on earth are IgG Fc receptors? Well, basically they’re another type of antibodies. They are found sticking out of B-cells or any cells that have the Fc receptor specifically.
What does ADE of viral entry mean for coronaviruses specifically? These researchers discovered a novel mechanism in which fully neutralizing antibodies can mimic the function of a viral receptor when coronaviruses try to get into Fc-receptor expressing cells. As we know, two highly pathogenic coronaviruses have emerged before SARS-CoV-2. These 2 coronaviruses are SARS-CoV, which uses its spike glycoprotein to bind ACE-2 expressing cells (like SARS-CoV-2), and MERS-CoV, which uses its spike glycoprotein to bind to cells expressing a different cell surface receptor called DPP4.
These receptors are found in different cells in our body. The mechanism which SARS-CoV and MERS-CoV use (this means using the S1 and S2 site of the spike glycoprotein) to enter our cells remains the same.
Now with our brains charged and fueled with the knowledge of ADE of viral entry, let’s dive into what these researchers found with their research. Interestingly, ADE has been seen for coronaviruses before this paper. The issue, however, was that we didn’t know the mechanism in which ADE of coronaviruses worked.
To close this gap, these researchers found a monoclonal antibody (MAbs) that can neutralize MERS-CoV entry (coined Mesmab1). A monoclonal antibody is a lab made antibody. Its sole job is to recognize and bind specific proteins or receptors of any cell or virus. They studied how it affects viral entry of MERS-CoV and shine light on the mechanism of ADE of viral entry.
The main thing these researchers found was that by using monoclonal antibodies and testing ADE on coronaviruses, a model system was designed to effectively understand ADE. This means that this system of testing could potentially be used for other viruses, like SARS-CoV-2. The model system this team created can be used to understand how the antibodies our body makes can affect re-infection and ADE of viral entry, and they revealed a novel molecular mechanism by which ADE of coronaviruses acts .
The researchers also showed that if a specific monoclonal antibody can be made, it can reduce the risk of ADE by mimicking the actual receptors that a virus would use to enter our cells. These types of monoclonal antibodies are known as neutralizing antibodies (as we discussed above!). In order to use them as a drug to prevent viral entry, a correct dose of the antibody must be used. If this dosage isn’t correct, there is a potential to cause ADE.
We want to ensure that ADE isn’t induced as this would trigger viral entry into other cells that contain the Fc receptor. This is bad and we don’t want it happening. We just want to ensure that we can use the neutralizing antibodies to prevent entry of the viruses into our cells.
To come to all of those findings, the researchers had to first characterize Mersmab1 interaction with the MERS-CoV virus using a variety of biochemical tests. These tests showed them what components of the spike glycoprotein of MERS-CoV binds to Mersmab1. It also showed them how to continue with their experiments. Specifically, they found that the ectodomain (a component of the MERS-CoV spike protein) binds Mersmab1 really well.
Now these researchers looked at if Mersmab1 helps with the entry of MERS-CoV into cells that expresses the receptor used in ADE of viral entry (the Fc receptor). They found that without Mersmab1, MERS-CoV can’t enter the Fc receptor expressing cells. But with Mersmab1, MERS-CoV can enter cells that express the Fc receptor but can’t get into cells that express DPP4 (the normal receptor used for MERS-CoV entry).
The novel molecular mechanism of ADE was seen when they looked how Mersmab1 causes the MERS-CoV spike protein to change. The changes to the spike protein happen when they are cleaved. They saw that with Mersmab1 there are 2 cleavage events that happens to the spike protein. See our sidebar to learn more about the spike protein cleavage.
They also did experiments looking into the binding between Mersmab1 and the MERS-CoV spike ectodomain. The spike ectodomain is where the receptor binding domain of the MERS-CoV spike glycoprotein is positioned. They found some clues that a monoclonal antibody, like Mersmab1, can bind a spot on a spike protein on a coronavirus, like MERS-CoV or SARS-CoV. This allows for the receptor binding domain to form.
But we still need to know the pathways of Mersmab1-dependent MERS-CoV entry. The researchers investigated this by studying how different proteases (these are proteins that can cleave other proteins, like the spike glycoprotein) are involved. The researcher’s experiments showed that both DPP4 and Mersmab1-dependent MERS-CoV entry are activated by three classes of proteases. This essentially means that the exact same pathway for viral entry is used both when Mersmab1 is used and when it isn’t.
So now the researchers wanted to investigate what doses of Mersmab1 can be given to induce ADE of MERS-CoV entry into Fc receptor presenting cells. To determine the possible doses they could give, they tested three different receptor variations. That lead them to confirm that ADE of MERS-CoV entry into cells depends on dosage of Mersmab1 and the type of receptors present on the cells.
This tells us quite a lot about ADE of viral entry for coronaviruses!
Of course, all this work was done in vitro, which means it was done outside of a living organism. To confirm all these interesting findings, the researchers must also perform in vivo studies. While we are still unsure about the occurrence of ADE in vivo for coronavirus infections, this research team’s previous studies show that it can occur in certain conditions for MERS-CoV infection.
This study shows us just how complicated the role of antibodies can be during viral entry of viruses. It also shines a light on the antibody-dependent enhancement of viral entry for SARS-CoV and even feline coronavirus. This study could also give us some clues on the potential of ADE of viral entry for SARS-CoV-2 too. Of course, we still need to do a lot of work to find out if this phenomenon occurs with SARS-CoV-2. If it does, it could provide some insight into different vaccine designs or even into possible of antibody-based drug therapies to help combat the global pandemic.
In the end, the work these researchers did provides a guideline that could be used for a viral vaccine and antibody-based anti-viral drug design This study could prove to be one step closer to helping us understand coronaviruses and how they can use other mechanisms to enter our cells and start the process of infection. By looking at past research, we could take a step in the right direction towards increasing our understanding SARS-CoV-2.
