Scientists have identified a new way in which antibodies disable viruses. In addition to latching on to antigenic proteins on the surface of a virus and blocking the virus from infecting host cells, a new study shows antibodies can also burrow in and distort the viral surface, effectively preventing the virus from docking on and entering host cells.

An international team of researchers led by scientists at Penn State report this new finding in an article in the journal Cell, titled “Human antibody C10 neutralizes by diminishing zika 2 but enhancing dengue dynamics.” The authors show the same antibody can neutralize zika and dengue in two different ways.

The same antibody can neutralize zika and dengue viruses in two different ways — one where it binds to the virus and deactivates it (left), which is the traditional way we think about antibody activity, and the other where it burrows in and distorts the virus (right). [Source: Ganesh Anand, Penn State]
“This study reveals a new mode of virus neutralization by antibodies. Antibodies have been traditionally assumed to neutralize their targets by a sole mechanism of blocking the surface, so that the virus cannot access its target receptor site. We have demonstrated that antibodies show additional mechanisms of virus neutralization. They distort their virus targets by burrowing into the target surface,” says Ganesh Anand, PhD, associate professor of chemistry at Penn State and co-corresponding author of the paper.

“Furthermore,” says Anand, “different concentrations of antibodies elicit different conformational changes in the entire virus particle. This study emphasizes how a virus represents a moving target for antibodies, which in turn alters the modes of antibody engagement. This also reveals that viruses offer a nonuniform epitope landscape for antibodies to bind and neutralize.”

Anand and his colleagues investigate the interactions between human monoclonal antibody (HMAb) C10 and the two disease-causing viruses that the antibody strongly neutralizes.

Combining cryogenic electron microscopy (cryo-EM) and hydrogen/deuterium exchange mass spectrometry (HDXMS), the researchers visualize the two viruses and explore their movement in presence of the antibody.

“Cryo-EM involves flash-freezing a solution containing molecules of interest and targeting them with electrons to generate numerous images of individual molecules in different orientations,” says Anand. “These images are then integrated into one snapshot of what the molecule looks like. The technique provides more accurate pictures of molecules than other forms of microscopy.” The team collected high resolution, static cryo-EM snapshots of the viruses in increasing concentrations of the HMAb C10 antibody.

“The notion that viruses are highly mobile targets, that certain antibodies enhance the virus’ mobility and achieve neutralization through distortion came from amide HDXMS. HDXMS is a method that can be leveraged to measure the movements of virus surface proteins,” says Anand. HDXMS involves submerging molecules of interest — in this case zika and dengue virus with HMAb C10 antibodies—in “heavy water” that has had its hydrogen atoms replaced with deuterium, hydrogen’s heavier isotope.

“When you submerge a virus in heavy water, the hydrogen atoms on the surface of the virus exchange with deuterium,” says Anand. “You can then use mass spectrometry to measure the heaviness of the virus as a function of this deuterium exchange. By doing this, we observed that dengue virus, but not zika virus, became heavier with deuterium as more antibodies were added to the solution. This suggests that for dengue virus, the antibodies are distorting the virus and allowing more deuterium to get in. It’s as if the virus is getting squished and more surface area becomes exposed where hydrogen can be exchanged for deuterium.”

Although HMAb C10 effectively neutralizes both zika and dengue viruses, this HDXMS data shows while the antibody squishes the dengue virus, it does not result in increased incorporation of heavy water in the zika virus, indicating the antibody does not squish the zika surface.

“The combination of static imaging with dynamic visualization by mass spectrometry is novel and provided orthogonal complementary insights into virus behavior in solution,” says Anand. “It’s like those cartoon flipbooks, where each page has a slightly different image, and when you flip through the book, you see a short movie. Imagine a flipbook with drawings of a racehorse. Cryo-EM shows you what the racehorse looks like and HDXMS shows you how fast the racehorse is moving. You need both techniques to be able to describe a racehorse in motion. This complementary set of tools enabled us to understand how one type of antibody differentially affects two types of viruses.”

The researchers note, the more antibodies they add, the more distorted the dengue virus particles become, suggesting that the relationship between the quantities of interacting molecules determines the extent of neutralization. At saturating conditions, in which antibodies are added at high enough concentrations to fill all available binding locations on the dengue viruses, the researchers show, 60% of the virus’ surface is distorted. This distortion is enough to protect host cells from infection.

“If you have enough antibodies, they will distort the virus particle enough so that it’s preemptively destabilized before it even reaches its target cells,” Anand says.

When the scientists expose BHK-21 cells—a cell line derived from the kidneys of baby hamsters–to antibody-bound dengue viruses, they see 50–70% fewer cells are infected.

Anand explains, while “antibodies can work by jamming the exits so the passenger cannot get out of the car, we have found a new mechanism in dengue virus where antibodies basically total the car so it cannot even travel to a cell.”

Unlike SARS-CoV-2, which has spike proteins protruding in all directions, Anand explains, the surfaces of both zika and dengue are smoother with easily accessible “peaks” known as five-fold vertices and progressively inaccessible “valleys” known three-fold and two-fold vertices.

“Antibodies do not like two-fold vertices because they are very mobile and difficult to bind to,” says Anand. “We found that once the five- and three-fold vertices have been fully bound with antibodies, if we add more antibodies to the solution, the virus starts to shudder. There’s this competition taking place between antibodies trying to get in and the virus trying to shake them off. As a result, these antibodies end up burrowing into the virus rather than binding onto the 2-fold vertices, and we think it’s this digging into the virus particle that causes the virus to shake and distort and ultimately become non-functional.”

Zika is a more stable and less dynamic virus than dengue, which has a lot of moving parts, explains Anand. “Dengue and Zika look similar but each one has a different give. Dengue may have evolved as a more mobile virus as a way of avoiding being caught by antibodies. Its moving parts confuse and throw off the immune system. Unfortunately for dengue, antibodies have evolved a way around this by burrowing into the virus and distorting it.”

The distortion strategy of virus neutralization is not unique to antibody engagement with dengue and zika viruses. “Dengue is just a model virus that we used in our experiments, but we think this preemptive destabilization strategy may be broadly applicable to any virus,” says Anand. “It may be that the antibodies first attempt to neutralize viruses through the barrier method and if they are unsuccessful, they resort to the distortion method.”

These new findings could be useful in designing therapeutic antibodies, Anand says. “HMAb C10 antibodies are specific to dengue and zika viruses and happen to be capable of neutralizing zika and dengue viruses in two different ways, but you could potentially design therapeutics with the same capabilities for treating other diseases, such as COVID-19. By creating a therapeutic with antibodies that can both block and distort viruses, we can possibly achieve greater neutralization.”

He adds, “You don’t want to wait for a virus to reach its target tissue, so if you can introduce such a therapeutic cocktail as a nasal spray where the virus first enters the body, you can prevent it from entering the system. By doing this, you may even be able to use less antibody since our research shows that it takes less antibody to neutralize a virus through the distortion method. You can get better bang for the buck.”

The study uncovers a new strategy that antibodies use to disable viruses. Future studies by the group will probe deeper into the terms of this novel engagement. “We are trying to define the first principles of antibody engagement on virus surfaces. We are particularly interested in identifying the rules for destabilizing or distorting antibody design. This novel mechanism would add to the antiviral arsenal. We wish to develop targeted virus-distorting antibodies. We are also interested in correlating virus mutational hotspots with mobile loci on the surface,” says Anand.