“Cryo-EM involves flash-freezing a solution containing molecules of interest and then targeting them with electrons to generate numerous images of individual molecules in different orientations,” explained Anand. “These images are then integrated into one snapshot of what the molecule looks like. The technique provides much more accurate pictures of molecules than other forms of microscopy.”

To document the effects of antibodies on Zika and dengue viruses, the team collected cryo-EM snapshots of the viruses under conditions of increasing concentrations of antibodies.

In parallel, the team applied HDXMS, a technique in which molecules of interest — in this case Zika and dengue virus, along with HMAb C10 antibodies — are submerged in heavy water. Heavy water, Anand explained, has had its hydrogen atoms replaced with deuterium, hydrogen’s heavier isotopic cousin.

“When you submerge a virus in heavy water, the hydrogen atoms on the surface of the virus exchange with deuterium,” he said. “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.”

In contrast, Zika virus did not become heavier when placed in heavy water, suggesting that its surface, while fully occupied by antibodies, is not distorted by the antibodies.

Anand explained that by combining cryo-EM and HDXMS, the team was able to get a comprehensive picture of what happens when antibodies attach to Zika and dengue viruses.

“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,” he said. “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.”

He noted that the fact that the more antibodies they added, the more distorted the dengue virus particles became, suggests that stoichiometry — the relationship between the quantities of the reactants and the products before, during and after a chemical reaction — matters.

“It’s not enough to just have antibodies present,” he said. “How much antibody you add determines the extent of neutralization.”

In fact, the team found that at saturating conditions, in which antibodies were added at high enough concentrations to fill all the available binding locations on the dengue viruses, 60% of the virus’ surfaces became distorted. This distortion was enough to protect the 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 said.

Indeed, when the scientists incubated the antibody-bound dengue viruses with BHK-21 cells, a cell line from the kidneys of baby hamsters that is often used in viral infection research, they found that 50-70% fewer cells were infected.

Anand explained that with some viruses, including Zika, antibodies work by jamming the exits so the passenger cannot get out of the car. We have found a new mechanism in dengue virus whereby antibodies basically total the car so it cannot even travel to a cell.”

How are the antibodies distorting the dengue virus particles?

Anand explained that contrary to the now-familiar SARS-CoV-2, which has spike proteins protruding in all directions, the surfaces of both Zika and dengue is a smoother surface with peaks and valleys.

Once all the proteins on the 5-fold vertices have been bound, antibodies will turn to their second-favorite peaks — the 3-fold vertices. Finally, they are left with only the 2-fold vertices.

“Antibodies do not like two-fold vertices because they are very mobile and difficult to bind to,” said Anand. “We found that once the 5- and 3-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.”

What is the difference between Zika and dengue?

Anand explained that Zika is a much more stable, less dynamic virus than dengue, which has a lot of moving parts.

“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.”

It appears, he said, that the same type of antibody can neutralize Zika and dengue in two different ways — one where it binds to the virus and deactivates it, which is the traditional way we think about antibody activity, and the other where it burrows in and distorts the virus.

What about other viruses?

Anand said the distortion strategy his team discovered may be used by antibodies when they are confronted with other types of viruses as well.

“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,” he said. “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.”

Are there any potential applications of the findings?

The findings could be useful in designing therapeutic antibodies, Anand said.

“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,” he said. “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 added, “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 even 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.”

Overall, Anand stressed that the importance of the study is that it reveals an entirely new strategy that some antibodies use to disable some viruses.

“Previously, all we knew about antibodies was that they bind and neutralize viruses,” he said. “Now we know that antibodies can neutralize viruses in at least two different ways, and perhaps even more. This research opens the door to a whole new avenue of exploration.”

Other authors on the paper include Xin-Xiang Lim, graduate student; Jian Shi, manager, Cryo-EM Facility; and Shee-Mei Lok, professor, National University of Singapore. Co-authors also include Bo Shu, research fellow; Shuijun Zhang, assistant professor; Aaron W.K. Tan, graduate student; Thiam-Seng Ng, graduate student; Xin-Ni Lim, graduate student; and Valerie Chew, assistant professor, Duke-National University of Singapore Medical School. Gavin R. Screaton, head of the Medical Sciences Division, Oxford University, also is an author.

This research was supported by the National Research Foundation of Singapore, the Ministry of Health of Singapore and by Penn State.

Source: Eurekalert