Using a cutting-edge approach, scientists at Fred Hutchinson Cancer Research Center constructed an atlas of mutations HIV uses to escape broadly neutralizing antibodies, potent immune molecules that form our body’s first line of defense against the virus. The information could help guide researchers who are testing broadly neutralizing antibodies’ potential to prevent or treat HIV infection, as well as those working to develop more effective preventive vaccines.
Published today in the journal Immunity, the work shows that mutations that affect whether antibodies can block HIV occur in areas of the virus’ envelope protein that directly touch antibodies, and in areas beyond antibodies' reach.
“This is a game-changer in saying, what’s important are the functional interactions [between antibodies and HIV], and they’re what’s going to help us understand how the virus can escape in a person if we do have a vaccine, or we do use antibodies as therapy,” said Hutch HIV researcher Dr. Julie Overbaugh, who holds the Endowed Chair for Graduate Education. She participated in the research with Hutch colleagues Dr. Jesse Bloom and graduate student Adam Dingens. “And we weren’t getting that picture before — we were just getting pieces of it.”
Antibodies, specialized immune proteins that can bind and block viral infection, are among the body’s first line of defense against germs. Some, known as broadly neutralizing antibodies, are able to block many variants of a virus. HIV researchers have long studied the sites on HIV’s external protein, where antibody meets virus, in the hope of understanding what makes these special antibodies able to foil many viral strains.
Vaccine designers hope to use this understanding to make HIV vaccines that trigger protective broadly neutralizing antibody responses. And broadly neutralizing antibodies themselves are being tested as potential therapeutics to prevent and even treat HIV.
“In these immunotherapy clinical trials, there’s been viral escape from each antibody, and it’s critical to understand how that escape occurs,” said Dingens, who spearheaded the study.
The location on the virus that’s directly contacted by the antibody — in this case, a segment of HIV’s envelope protein — is called the structural epitope. But the structural epitope isn’t the whole picture, Dingens noted. What’s really important is the functional epitope. This is the area that affects whether an antibody can block HIV, even if it doesn’t directly contact the antibody.
HIV's envelope proteins are essentially carefully folded, yet pliable, strings of different amino acids. A mutation that results in one amino acid being swapped for another can have ripple effects, subtly altering the structure of the folds so that a once-neutralizing antibody can no longer recognize that region of the virus.
But HIV is an ever-changing pathogen that is up against our immune system, which can generate a mind-boggling array of different antibodies. Looking at the problem one epitope at a time is a little like trying to understand a puzzle by studying just one piece.
Dingens wanted to get a big-picture view of broadly neutralizing antibodies’ functional epitopes.
To put the puzzle together, he used an approach called deep mutational scanning, which computational biologist Bloom had adapted for HIV and other viruses. Dingens had already used the method to examine the functional epitope of a single antibody against HIV. Since then, he extended it to study several antibodies at once.
Dingens used deep mutational scanning to create a library of viruses containing all possible mutations in the gene that encodes HIV’s envelope protein. He then passed the mutated viruses through a gauntlet of broadly neutralizing antibodies and allowed those that escaped neutralization to infect cells. Finally, he measured how much each mutation affected HIV’s ability to skirt the antibody blockade.
No broadly neutralizing antibody can block all variants of HIV. To cover their bases, the researchers chose to test the evasion of nine well-characterized broadly neutralizing antibodies.
Using his method, Dingens outlined each antibody’s functional epitope, defined as the site where mutation allowed the viruses to escape neutralizing antibodies and infect cells. The team was also able to learn which regions of the structural epitope contribute the most to each antibody’s ability to block HIV.
“The fact that he could do it for all the main classes of antibodies is a bonus,” Overbaugh said. “They’re all heavily studied, to the point it was surprising there was so much information to add in one study.”
As HIV evolves to dodge broadly neutralizing antibodies, it builds up mutations — but without functional information, it’s not always clear which mutations are affecting virus escape.
“The findings could be very useful,” Bloom said. “When you look at a viral genetic sequence, you want to actually know what a mutation does.”
The team also gained important insights about an HIV mutation that researchers have already seen in a clinical trial of broadly neutralizing antibodies. Using the atlas, they singled out a specific mutation, outside the antibody’s structural epitope, as a key stop on the virus’ path to escape.
Assessing the importance of mutations seen in clinical trials is just the beginning, Bloom said. The researchers hope that deep mutational scanning, and the atlases of functional epitopes it will allow scientists to build, will help guide virologists by enabling them to think beyond the structural epitope.
The findings suggest that as scientists choose antibodies to develop as therapeutics, they should take into account how easily viruses could mutate to escape a specific antibody, Dingens said.
The data from this study will be immediately applicable for this purpose, Overbaugh said. Understanding potential overlap between functional epitopes will help them pick the right combination of antibodies that block all of HIV’s exit routes.
Researchers studying other viruses, including influenza, also seek out broadly neutralizing antibodies and the clues they hold for understanding viruses’ vulnerabilities. The current work suggests a totally different way to map viral epitopes of newly discovered broadly neutralizing antibodies, Overbaugh said. Instead of working backward from a new antibody’s structure, scientists will learn more direct information by performing deep mutational scanning to chart its functional epitope, she said.
Dingens' strategy could be applied to viruses beyond HIV, said Bloom. Influenza is another virus for which researchers are hoping to design a broadly neutralizing antibody–triggering vaccine. A deeper understanding of the functional epitopes of already characterized antibodies against the virus would aid that quest.
Bloom also noted that the enormous variability in both HIV and antibodies means that there’s a much larger landscape of functional epitopes and HIV-escape mutations that has yet to be charted. For example, in order to map the functional epitopes of more antibodies, a collection of libraries covering more HIV strains must be created, Overbaugh said.
“The atlas allows us to better understand the relationship between structure and function,” Dingens said. “It opens the door.”
Sabrina Richards, a staff writer at Fred Hutchinson Cancer Center, has written about scientific research and the environment for The Scientist and OnEarth Magazine. She has a PhD in immunology from the University of Washington, an MA in journalism and an advanced certificate from the Science, Health and Environmental Reporting Program at New York University. Reach her at srichar2@fredhutch.org.
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