One of the most challenging aspects of combatting HIV-1 infection is that the virus continually evades neutralizing antibodies. However, one consequence of this is that a small percentage of people with HIV-1 (1-5%) develop rare, broadly neutralizing antibodies (bNAbs) that can neutralize a large fraction of global HIV-1 isolates. These broadly neutralizing antibodies are among the most promising new long-acting HIV treatments, offering the potential to forego traditional daily dose of antiretroviral drugs. Indeed, a recent trial found that participants who received a single dose of two bNAbs maintained a nearly undetectable viral load for up to 20 weeks, and a third did so for about a year.

Despite the known promise of bNAbs, the pathways through which the virus escapes these antibodies remain incompletely understood across diverse HIV-1 strains.

“Knowing how different strains of the virus respond to leading bNAb therapies will greatly improve our ability to anticipate whether a particular therapy will be effective for individual patients,” says Paul Bieniasz, PhD, professor at The Rockefeller University and an HHMI Investigator. “And if we can identify broadly neutralizing antibodies that the majority of strains have great difficulty escaping from, we can create more robust treatments.”

Now scientists have established the most comprehensive view to date of how HIV-1 can escape bNAbs. Using thousands of parallel viral selection experiments combined with bioinformatic analysis and experimental validation, the team discovered viral mutations that make HIV-1 strains resistant to two bNAbs: 3BNC117 and 10-1074.

This work is published in Nature Microbiology in the paper, “Diverse paths to broadly neutralizing antibody escape among HIV-1 strains.“

The researchers sought to investigate the relationship between different HIV-1 strains and bNAbs collected from HIV infected persons. Only a handful of resistance mutations have been identified in a limited number of viral strains. The researchers wanted to expand that number to represent global viral diversity.

“No one has attempted to do this at such a scale before,” said Theodora Hatziioannou, PhD, research professor at The Rockefeller University.

The team developed an approach that would allow them to study the mutational pathways to escape among 15 strains of HIV-1 sourced from around the globe. The goal was to pinpoint the mutations that were contributing to each strain’s propensity to develop resistance.

“We found that most viral strains can escape bNAb neutralization, but there’s substantial variation in the likelihood that they will and the mechanisms that enable it,” says Alex Stabell, MD, PhD, an infectious disease physician and clinical scholar at The Rockefeller.

Stabell devised a pipeline that began by growing large amounts of virus in cell culture. The bulk populations were used to seed thousands of parallel selection experiments with varying concentrations of bNAbs. Viruses that were able to spread in the presence of the bNAbs were isolated and sequenced. Custom bioinformatic processing gave a list of putative resistance mutations, which were subsequently experimentally validated for each viral strain.

Using this method, called RISC (resistance identification via selection and cloning), the team found more than 100 bNAb escape mutations across the 15 viral strains tested, dramatically expanding the known number. Surprisingly, they found that in most cases, a single amino acid change may be enough to confer resistance. That turned out to be true for 12 of the 15 viruses tested against the 3BNC117 antibody and for all nine tested against 10-1074.

“It was striking that it’s actually quite easy for most HIV strains to escape these special antibodies,” Bienasz says. “But it’s not true for all strains—a handful Alex worked with needed multiple amino acid substitutions or unusual ways to replicate in order to escape.”

“The genetic barrier to resistance was higher for these viruses,” Stabell adds. “One of the goals of therapy these days is not simply to have therapies that are transiently effective, but to have this high genetic barrier.”

They also identified a surprising number of mutations occurring outside the epitope on the viral envelope recognized by bNabs that target the CD4 binding site, such as 3BNC117. (10-1074 aims for a more mutable envelope target, which may help explain why it’s easier to escape.) “These were quite prominent and unexpected,” says Hatziioannou. “No one would have predicted these would affect bNAb sensitivity.”

In the future, the team will use Stabell’s method to identify to discover resistance mutations to other bNAbs as well as to combinations of them.

“HIV-1 mutates so fast and the diversity in the population is already quite enormous, so we’ve long known that a multidrug approach is the best course of treatment,” Hatziioannou says. “We hope to identify combinations that potentially raise the genetic barrier to resistance and are therefore more effective.”

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