When some viruses infect our bodies, “our immune system can create a type of ‘permanent immunity’, meaning that once you’ve been exposed to that virus, you’ve got protection from it and are typically good for life,” explained Dr. Caelan Radford, a recent PhD graduate from Dr. Jesse Bloom’s lab. This is what happens with measles for example, but it is the best case scenario. Other viruses, like HIV, work pretty hard to slide past the immune system’s surveillance to infect cells even in previously infected individuals. A major reason HIV is so challenging to build immunity against, is that it “has evolved many strategies to prevent the immune system from recognizing various epitopes, or regions” of its viral proteins, especially regions conserved across HIV strains, stated Radford. The virus’ envelope (Env) protein is particularly guilty of continuous diversification across strains of HIV, amplifying this problem. Env is a viral entry protein, which enable viruses like HIV to attach and invade its host’s cells by binding to receptors and fusing the virus and cell membranes. The immune system can prevent viruses from entering cells by producing antibodies that block the virus’ infectious ability, referred to as virus neutralization. While HIV makes it hard for the immune system to produce such antibodies, there are some individuals with HIV that do naturally produce broadly neutralizing antibody responses with neutralizing activity against most of the diverse strains of HIV, highlighting that studying these responses might inform development of HIV therapies like vaccines. In a recently published Cell Host & Microbe study led by Dr. Radford, the Bloom team sought to investigate the neutralizing specificity of human anti-HIV serum to better understand HIV immune responses and inform prevention strategies.
To approach this goal, the Bloom researchers had a few points to consider. First, they needed a way to screen for Env mutations that allow for immune escape. However, since these viruses are rapidly evolving to evade immunity targeting various epitopes on their viral entry proteins, the Bloom team figured that it might not just be individual mutations that enable the virus to put on its invisibility cloak, but rather combinations of mutations. Second, the researchers wanted to develop a much safer way to investigate why HIV is so good at escaping immunity that avoided actually working with HIV mutant viruses. For this, the team turned to deep mutational scanning, which is a versatile tool that allows mapping genotypes to phenotypes to screen the effect of many mutations on a protein of interest. Previously, Drs. Adam Dingens and Hugh Haddox set the groundwork for the current study. Dingens and Haddox used deep mutational scanning to map single HIV envelope mutations that could escape antibody neutralization. Building upon this work, researchers from the Bloom Lab worked to develop a new system that would be able measure the effect of multiple viral mutations simultaneously. Tackling the safety point, the authors realized they only needed to work with a very small portion of the HIV virus, the envelope protein. On its own, the Env protein is harmless and allows scientists to study infection of cells without risk. They chose to use a safe and easily controllable lentiviral system to create barcoded libraries of singly or multiply mutated Env proteins, that can integrate into the genomes of cells that they infect. To generate genotype-phenotype linked lentivirus variant libraries, expression of the barcoded Env mutant genes can be reactivated while making lentiviruses again after the initial infection, enabling the researchers to identify which Env mutants are able to infect cells through a combination of long-read and short-read sequencing. After building this system, Radford et al. could map how human sera that have been shown to have broad HIV neutralizing antibodies target the virus by determining which Env mutants can overcome this neutralization and infect cells.
After developing this system, Radford first gave it a test run with a well-characterized, broadly neutralizing antibody. Their new deep mutational scanning method identified Env mutations at or near the sites where this antibody bound to the envelope protein that enabled escape. Their results correlated well with neutralization assays, thus validating the efficacy of their method. Radford then started to test various human anti-HIV sera that contain antibodies capable of neutralizing the test HIV strain to identify the mutations capable of escaping neutralization. Here he ran into a bit of a roadblock: he found that this strain of HIV was extremely hard to neutralize! Luckily, the Bloom researchers teamed up with colleagues in Germany, who sent some very valuable and particularly potent neutralizing sera from several individuals living with HIV. To Radford’s relief and excitement, he tested the neutralizing ability of these sera for his particularly stubborn HIV strain and it worked! Back on track for discovery of mutations that can escape neutralization, he then added the sera to the mutant Env lentiviral system and used deep mutational scanning to identify which envelope mutations were able to escape neutralization. Most sera tested had neutralizing activity resembling that of a monoclonal antibody. However, serum tested from one individual appeared to target “multiple epitopes simultaneously, and worked very well during the validation neutralization assays, working like two different sera combined,” Radford exclaimed.
Through the development of this deep mutational scanning system, this work will allow the study of naturally elicited responses against HIV and lead to a better understanding of how those types of responses develop and neutralize viruses. Radford emphasized that it took a dedicated team of researchers from the Bloom Lab many years to develop this system and importantly, they took the time to carefully annotate and document this system so that others can apply it to their own questions. While this lentiviral system lives on in the Bloom lab where researchers are using it for their own research curiosities, Radford is excited to see this project expand to “investigate more strains of HIV to understand the differences between these strains and how they escape antibodies.” Radford concluded by acknowledging that many researchers made this important work possible, specifically thanking Drs. Katharine Crawford and Bernadeta Dadonaite who worked with him to initially develop this deep mutational scanning and lentiviral system to study SARS-CoV-2 spike mutations. He also thanks colleagues in Germany, Drs. Florian Klein, Philipp Schommers and Lutz Gieselmann who provided the human anti-HIV sera used in this study.
This work was supported by the National Institutes of Health and the National Institute of Allergy and Infectious Disease.
UW/Fred Hutch/Seattle Children’s Cancer Consortium member Julie Overbaugh contributed to this work.
Radford CE, Schommers P, Gieselmann L, Crawford KHD, Dadonaite B, Yu TC, Dingens AS, Overbaugh J, Klein F, Bloom JD. Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning. Cell Host Microbe. 2023 Jul 12;31(7):1200-1215.e9. doi: 10.1016/j.chom.2023.05.025. Epub 2023 Jun 15. PMID: 37327779