Phage display libraries and deep mutational scanning to map antibody epitopes in HIV-1

From the Human Biology and Basic Sciences Division

Mapping the individual amino acids where antibodies bind (epitopes) to target viral proteins (antigens) is crucial to predicting how target proteins may evolve to escape antibody binding, improve antigen design for vaccines, and understand antibody function. For HIV-1, multiple structural and site-directed mutagenesis approaches have provided detailed information about important antibody epitopes. Recently, deep mutational scanning (DMS), a methodology that simultaneously evaluates the functional consequences of every possible single-point mutation across a protein sequence, has revealed key epitopes that impact neutralization in of HIV-specific antibodies.

In a quest to detect the effects of mutations on non-neutralizing functions, researchers in the Overbaugh (Human Biology) and Bloom (Basic Sciences) labs, led by Molecular and Cell Biology graduate student Meghan Garrett, developed Phage-DMS, a method that combines phage display technology and DMS to identify antibody epitopes. In the study, Phage-DMS was applied to two key immunodominant regions of the HIV envelope protein (Env), the V3 region of gp120 and the gp41 ectodomain. The researchers identified the epitopes of four well-characterized HIV monoclonal antibodies. Their results, now published in the journal iScience, confirmed previously determined escape sites from other approaches and identified novel epitope sites.

To generate the phage display libraries, synthesized gene fragments encoding wild-type and mutant peptides from the V3 region of gp120 or the gp41 ectodomain are cloned into a phage display vector. Each phage in the library displays a particular variant, which then is incubated with the antibody of interest. The phage-antibody complexes are then immunoprecipitated with magnetic beads. DNA is extracted and amplified from the selected phages, sequenced, and computationally analyzed to determine the effects of single mutations in antibody binding. Meghan Garrett explained the potential applications of the technology: “Isolating antibodies from people who have been infected with a virus has been getting faster these days but taking those antibodies and figuring out where they bind is still a slow process. The method we developed speeds up this epitope mapping process and is a lot more precise than most other methods out there. Phage-DMS will tell you exactly which mutations result in loss of antibody binding, which could be useful in predicting escape mutations the virus could use to escape the immune response.”

Schematic for Phage-DMS approach.
Schematic of Phage-DMS Library Design and Experimental Approach Image provided by Meghan Garrett.

A surprising finding from the study is the observation that mutating residues near the antibody-antigen interface (as previously determined in structural studies) do not always result in functional changes. Equally surprising is the finding that mutations in distant sites can impact binding. “For example, previous studies showed that residues 595 to 609 on HIV Envelope were in close proximity to the F240 antibody when it binds to the protein. However, we showed that mutating several of these residues doesn’t significantly change the antibody's ability to bind. On the flip side, there are sites on Env not in close proximity to the antibody when you look at a crystal structure, but if you make specific mutations at those more distant sites, you can get loss of antibody binding. This all goes to show that it is important to focus on the “functional” epitope, which is different from the structural epitope,” said Garrett.

Finally, Garrett shared future applications for the Phage-DMS technology. “The next step is to finely map the antibody responses. Whereas our previous work was with monoclonal antibodies, we are interested in next mapping the polyclonal antibody response in people. We have been using Phage-DMS to study where antibodies in COVID patient plasma bind, which is an exciting use of this new technology,” Garrett said.

Garrett ME, Itell HL, Crawford KHD, Basom R, Bloom JD, Overbaugh J. (2020). Phage-DMS: A Comprehensive Method for Fine Mapping of Antibody Epitopes. iScience. 29;23(10):101622. doi: 10.1016/j.isci.2020.101622.

This work was supported by grants from the National Institutes of Health.

Fred Hutch/UW Cancer Consortium member Julie Overbaugh contributed to this study.