Complete mapping of viral escape from neutralizing antibodies

Abstract

Identifying viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a high-throughput approach to quantify the selection that monoclonal antibodies exert on all single amino-acid mutations to a viral protein. This approach, mutational antigenic profiling, involves creating all replication-competent protein variants of a virus, selecting with antibody, and using deep sequencing to identify enriched mutations. We use mutational antigenic profiling to comprehensively identify mutations that enable influenza virus to escape four monoclonal antibodies targeting hemagglutinin, and validate key findings with neutralization assays. We find remarkable mutation-level idiosyncrasy in antibody escape: for instance, at a single residue targeted by two antibodies, some mutations escape both antibodies while other mutations escape only one or the other. Because mutational antigenic profiling rapidly maps all mutations selected by an antibody, it is useful for elucidating immune specificities and interpreting the antigenic consequences of viral genetic variation.

Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants

“…Our work did not characterize the antigenic effects of mutations, which also play an important role in determining strain success in nature (13,14). However, our basic selection and deep-sequencing approach can be harnessed to completely map how mutations affect antibody recognition (57,80). But so far, experiments using this approach have not examined antibodies or sera that are relevant to driving the evolution of H3N2 influenza (57,80), or have used relevant sera but examined a non-comprehensive set of mutations (16).…”

“…However, our basic selection and deep-sequencing approach can be harnessed to completely map how mutations affect antibody recognition (57,80). But so far, experiments using this approach have not examined antibodies or sera that are relevant to driving the evolution of H3N2 influenza (57,80), or have used relevant sera but examined a non-comprehensive set of mutations (16). Future experiments that completely map how HA mutations affect recognition by human sera seem likely to be especially fruitful for informing viral forecasting.…”

Comprehensive profiling of translation initiation in influenza virus infected cells

“…Virus titering 859 By HA staining 860 We infected A549 cells in WSN growth media for 10 hours, collected cells, and stained 861 for HA using H7-L19 antibody [115,116] at 10 ug/ml, followed by goat anti-mouse IgG 862 secondary antibody conjugated to APC, and analyzed by flow cytometry to determine 863 the fraction of cells that were HA positive. We used the Poisson equation to calculate 864 viral titer with respect to HA expressing units.…”

Precision epidemiology for infectious disease control

“…1). Applying genomic technologies during the development and usage of immunotherapeutics (for example, monoclonal antibody cocktails 25 ) and vaccines can also provide insights into pathogen strategies for immune response evasion 26,27 and mechanisms of virulence 28,29 . By characterizing longitudinal samples from the same patients, pathogen sequencing also provides the potential for identifying genetic components involved in driving disease progression, thus providing novel drug targets 30 .…”