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Within-host genetic sequencing from samples collected over time provides adynamic view of how viruses evade host immunity. Immune-driven mutations mightstimulate neutralization breadth by selecting antibodies adapted to cycles ofimmune escape that generate within-subject epitope diversity. Comprehensiveidentification of immune-escape mutations is experimentally and computationallychallenging. With current technology, many more viral sequences can readily beobtained than can be tested for binding and neutralization, makingdown-selection necessary. Typically, this is done manually, by picking variantsthat represent different time-points and branches on a phylogenetic tree. Suchstrategies are likely to miss many relevant mutations and combinations ofmutations, and to be redundant for other mutations. Longitudinal AntigenicSequences and Sites from Intrahost Evolution (LASSIE) uses transmitted-founderloss to identify virus "hot-spots" under putative immune selection and choosessequences that represent recurrent mutations in selected sites. LASSIE favorsearliest sequences in which mutations arise. With well-characterizedlongitudinal Env sequences, we confirmed selected sites were concentrated inantibody contacts and selected sequences represented diverse antigenicphenotypes. Practical applications include rapidly identifying immune targetsunder selective pressure within a subject, selecting minimal sets of reagentsfor immunological assays that characterize evolving antibody responses, and forimmunogens in polyvalent "cocktail" vaccines.

Longitudinal Antigenic Sequences and Sites from Intra-Host Evolution (LASSIE) Identifies Immune-Selected HIV Variants