A major goal of HIV-1 vaccine development is induction of broadly neutralizing antibodies (bnAbs). While success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knock-in mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof-of concept and milestone towards development of an HIV vaccine.

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Antibodies are generated by B cells that evolve receptor specificity to pathogens through rounds of mutation and selection in a process called affinity maturation. Somatic hypermutation is mediated by an enzyme with DNA sequence context-dependent targeting and substitution resulting in variable probabilities of amino acid substitutions during affinity maturation. We have previously developed a program called Antigen Receptor Mutation Analyzer for the Detection of Low Likelihood Occurrences (ARMADiLLO) that performs simulations of the somatic hypermutation process to estimate the probabilities of observed antibody mutations. Here we describe the ARMADiLLO web server (https://armadillo.dhvi.duke.edu), an easy-to-use web interface that analyzes input antibody sequences and displays the probability estimates for all possible amino acid changes over the full length of an antibody sequence. The probability of antibody mutations can be used by immunologists studying B cell ontogenies and by vaccine designers that are pursuing strategies to elicit broadly neutralizing antibodies which are enriched with developmentally rate-limiting improbable mutations. The ARMADiLLO web server also contains precomputed results reporting the probability of amino acid substitutions in all human V gene segments and in a collection of HIV broadly neutralizing antibodies.