Humanized V(D)J-rearranging and TdT-expressing Mouse Vaccine Models with Physiological HIV-1 Broadly Neutralizing Antibody Precursors

Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through non-templated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs V{kappa}3-20 or V{kappa}1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had non-physiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as V{kappa}1-33 and/or V{kappa}3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated V{kappa}-to-J{kappa} joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center (GC) B cell responses. V{kappa}3-20-based responses were enhanced by N-region addition, which generates V{kappa}3-20-to-J{kappa} junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs. Significance StatementMouse models that express human precursors of HIV-1 broadly neutralizing antibodies (bnAbs) are useful for evaluating vaccination strategies for eliciting such bnAbs in humans. Prior models were handicapped by non-physiological frequency and/or diversity of B lymphocytes that express the bnAb precursors. We describe a new class of mouse models in which the mice express humanized bnAb precursors at a more physiologically relevant level through developmental rearrangement of both antibody heavy and light chain gene segments that encode the precursors. The model also incorporated a human enzyme that diversifies the rearranging gene segments and promotes generation of certain variable region sequences needed for the response. This new class of mouse models should facilitate preclinical evaluation of candidate human HIV-1 vaccination strategies.