Plates were washed 5, and the secondary Ab conjugated with HRP diluted in dilution buffer was added to each well. and functional bnAb features, after a single immunization. Keywords:human immunodeficiency virus, germline-targeting vaccine, envelope trimer, broadly neutralizing antibody == Graphical abstract == == Highlights == VRC01-class naive B cells can acquire many bnAb-like mutations after 1 immunization VRC01-class Abs neutralizing multiple N276 HIV pseudoviruses were elicited Trimer-based but not minimal GT immunogen priming elicited key trimer-binding SHMs Protective broadly neutralizing antibodies (bnAbs) to HIV are difficult to elicit by vaccination because they require mutations that have been observed to take years in HIV-infected humans. Lee Ruxolitinib Phosphate et al. show that several key mutations required for Env trimer binding and bnAb development can be acquired after a single immunization. == Introduction == Discovery of an efficacious vaccine against the human immunodeficiency virus (HIV) has been difficult because of the high mutability and diversity of HIV. For an antibody-based HIV vaccine to offer protection, it Ruxolitinib Phosphate would need to elicit broadly neutralizing antibodies (bnAbs) that can potently neutralize a wide range of primary HIV isolates. HIV bnAbs are uncommon at least in part because naive B cells with bnAb-class epitope specificities are rare (Briney et al., 2019;Havenar-Daughton et al., 2018;Jardine et al., 2016a;Steichen et al., 2019) and have features atypical of other antibodies, such as unusually long heavy-chain (HC) complementarity-determining regions (CDRs) (Doria-Rose et al., 2014;Falkowska et al., 2014;Freund et al., 2017;Landais et al., 2017;MacLeod et al., 2016;Sok et al., 2014;Walker et al., 2009,2011) or a short light-chain (LC) CDR loop (Zhou et al., 2013). Potent bnAbs are typically found in individuals chronically infected with HIV after years of infection and have more somatic hypermutations (SHMs) than usually elicited in response to vaccinations (Jardine et al., 2016b;Sok and Burton, 2018). Some key mutations in bnAbs are not commonly observed in typical antibodies, including insertions, deletions, and disulfide bonds (Doria-Rose et al., 2014;Landais and Moore, 2018;Zhou et al., 2010). An HIV vaccine research approach called structure-based rational vaccine design strives to design immunogens that recapitulate naturally occurring bnAb responses (Burton, 2017;Stamatatos et al., 2017). One strategy involves priming with an immunogen that has a high binding affinity for predicted naive precursors of known bnAb-class B cells (Jardine et al., 2013). Theoretically, these germline-targeting (GT) immunogens would activate rare bnAb precursor B cells and induce germinal center (GC) responses (Mesin et al., 2016) to expand the population of bnAb precursors and initiate affinity maturation. Booster immunizations are administered sequentially to deliver immunogens more closely resembling the native Env trimer, guiding acquisition of SHMs needed for breadth and potency (Briney et al., 2016;Escolano et al., 2016;Jardine et al., 2016b;Steichen et al., 2016;Tian et al., 2016). One broadly neutralizing epitope that is promising for epitope-specific vaccine design is the CD4 binding site (CD4bs). As the receptor binding site on Env, the CD4bs is highly conserved across HIV Ruxolitinib Phosphate isolates, and bnAbs recognizing this epitope have been identified in multiple HIV-positive donors (Bonsignori et al., 2016;Huang et al., 2016;Kong et al., 2016;Umotoy et al., 2019;Zhou et al., 2015). VRC01 is a representative CD4bs bnAb with high breadth and potency. VRC01-class bnAbs utilize an IGHV1-2 HC gene that is found in approximately 2%4% of the human B cell repertoire (Arnaout et al., 2011;DeKosky et al., 2015). Epitope recognition by VRC01-class bnAbs relies on several conserved features arising from their IGHV1-202 gene. The HC CDR3 (H-CDR3) can Rabbit Polyclonal to HTR7 be variable but uses a tryptophan residue at the fifth to the last residue (Trp100B in VRC01). Various LC V-genes are observed but have a short, 5-amino acid (aa) L-CDR3 (Diskin et al., 2011;Huang et al., 2016;Umotoy et al., 2019;West et al., 2012;Zhou et al., 2015). Strategies to elicit VRC01-class bnAbs by vaccination have been investigated extensively (Briney et al., 2016;Chen et al., 2021;Duan et al., 2018;Jardine et al., 2013,2016a;McGuire et al., 2014;Stamatatos et al., 2017). Several GT immunogens exist for VRC01-class bnAbs, at least two of which are currently being tested in phase 1 clinical trials (ClinicalTrials.gov:NCT03547245,NCT04224701;Jardine et al., 2016a;Medina-Ramrez et al., 2017). If rare VRC01-class naive precursors can be primed, then one of the biggest hurdles of achieving neutralization potency and breadth is accommodation of a conserved N-linked glycan at position N276 of Env near the CD4bs. To do so, VRC01-class bnAbs typically introduce glycine residues and/or acquire deletions in the L-CDR1 to promote loop flexibility Ruxolitinib Phosphate (Zhou et al., 2015) or expand the epitope contact surface area via H-CDR3 to enhance binding (Bonsignori et al., 2018). Animal immunization models to assess VRC01-class B cell targeting immunogens have shown that the ability to accommodate.