Similarly, the low affinity variants for each antibody could be selectively enriched up to 237-fold. of Env and enhanced green fluorescent protein expression to normalize for Env protein levels, and 4) display on HEK cells to ensure native folding and mammalian glycosylation. For proof of concept, we applied our method to a chimeric HIV-1 Env model library comprising variants with differential binding affinities to the V3-loop-directed mAbs 447C52D and HGN194. Fluorescence-activated cell sorting selectively enriched a high affinity variant up to 56- and 55-fold for 447C52D and HGN194, respectively, after only a single round of panning. Similarly, the low affinity variants for each antibody could be selectively enriched up to 237-fold. The binding profiles of membrane-bound gp145 and soluble gp140 chimeras showed identical affinity ranking, suggesting that the technology can AZ-960 guide the identification of Env variants with optimized antigenic properties for subsequent use as vaccine candidates. Finally, our mAb-based cellular display and selection strategy may also prove useful for the development of prophylactic vaccines against pathogens other than HIV. virus peptide 2ATERTtelomerase reverse transcriptaseTettetracyclineTOtetracycline operatorTRtetracycline repressorT-RExtetracycline-regulated expressionV1-V5HIV envelope variable loops 1C5 Introduction With approximately 35 million infected individuals worldwide and about 39 million AIDS-related deaths so far, the human immunodeficiency virus (HIV)-1 pandemic continues to be a major global public health challenge. According to the World Health Organization, only 49% of HIV-1 positive individuals have access to anti-retroviral therapy, affirming the need for an efficient vaccine.1 Regarding the humoral immune response, the HIV-1 surface protein envelope (Env) is the only virus-encoded determinant present on the virus surface, and thus accessible to antibodies.2 Env BSPI is expressed as a gp160 precursor protein that is proteolytically cleaved into gp120 and gp41 by the Golgi-associated furin protease. 3 gp120 and 3 gp41 subunits assemble into the final trimeric (heterohexameric) Env spike, with each gp41 transmembrane subunit being non-covalently associated with the external gp120 subunit, respectively. Whereas gp41 is more conserved, the gp120 subunit has a highly variable and heavily glycosylated surface that includes 5 variable loops (V1CV5).3 The resulting vast number of variants circulating in the human population represents a major challenge for vaccine development, and mainly accounts for the failure of classical vaccine development approaches like chemical inactivation or live attenuation.2 AZ-960 However, after several years of infection, 10C50% of patients develop broadly neutralizing antibodies (bnAbs),4,5 which recognize conserved, mostly conformational or quaternary-structure-dependent epitopes on Env.6 Some of these bnAbs can neutralize up to 80C90% of virus strains.5 Prior to 2009, only a few bnAbs were known, and all targeted either the membrane-proximal external region (MPER), the CD4 binding site, or a glycan-dependent epitope in gp120. Recently, however, the development of AZ-960 highly efficient screening methods has resulted in the isolation of a multitude of new bnAbs targeting multiple sites of vulnerability on the trimer.7 Passive immunization of macaques with bnAbs provided complete protection from infection,8,9,10 offering an important starting point for the development of an efficient vaccine. However, due to the high variability of Env,11 an extensive glycan shield,12 conformational masking of target sites,13,14 and conformational instability of Env,3 the elicitation of bnAbs by active immunization is still a major obstacle in vaccine design. To counter these escape strategies, bnAbs often exhibit unique features like high rates of somatic hypermutation and long CDR3-loops, as a result of years of complex co-evolution between virus escape and immune adaptation.15 To properly instruct the development of antibody responses with a broader neutralization profile, several promising, and not mutually exclusive, approaches have been investigated recently, resulting in new generations of envelope immunogens. The approaches include: 1) a directed evolution approach, which identified a chimeric gp120 Env variant (ST-008) eliciting neutralizing antibody responses in rabbits,16 2) the heterologous substitution or deletion of the V1 loop or hyperglycosylation of variable loops to focus antibody responses to more conserved epitopes like the CD4 binding site,17,18 3) chemical cross-linking of Env aiming toward stabilizing the Env trimer in its closed conformation and directing the humoral immune response to neutralizing epitopes,19 and 4) soluble recombinant Env trimers genetically engineered to form stable, closed and well-folded trimers.20,21,22 Notably, such trimers for the first time enabled the induction of neutralizing antibodies against the sequence-matched tier 2 virus in rabbits and macaques.23,24 To mimic the complex co-evolution between Env and the immune system in AZ-960 vivo, sequential immunization with different Env immunogens was applied to first prime naive B cells with a germline B cell receptor, and then enable generation of the.