Direct probing of germinal center responses reveals immunological features and bottlenecks for nAb responses to an engineered HIV trimer

Generation of Tier 2 HIV neutralizing antibody (nAb) responses by immunization remains a challenging problem, and the immunological barriers to induction of such responses by Env immunogens remain unclear. We explored these barriers by combining a suite of innovative techniques, including longitudinal lymph node fine needle aspirates, germinal center (GC) B cell lineage tracking, and a new method for detecting and quantifying GC T follicular helper (GC Tfh) cells, in non-human primates immunized with a native-like HIV-1 Env trimer protein (BG505 SOSIP.v5.2). A majority of immunized animals (9/12) developed Tier 2 neutralizing antibodies (nAb). Tier 2 nAb development best correlated with GC B cell magnitude in response to later booster immunizations and the quality of the Tfh help. Notably, these immunological factors distinguished between qualitatively successful and unsuccessful vaccine Ab responses, as they correlated with nAb development but did not correlate with simple Env Ab binding titers. Therefore, direct probing of germinal centers in future vaccine trials is key, as this suite of technically robust approaches provides quantitation of the proximal immune correlates of neutralizing antibody development and could allow redesign of optimal multi-stage vaccination schedules. RNA-sequencing of circulating HIV Env-specific CD4 cells isolated from a cohort of rhesus macaques immunized with BG505 SOSIP.v5.2 HIV Env trimer. Cells were directly isolated from blood by flow cytometry using the AIM assay. Total RNA was extracted, messenger RNA was selected and cDNA was amplified linearly with a PCR based method (Picelli et al. 2014). Libraries were prepared. using the NexteraXT Illumina sequencing platform.