Lymph node fine needle aspirates reveal germinal centers

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Lymph node fine needle aspirates reveal germinal centers as predictors of HIV trimer-induced neutralizing

Lymph node fine needle aspirates reveal germinal centers as predictors of HIV trimer-induced neutralizing antibodies Colin Havenar-Daughton Laboratory of Shane Crotty La Jolla Institute for Allergy and Immunology IAS 2017 July 24, 2017

Germinal centers are the site of somatic hypermutation/affinity maturation and lead to B cell

Germinal centers are the site of somatic hypermutation/affinity maturation and lead to B cell memory and plasma cell generation Tfh CXCR 5+CD 4+ Germinal Center Tfh CXCR 5

How can we measure GC Tfh and GC B cell responses? Indirect: Blood Tfh

How can we measure GC Tfh and GC B cell responses? Indirect: Blood Tfh cells Plasma CXCL 13 biomarker of GC response Direct: Lymph Node Fine needle aspirates (LN FNA) Havenar-Daughton, Lee, Crotty, 2017, Imm. Reviews Locci, et al. 2013, Immunity Havenar-Daughton, et al. 2016, PNAS Havenar-Daughton, et al. 2016, Cell Reports

LN FNA cell count LN FNAs are non-disruptive 106 cells FNA flow cytometry is

LN FNA cell count LN FNAs are non-disruptive 106 cells FNA flow cytometry is quantitative for germinal center B cells and Tfh GCLN B cells GC Tfh cells % GC Tfh cells Havenar-Daughton, et al. 2016, Cell Reports % GC B cells

Innovative tools to study T and B cell immunology relevant to long-term protective humoral

Innovative tools to study T and B cell immunology relevant to long-term protective humoral immunity in non-human primate models (and humans) Lymph Node Fine Needle Aspirates (FNA) v Widely used in humans (oncology) v Draining lymph nodes stay intact v Sequential analysis following multiple immunization Havenar-Daughton, Lee, Crotty, 2017, Imm. Reviews

Antigen: BG 505 SOSIPv 5. 2 (V 3 stabilized) Adjuvant: 1) PLGA NP (R

Antigen: BG 505 SOSIPv 5. 2 (V 3 stabilized) Adjuvant: 1) PLGA NP (R 848+MPL) 2) Iscomatrix BG 505 neutralizing antibody BG 505 SOSIP. v 5. 2 immunized rhesus macaques generate autologous HIV neutralizing Abs BG 505 v 5. 2 Havenar-Daughton, et al. 2016, Cell Reports

Top neutralizers Non/Low neutralizers % GC B cells BG 505 neutralizing Ab titer Late

Top neutralizers Non/Low neutralizers % GC B cells BG 505 neutralizing Ab titer Late GC B cell frequencies are associated with development of autologous BG 505 neutralizing antibodies 1 st 2 nd 3 rd 4 th Immunization Havenar-Daughton, et al. 2016, Cell Reports

GC longevity and Immunization Interval GC longevity 6 week booster interval Havenar-Daughton, et al

GC longevity and Immunization Interval GC longevity 6 week booster interval Havenar-Daughton, et al 2016 Cell Reports

Scripps CHAVI-ID Large NHP Immunization study timeline 3 immunizations at weeks 0, 8, and

Scripps CHAVI-ID Large NHP Immunization study timeline 3 immunizations at weeks 0, 8, and 24 • • 100 mg BG 505 SOSIP. 664 (total per immunization) Iscomatrix adjuvant Subcutaneous injection (Sub. Q) Mid-thigh in both legs (50 mg each) Pauthner and Havenar-Daughton et al, 2017, Immunity

Strong BG 505 neutralizing titers generated after BG 505 SOSIP. 664 immunization week: •

Strong BG 505 neutralizing titers generated after BG 505 SOSIP. 664 immunization week: • All animals (n=12) generated BG 505 n. Ab titers after 3 immunizations • Substantial BG 505 NAb titers in many animals already at wk 10 Pauthner and Havenar-Daughton et al, 2017, Immunity

Early GC B cell frequencies predict BG 505 neutralization LN FNA • ELISA binding

Early GC B cell frequencies predict BG 505 neutralization LN FNA • ELISA binding titers do NOT correlate the n. Ab titers. Pauthner and Havenar-Daughton et al, 2017, Immunity

GC B cell frequencies correlate with BG 505 neutralization Post 2 nd Immunization GC

GC B cell frequencies correlate with BG 505 neutralization Post 2 nd Immunization GC B cells Post 3 rd Immunization GC B cells Pauthner and Havenar-Daughton et al, 2017, Immunity

Immunization Intervals: 8 wk boost is preferable Timing of booster Immunization: 8 week interval

Immunization Intervals: 8 wk boost is preferable Timing of booster Immunization: 8 week interval generates increased GCs 6 week booster interval 8 week booster interval • Increased GC B cell responses after the 2 nd immunization (P = 0. 001) may drive greater SHM and better neutralizing Ab responses Havenar-Daughton, et al 2016 Cell Reports Pauthner and Havenar-Daughton et al, 2017, Immunity

Summary • Germinal centers predict HIV neutralization antibody responses after Env trimer immunization •

Summary • Germinal centers predict HIV neutralization antibody responses after Env trimer immunization • Monitoring GCs informs an immunological understanding of how to improve immunization regimens: • • The importance of the primary immunization Optimal booster scheduling (8 wks preferable to 6 wks) Selection of optimal route of immunization LN FNAs provide direct access to GC Tfh cells and GC B cells • • • Simple, highly successful technique Immunophenotyping Ag-specific T cells Ag-specific B cells Gene expression BCR and TCR sequencing

Acknowledgments Dennis Burton Sal Butera Shane Crotty Bill Schief Andrew Ward Ian Wilson Rich

Acknowledgments Dennis Burton Sal Butera Shane Crotty Bill Schief Andrew Ward Ian Wilson Rich Wyatt Guido Silvestri Diane Carnathan Jennifer Wood Yerkes Vet Staff NHPs: Data Analysis: Dan Barouch Joseph Nkolola Jinyan Liu Matthias Pauthner Colin Havenar-Daughton Devin Sok Immunogen Providers: Rogier Sanders - Alba Torrents de la Pena Andrew Ward - Gabe Ozorowski, Chris Cottrell Bill Schief - Jon Steichen, Dan Kulp Richard Wyatt - Javier Guenaga Darrell Irvine - Talar Tokatlian