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 IAS2017 July 24, 2017

Germinal centers are the site of somatic hypermutation/affinity maturation and lead to B cell memory and plasma cell generation Tfh CXCR5+CD4+ Germinal Center Tfh Tfh CXCR5

How can we measure GC Tfh and GC B cell responses? Indirect: Blood Tfh cells Plasma CXCL13 biomarker of GC response Direct: Lymph Node Fine needle aspirates (LN FNA) Germinal centers are an essential aspect of mounting most antibody responses and are critical for the generation of HIV bnAbs However, we know little about GC activity in current human or nhp immunization studies. We’ve spent the last few years building a set of assays to monitor GC activity. 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 FNAs are non-disruptive LN FNA cell count LN FNAs are non-disruptive 106 cells % GC B cells LN FNA flow cytometry is quantitative for germinal center B cells and Tfh % GC Tfh cells GC B cells GC Tfh cells Are the % of GC B and GC Tfh cells representative of their frequencies in the whole LN? Havenar-Daughton, et al. 2016, Cell Reports

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) Widely used in humans (oncology) Draining lymph nodes stay intact Sequential analysis following multiple immunization Havenar-Daughton, Lee, Crotty, 2017, Imm. Reviews

BG505 SOSIP.v5.2 immunized rhesus macaques generate autologous HIV neutralizing Abs BG505v5. BG505v5.2 BG505 neutralizing antibody Antigen: BG505 SOSIPv5.2 (V3 stabilized) Adjuvant: 1) PLGA NP (R848+MPL) 2) Iscomatrix All animals make high binding Ab titers 4 animals with each adjuvant will undergo FNA repeatedly 2 additional animals will undergo whole LN biopsy at each time point (PLGA NP) Animals are immunized in both legs Subcutaneous in the medial, lower quadriceps Each FNA/biopsy consists of sampling at both inguinal and an axillary LN 2 animals with adjuvant only: PLGA NP (R848+MPL) Havenar-Daughton, et al. 2016, Cell Reports

Late GC B cell frequencies are associated with development of autologous BG505 neutralizing antibodies Top neutralizers Non/Low neutralizers BG505 neutralizing Ab titer % GC B cells 1st 2nd 3rd 4th Immunization % GC B does not correlate with ELISA binding Ab titers And ELISA titers do NOT correlate the nAb titers. Havenar-Daughton, et al. 2016, Cell Reports

GC longevity and Immunization Interval 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 24 100 mg BG505 SOSIP.664 (total per immunization) Iscomatrix adjuvant Subcutaneous injection (SubQ) Mid-thigh in both legs (50 mg each) This is the standard immunization protocol that we used … Pauthner and Havenar-Daughton et al, 2017, Immunity

Strong BG505 neutralizing titers generated after BG505 SOSIP Strong BG505 neutralizing titers generated after BG505 SOSIP.664 immunization week: All animals (n=12) generated BG505 nAb titers after 3 immunizations Substantial BG505 NAb titers in many animals already at wk10 Pauthner and Havenar-Daughton et al, 2017, Immunity

Early GC B cell frequencies predict BG505 neutralization LN FNA If the outlier animal (1:20,000 nAb titer) is excluded, P = 0.001 and r=0.8. "This matters because the ELISA titers do NOT predict the nAb titers." Which you should also state much earlier) ELISA binding titers do NOT correlate the nAb titers. Pauthner and Havenar-Daughton et al, 2017, Immunity

GC B cell frequencies correlate with BG505 neutralization Post 2nd Immunization GC B cells Post 3rd 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 generates increased GCs 6 week booster interval 8 week booster interval Increased GC B cell responses after the 2nd 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 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 Immunogen Providers: Rogier Sanders NHPs: Dennis Burton Sal Butera Shane Crotty Bill Schief Andrew Ward Ian Wilson Rich Wyatt NHPs: Dan Barouch Joseph Nkolola Jinyan Liu Guido Silvestri Diane Carnathan Jennifer Wood Yerkes Vet Staff 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 Data Analysis: Matthias Pauthner Colin Havenar-Daughton Devin Sok