1. 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 July 24, 2017

2. 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

3. 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

5. 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

6. 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

7. 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

8. 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

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

11. 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

12. 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

13. Early GC B cell frequencies predict BG505 neutralization
LN FNA
If the outlier animal (1:20,000 nAb titer) is excluded, P = 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

14. 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

15. 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

16. 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

17. 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