Optimized HIV Env trimer immunizations in nonhuman primates
Optimized HIV Env trimer immunizations in nonhuman primates to elicit Tier 2 neutralizing antibodies IAS 2017 - Paris Matthias Pauthner PhD Student Burton Lab The Scripps Research Institute La Jolla, USA July 24th, 2017 Acknowledgments Planning and Organization: Immunogen Providers:
Dennis Burton Sal Butera Shane Crotty Bill Schief Andrew Ward Ian Wilson Rich Wyatt 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 NHPs: Data Analysis: Dan Barouch Joseph Nkolola Jinyan Liu
Matthias Pauthner Colin Havenar-Daughton Devin Sok Scripps CHAVI-ID Large NHP Immunization study timeline 3 immunizations at wk 0, 8 and 24 (unless otherwise noted): Groups of 6 12 rhesus macaques 100 mg SOSIP (total per immunization) Iscomatrix adjuvant Subcutaneous injection (SubQ) Mid-thigh in both legs (50 mg each)
Data Analysis: Legend: Red = Neutralization IC50, Blue = Binding EC50 BG505 = BG505 N332, unless stated otherwise Pauthner et al., Immunity 20 Strong BG505 neutralizing titers after BG505 SOSIP.664 immunization Week 10 Week 26
Limit Of Detection week: All animals (n=12) generated BG505 nAb titers after 3 immunizations Why did 100% of BG505 SOSIP.664 immunized animals generate neutralizing responses in this study? Strong adjuvant - Iscomatrix (used in some but not all previous studies) Bilateral immunizations. This engages more individual
lymph nodes, increasing the likelihood of recruiting antigenspecific CD4 T cells, and B cells with neutralizing epitope specific B cells. A shift to an immunization regimen with longer intervals, wk 0, 8, 24. Presentation by Colin Havenar-Daughton Subcutaneous immunization route Immunization Route: SubQ generates consistent nAb titers Increased GC B cell frequencies in inguinal LNs SubQ immunization shows more rapid drainage to more LNs compared to IM SubQ immunization is superior to intramuscular (IM) immunization
(P = 0.02) in generating consistent autologous Tier 2 Effects of trimer design and stabilization on immunogenicity Design: SOSIP and NFL induce similar responses Week 10 Week 26 NFL immunized animals show delayed but comparable peak nAb responses Stabilization: Overview of design concepts
WT v4.1/ v5.2 Olio6 V3-loop sequence Modified constructs contain additional mutations outside the V3 loop, designed to increase yield, stability and trimerization BG505 SOSIP v4.1/v5.2 from Rogier Sanders BG505 Olio6 and Olio6 CD4-KO from Bill Schief
Stabilization: Successful reduction of V3-loop binding titers V3 peptide binding SOSIP binding V3 loop lockdown reduced V3 peptide binding titers to varying degrees Olio6 showed greater V3 antibody reduction than other WT vs. Olio6 V3 binding
Stabilization: Tier 1 NAbs are significantly reduced, Tier 2 is maintained SF162 BG505 BG505 / SF162 Olio6 abrogated SF162 neutralization and reduced MW965 titers Stabilized trimers induced similar peak BG505 NAb responses as SOSIP.664 immunized animals Effects of antigen dose and delivery on
immune response Delivery: Osmotic Pumps vs. Conventional immunizations Pump group immunizations: 100 mg BG505 SOSIP.v5.2 + Iscomatrix delivered by pump over 14 days 100 mg BG505 SOSIP.v5.2 + Iscomatrix bolus injection on day 14 200 g total dose compared to 100g total dose in conventional immunizations Delivery: Pump vs. Conventional immunizations Weeks:
Pump immunization generated significantly higher neutralizing titers 3 animals from the pump group generated neutralizing titers >1:1000 after only 2 immunizations Mapping of the neutralization response in NHPs with high autologous neutralization titer BG505 Neut Titer 10000 9 High-Titer NHPs 1000
100 10 All Animals (n = 78) Mapping: Autologous neutralization is gp120 specific BG505 neutralization competes with soluble BG505 D368R gp120 BG505 neutralization is not affected by soluble BG505 Mapping: N241/N289 glycan hole reactivity
4 of 9 animals show 2-4 fold change in serum titer on BG505 N241 N289 glycan knock-in mutant Absence of N332 glycan had no strong effect on serum nAb titers Mapping: High Neutralizers have some Tier 2 breadth Montefiori 12-virus global panel: Other viruses tested: Major Findings Autologous Tier 2 nAbs reproducibly generated in BG505
SOSIP.664-immunized animals Rapid development of autologous Tier 2 nAbs (10 weeks) with the new immunization schedule and design NFL and SOSIP elicit similar autologous nAb responses Stabilized trimer antigens reduced non-neutralizing Abs, but did not increase autologous nAbs Extended immunogen delivery can significantly enhance nAb generation Some Tier 2 neutralizing breadth consistently generated among the top BG505 neutralizers on global 12 virus panel Acknowledgments Planning and Organization:
Immunogen Providers: Dennis Burton Sal Butera Shane Crotty Bill Schief Andrew Ward Ian Wilson Rich Wyatt 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 NHPs: Data Analysis: Dan Barouch Joseph Nkolola Jinyan Liu
Matthias Pauthner Colin Havenar-Daughton Devin Sok Extra Slides Stabilization: WT vs. Olio6 V3-peptide binding is nearly identical BG505 WT and Olio6 V3 peptide binding are nearly identical. Stabilization: Olio6 knocks out V3 peptide response
MW965 neutralization in Olio6 immunized animals does not compete with the BG506 V3 peptide. Mapping: BG505 vs. MG505 A2 neutralization Most animals did not cross-neutralize MG505 A2 Geometric mean BG505 neutralization is higher for MG505 A2 neutralizers, and BG505 / MG505 neutralization roughly correlates Mapping: Some NHPs target the V1-loop base Immunization Route: SubQ Tier 1 Neut / V3-binding
Weight [kg] 9 10 0 Well controlled cohort: Age, weight and gender are tightly matched 6-12 animals per group e 4 5 6 7 8 Age at prime [a]
0% 10 al 3 10% 20 M
2 20% 30 e 0% 30% al
20% 40 Fe m 40% 40% Animal Count 60%
of all animals (n=78 of all animals (n=78) 50 BG505 N332 neutralization assay comparison TSRI and Duke Labs Excellent correlation between TSRI and Duke Labs TSRI Lab neutralization assays are more sensitive, because of differences in curve fitting for incomplete curves at low serum dilutions.
Identification of GC B cells by LN FNA and flow cytometry Ki67 CD20 Fine Needle Aspirate (FNA) lymph node (LN) Biopsies: CD3 GC Tfh cells: PD-1HI CXCR5HI
GC B cells Bcl6 GC B cell frequencies predict BG505 neutralization GC B cell % does not correlate with BG505 Ab binding titers, consistent with the1st LN FNA rhesus study Similar result to previous FNA SOSIP study Havenar-Daughton et al., Cell Reports 2016 Stabilization: Immunodominance of V3 loop tip and trimer breakdown products
Week 11 gp120 ELISA titer Week 3 Reduced GC activity in v4.1 and Olio6 is likely due to reduced V3 specific GC B cell responses Reduced GC activity in v5.2 and Olio CD4ko, may be due to reduced monomeric gp120 and/or partial gp41 breakdown Stabilization: GC B cells correlate with NAb titers V4.1
V5.2 Olio6 Olio6 CD4-KO GC B cell frequency correlates with BG505 neut titer GC B cell frequency also correlates with BG505 binding titer (unlike SOSIP.664 controls), possibly due to reduction of the V3 and other off-target responses Delivery: Liposome Olio6-CD4ko immunizations Similar responses at weeks 10 and 12, following one boost.
PD-1 PD-1 Delivery: Pump vs. Conventional immunizations week 11 Ki67+ GC Tfh control vs pump 20 CXCR5 15
0.04 Pumps Conv 0.02 10 5 80 < 0.0001
60 40 20 pu m ps Week 27 nv Week 11
. 0 Week 3 co 0 Ki67 % Ki67+ of GC Tfh cells
% GC Tfh cells Week 11 GC Tfh cells are increased after pump immunization s Proliferation of GC Tfh cells (Ki67+) is increased after pump Pumps 40immunization Conv 30
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