Burton Lab – The Scripps Research Institute 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 Introduce Colin and me. In this audience probably know us, but for those who don’t … The reason to do this study was 1) There were great concerns regarding the reliable induction of autolgous neut titers following protein immunization in NHPs based on multiple cohorts – therefore we set out to optimize and compare parameters 2) to do many head to head comparisons, sharing control groups and condensed into a period of one year

Acknowledgments Planning and Organization: NHPs: Dennis Burton Sal Butera Shane Crotty Bill Schief Andrew Ward Ian Wilson Rich Wyatt NHPs: Dan Barouch Joseph Nkolola Jinyan Liu 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

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) When we started ... .. only 2 sutdies testing SOSIP in NHPs Data Analysis: Legend: Red = Neutralization – IC50, Blue = Binding – EC50 BG505 = BG505 N332, unless stated otherwise Pauthner et al., Immunity 2017

BG505 binding Ab titers after BG505 SOSIP.664 immunization Limit Of Detection week: Animal colors correspond to the Neutralization time course on slide! The high neutralizer animal also had a SOSIP titer of ~1:18.000 at week 26. All animals (n=12) generated substantial BG505 SOSIP binding titers already at wk10. Animals show a very consistent, tightly grouped response.

Strong BG505 neutralizing titers after BG505 SOSIP.664 immunization Limit Of Detection week: Week 10 Week 26 Also most animals are still neutralizing at week 20 after third boost. Very encouraging, as nAbs at week 10 is a lot fast than natural infection which takes 3-6 months for nAbs to form. All animals (n=12) generated BG505 nAb titers after 3 immunizations Substantial BG505 NAb titers in many animals already at wk10

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 antigen-specific 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 You can discuss here also the much more substantial titers than we observed in previous studies.

Immunization Intervals: 8 week boost is preferable Timing of booster Immunization: 8 week interval generates increased GCs Increased GC B cell responses after the 2nd immunization (P = 0.001) may drive greater SHM and better nAb responses 8 wk booster interval (this study) 6 wk booster interval (previous FNA SOSIP study) Havenar-Daughton et al., Cell Reports 2016

Immunization Route: SubQ generates consistent nAb titers Increased GC B cell frequencies in inguinal LNs BG505 N332 neutralization at w10 and 12 are not significantly different between SubQ and IM. Differences are stronger after second boost at w26 and become significant. BG505 gp120 binding is not significantly different at w26, so trimer stability is not necessarily the reason for differences. Probably related to bioavailability in lymph nodes. 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 neutralization.

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    1)  Ab responses to the tag / tag-dependent neoepitopes on NFL versus SOSIP (NFL immunogen was tagged. SOSIP immunogen was not)    2)  Ab responses to the base of NFL versus SOSIP (Is that sequence different? Rich STILL does not have the NFL sequence in the CHAVI-ID database)    3) Ab responses to fine, but reproducible, differences between the NFL and SOSIP structure/glycosylation

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 Tier 1 responses may interfere with the induction of Tier 2 responses, because Tier 2 specificities may be rare or low affinity and disadvantaged in the competition for antigen and T cell help.

Stabilization: Successful reduction of V3-loop binding titers V3 peptide binding SOSIP binding WT vs. Olio6 V3 binding Trimer were able to reduce V3 titers to various degrees: V3 loop lockdown reduced V3 peptide binding titers to varying degrees Olio6 showed greater V3 antibody reduction than other approaches

Stabilization: Tier 1 NAbs are significantly reduced, Tier 2 is maintained SF162 BG505 BG505 / SF162 V3 peptide competitions indicate a residual non-V3 component to MW965 neutralization, which explains why SF162 is knocked out and MW965 is only reduced. 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

Antigen Dose: 100 µg vs 20 µg induce similar titers Similar peak responses at 20 µg dose 20 µg dose produced a lower response of neutralizing antibodies after the first boost No significant differences in GC B cell and Tfh frequencies (all FNA time points)

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 µg total dose compared to 100µg total dose in conventional immunizations

Delivery: Pump vs. Conventional immunizations Weeks: Pump delivered immunogen (before bolus) boosted neutralizing responses at week 10 and week 26 Neutralizing (and binding Ab titer) responses peaked later after each immunization Data shown in time course is from one representative run – neut was tested at least twice for each time point. 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 9 High-Titer NHPs (n = 78)

Mapping: Autologous neutralization is gp120 specific BG505 neutralization competes with soluble BG505 D368R gp120 BG505 neutralization is not affected by soluble BG505 V3-peptide

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: NHPs: Dennis Burton Sal Butera Shane Crotty Bill Schief Andrew Ward Ian Wilson Rich Wyatt NHPs: Dan Barouch Joseph Nkolola Jinyan Liu 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

Extra Slides

Stabilization: WT vs. Olio6 V3-peptide binding is nearly identical V3 peptide competitions indicate a residual non-V3 component to MW965 neutralization, which explains why SF162 is knocked out and MW965 is only reduced. BG505 WT and Olio6 V3 peptide binding are nearly identical.

Stabilization: Olio6 knocks out V3 peptide response V3 peptide competitions indicate a residual non-V3 component to MW965 neutralization, which explains why SF162 is knocked out and MW965 is only reduced. 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 Done under the assumption that MG505 and BG505 neutralizing lineages are cross-reactive – we can test that by doing a MGt505 neutralization competition with BG505 SOSIP

Immunization Route: SubQ Tier 1 Neut / V3-binding BG505 N332 neutralization at w10 and 12 are not significantly different between SubQ and IM. Differences are stronger after second boost at w26 and become significant. BG505 gp120 binding is not significantly different at w26, so trimer stability is not necessarily the reason for differences. Probably related to bioavailability in lymph nodes.

Design: SOSIP and NFL – Tier 1 Neut / V3-binding We repeated the NFL group binding on NFL His-captured ELISA plates, with a new batch of protein from the Wyatt lab, and the titers turned out much higher than the original NFL on NFL binding. I asked Devin to please repeat both NFL on SOSIP and NFL on NFL binding one more time to confirm – for now, it looks like NFL induced Ab titers come out a little lower on SOSIP trimers than on NFL trimers.

NHP Cohort Overview Well controlled cohort: Economy of scale and time, to do this study in one big experiment. Well controlled cohort: Age, weight and gender are tightly matched 6-12 animals per group

BG505 N332 neutralization assay comparison – TSRI and Duke Labs Some brief remarks regarding the quality of the BG505 neutralization 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 Fine Needle Aspirate (FNA) lymph node (LN) Biopsies: CD20 Ki67 CD3 Bcl6 GC B cells Gated on CD3/CD4 T cells GC Tfh cells: PD-1HI CXCR5HI

GC B cell frequencies predict BG505 neutralization GC B cell % after 1st immunization correlates well with week 10 neutralization; If the outlier animal (1:20,000 nAb titer) is excluded, P = 0.001 and r=0.8. GC B cell % after 2nd immunization correlates well with week 10 neutralization GC B cell % after 3nd immunization correlates well with week 26 neutralization Similar results to previous FNA SOSIP study 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 3 Week 11 gp120 ELISA titer Non-neutralizing B cell specificities in the GC may out-compete neutralizing B cell specificities. Reducing V3 exposure and increased trimer stability may reduce the frequency of non-neutralizing B cells in the GC Immunogens with both V3-lockdown and increased stability (v5.2 and olio6cd4ko) show significant reductions in GC activity suggesting that the non-neutralizing B cell specificities found on in vivo breakdown products may be as large of a component of the non-neutralizing B cell response as the V3 loop tip. 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 products

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.

Delivery: Pump vs. Conventional immunizations PD-1 Ki67 PD-1 Week 11 CXCR5 FNA is 1 week after the pump immunization is completed. Considering the delayed Ab kinetics, the GC kinetics may also be delayed (under investigation) GC Tfh cells are increased after pump immunization Proliferation of GC Tfh cells (Ki67+) is increased after pump immunization