1. Winfried Weissenhorn, IBS Grenoble HIV envelope proteins
EHVA Satellite Meeting, IAS 2017 Conference
Non-ARV Based Interventions to Combat HIV/AIDS: New Insights and Initiatives
July 23rd 2017, Paris

2. HIV Env Apex CD4bs N332 V1, V2, V3 CD4bs N332
Quarternary structure epitopes
Various bNAb epitopes
Interface epitopes shielded
Germline BCR binding?
Apex
CD4bs
N332
Conformational dynamics/instability
Exposure of non neutralizing Ab
epitopes / CD4i
V1, V2, V3
CD4bs
N332

3. HIV Env SOSIP
Soluble cleaved Env ectodomain (without MPER), native prefusion conformation
BG505 SOSIP and others present the native env conformation present on virions
Several classes of bnAb have been isolated binding to Env SOSIPs
Most bnAbs bind or accommodate glycans
[Julien et al. Science 2013; Pancera et al, Nature, 2014]
[Haynes, Burton Science 2017]

4. Project Goals
Develop stable native Env gp140 trimers as immunogens to trigger naïve B cells to elicit bNAb
Reduce structural instability to avoid B cell responses to immunodominant non-bNAb Env epitopes
Guide bNAb B cell development
Design
Produce
Antigenicity Kd
Stability Tm EM
gl-targeting
293F
gp140

5. Env stabilization strategies
BG505 SOSIP
Specific chemical cross linking
Top and inter gp120 protomers
Without affecting local dynamics
BG505 SOSIP V2 cross-linked
TM 69.8 versus 67.8 of “wild-type” BG505 SOSIP
Interaction with bnAbs but not non-neutralising Abs
Rabbit studies initiated
W. Weissenhorn, IBS

6. Env stabilization strategies
BG505 SOSIP design: Stabilization of the C-terminus:
BG505 SOSIP_CTM
ConB SOSIP_CTM
TM 74.8 versus 67.8 of “wild-type” BG505 SOSIP
Interaction with bnAbs but not non-neutralizing Abs
Rabbit studies initiated
W. Weissenhorn, IBS

7. Env stabilization strategies
Clade C sequences and consensus clade C – SOSIP design
Engineering stabilizing mutations:
Based on sequence variants
Structure based
Variant
Tm [°C] DSF
Tm [°C] DSC
sC22 v4
70.0 ± 0.3
n.d.
sC23 v4
69.0 ± 0.0
71.1
ConC v4
70.7 ± 0.1
71.7
ConC v5
72.9 ± 0.1
75.1
BG505 SOSIP
65.7 ± 0.2
67.6
Increased thermostability
Binding to bnAbs
No binding to nnAbs
Rabbit studies initiated
sC23v4
ConCv5
sC22v4
R. Wagner, Univ; Regensburg

8. Env masking strategy
Gp120 core – masking non neutralizing antibodies
VRCO1
PGV04 +
3BNC117
VRC07
PEG 4, 8 or 12-mer with NHS ester linker
for amine (primarily lysine) coupling
sCD4
Q. Sattentau, Univ. Oxford

9. B cell immunogen design: SOSIP trimers
Induction of autologous Tier 2 NAbs
SOSIP.v5 trimers
Improved trimerization
Increased stability
Reduced V3 non-NAb epitope exposure
Reduced CD4i non-NAb epitope exposure
Improved bNAb exposure
Reduced V3 immunogenicity and Tier 1A NAb induction
Autologous neutralization titers
Uncleaved gp140
(Non-native trimers)
SOSIP.664 gp140
(Native-like trimers)
P <
50% protection
80% protection
Improves existing trimers
Allows making new trimers
AMC008 SOSIP.v4.2
bNAb PGV04
bNAb 35O22
Sanders et al PLoS Path. 9:e
Sanders et al Science 349:aac4223
De Taeye et al Cell
R. Sanders: AMC

10. B cell immunogen design: Trimer cross-linking
Stability under SDS PAGE
Positively-selected trimers have native
morphology and improved antigenicity
V3 negatively-selected trimers have native
morphology and improved antigenicity
Cryo-EM model of GLA cross-linked, PGT151-bound BG505 trimer
Summary
BG505 and B41 trimers have been cross-linked using two different chemistries (GLA and EDC)
Cross-linked trimers become more stable and have reduced non-NAbs binding
Positive and negative selection of cross-linked trimers optimizes antigenicity and morphology
Unmodified and cross-linked trimers have similar rabbit immunogencity and induce neutralizing antibodies
Schiffner et al J. Virol. 87:
Schiffner et al J. Virol. 90: 10
Q. Sattentau:UOXF

11. EAVI and ENV Manufacture of up to eight next generation trimers:
- ConS-SOSIP and ConS-linker versions with and without cross-linking
in GMP production (Polymun)
Additional candidates based on mosaic and lineage designs in preclinical assessment
First candidates selected (SDS-PAGE, BN-PAGE, DLS cryo-EM, X-ray,
crystallography, Antigenicity, Glycosylation profile, Immunogenicity rabbits/NHPs
GP41 anti MPER immunogens:
gp41 MPER TM proteoliposome formulations
Polymun, R. Sanders (AMC), Q. Sattentau (UOXF), R. Shattock (ICL) 11
W. Weissenhorn, IBS

12. Challenges Improving Env stability that the “top” can no longer “open”
Still retaining sufficient conformational flexibility
Stability in the vaccine formulation
What are the preferred structures/conformations of Env required
to induce bnAbs?
What are the preferred sequences of env immunogens to induce bnAbs?
Which Env can efficiently engage germline BCRs
How is the induction of bnAbs regulated compared to easily induced
non-neutralizing Abs?
bnAb generation requires extensive virus Env diversification
high somatic hypermutation
poor immunogenicity of glycans/diversity of glycans
bnAbs autoreactivity or polyreactivity
immune tolerance control may reduce the pool of reactive B cells

13. Acknowledgment Coordinators: Y. Levy, INSERM
G. Pantaleo, CHUV Lausanne
ENV design:
Q. Sattentau, Univ. of Oxford
R. Wagner, Univ of Regensburg
W. Weissenhorn, IBS , CEA-CNRS-UGA
This project has received funding from the European Union’s horizon research and innovation programme under grant agreement No as well as funding from the Swiss government (through the State Secretariat for Education, Research and Innovation, SERI) under grant agreement No The opinions expressed and arguments employed herein do not necessarily reflect the official views of the Swiss Government.
Coordinator: R. Shattock, Imperial College
Env design:
R. Sanders, Univ. Amsterdam
R. Shattock, Imperial College
Q. Sattentau, Univ. of Oxford
W. Weissenhorn, IBS, CEA-CNRS-UGA
This project has received funding from the European Union’s horizon research and innovation programme under grant agreement No and No 13

14. Acknowledgment Antibodies Dennis R Burton Barton F Haynes
Michel C. Nussenzweig
Mark Connors
John R Mascola
James Robinson
Antonio Lanzavecchia
Polymun 14