Epitope based HIV vaccine design Jonathan M. Gershoni
Current accepted hypothesis: The ultimate AIDS vaccine … faithfully reproduced envelope trimers … the closer to “native” is best.
see for example …
However, this current strategy might be wrong HIV co-evolved with humans precisely to evade immuno-surveillance & best conceal its most vulnerable neutralizing epitopes
Can we produce a gp 120 -based immunogen that HIV evolution can not anticipate? We need to work outside HIV’s comfort zone
OUT spike assemble/package HIV-1 virus V. genomes V. proteins gp 41 gp 120 conformational change CD 4 IN CCR 5 RNA Po. I Virus entry Membrane fusion RNA transcripts RNA to ds. DNA Chromosome Provirus
OUT spike assemble/package HIV-1 virus V. genomes V. proteins gp 41 gp 120 conformational change CD 4 IN CCR 5 RNA Po. I Virus entry Membrane fusion RNA transcripts RNA to ds. DNA Chromosome Provirus
CD 4 induced epitopes in gp 120 exposed epitope cryptic gp 120 quaternary epitope CD 4
The Working Hypothesis CD 4 binding to gp 120 induces neo-epitopes One should be able to produce CD 4 i defining m. Abs
Production of a CD 4 i m. Ab Immunize with CD 4/gp 120 complex Hybridomas Select m. Abs positive for CD 4/gp 120 Screen against CD 4 and gp 120 alone Keep m. Abs that prefer CD 4/gp 120
Production of a CD 4 i m. Ab Gershoni et al FASEB J 7: 1185 [1993]
17 b a human CD 4 i m. Ab
17 b a human CD 4 i m. Ab gp 120 Kwong PD et al 1998 Nature 393: 648
17 b a human CD 4 i m. Ab H 1 V 3 H 2 gp 120 Kwong PD et al 1998 Nature 393: 648
17 b a human CD 4 i m. Ab 17 b VL VH H 1 V 3 H 2 Bridging Sheet gp 120 Kwong PD et al 1998 Nature 393: 648
Bridging sheet dynamics
Bridging sheet dynamics outer domain inner domain HIV liganded bridging sheet Kwong PD et al 1998 Nature 393: 648
Bridging sheet dynamics outer domain inner domain SIV unliganded bridging sheet Chen B et al 2005 Nature 433: 834
Relaxed vs Stringent CD 4 i
At least 2 CD 4 i epitopes CG 10 Competition
Bridging Sheet 21 3 H 2 20 H 1 2 H 1 Huang CC 2005 SCIENCE 310: 1025
CD 4 affects the V 1 -V 2 conformation
17 b monitors the CD 4 effect on H 2 Bridging Sheet 17 b 21 3 H 2 20 DH 1 2 H 1 17 b does not require a Bridging-Sheet!
The sequence of CD 4 i conformations Unliganded trimer Bridging Sheet b 2 b 3 b 21 b 20 V 3 Julien JP 2013 SCIENCE 342: 1477
The sequence of CD 4 i conformations b 2 b 3 b 21 43 b 20 V 3 CD 4 Julien JP 2013 SCIENCE 342: 1477
The sequence of CD 4 i conformations b 3 b 21 Julien JP 2013 SCIENCE 342: 1477 Kwong PD et al 1998 Nature 393: 648
The sequence of CD 4 i conformations b 3 b 21 Julien JP 2013 SCIENCE 342: 1477 Kwong PD et al 1998 Nature 393: 648
The sequence of CD 4 i conformations b 3 b 21 b 2 Kwong PD et al 1998 Nature 393: 648
The sequence of CD 4 i conformations a functional co-receptor binding site b 3 b 21 b 2 Kwong PD et al 1998 Nature 393: 648
Conformational transitions of gp 120 CD 4 -F 43 drives H 2 in & downwards Disruption of b 21: b 3 Hbonds H 1 flips forward H 1 twists replacing b 3 with b 21: b 2 Hbonds zip up The bridging sheet is exposed and able to bind the co-receptor
HIV epitope based vaccines Two immunogen tactics: Locking gp 120 into a CD 4 i conformation Expression of reconstituted epitopes
Conformation-active peptides Incubate a phage display peptide-library with gp 120 Screen phage-bound-gp 120 with CG 10 Select CG 10 positive phages m 1 = C D R R D L P Q W A K R E C
m 1 -peptide induces the CG 10 epitope CD 4 m 1 fth 1 gp 120 + Dot-Blot experiment: Apply CD 4, F m 1 or F fth 1 to NC filter Incubate with or without gp 120 Probe with CG 10
m 1 -peptide is allosteric 2. 5 2. 0 1. 5 1. 0 0. 5 0 gp 120 CD 4 + + + CD 4 competition experiment: Apply F m 1 to ELISA wells Incubate with or without gp 120 Incubate with or without CD 4 Probe with anti-gp 120 ; or anti-CD 4
Optimizing m 1 to m 2 Biased Random Mutagenesis 97% / 3% Ophir & Gershoni 1995 Protein Eng 8: 143 G A TC G A T C G AT C Produce a BRM library of m 1 peptide Screen against gp 120 Picked preferred phages m 1 = C D R R D L P Q W A K R E C m 2 = C D R R D L P D W A I R A C
M 2 -peptide exposes both targets
Epitope Reconstitution Reverse Ig. Omics
TAU: Dr. Anna Roitburd – Berman Dr. Yael Weiss Ottolenghi Dr. Gilad Kaplan Smadar Neeman Arie Ryvkin Noy Motiee Prof. Tal Pupko Haim Ashkenazy Prof. George E. Lewis -- IHV – U Maryland Prof. Wayne Marasco -- Harvard University Dr. Avishay Lublin -- Kimron Dr. Shimon Perk -- Kimron Dr. David Swayne -- USDA Спасибо o
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