Download A VLP-based Platform for Vaccine Discovery

A VLP-based Platform for Vaccine Discovery
Dave Peabody & Bryce Chackerian
Department of Molecular Genetics and Microbiology
University of New Mexico
Some key issues in identification of new VLP vaccines:
•
How do we identify the important antigenic epitopes?
Particularly, structures that are hard to mimic using peptides
or epitopes that are poorly immunogenic in the context of
larger protein.
•
How to present these epitopes to the immune system
in a highly immunogenic format?
MS2 VLPs integrate into a single structural platform
the high immunogenicity of VLPs with the affinityselection capability of phage display.
PHAGE DISPLAY: A method to identify epitopes by affinity selection.
Phage Display (invented about 1985) is a method that allows
the selection of peptides from a complex random-sequence
population that have some arbitrarily chosen binding function.
M13 is a filamentous phage with a circular DNA genome inside
a long, skinny, helical capsid. Peptides can be genetically
fused to a phage structural protein so that each recombinant
phage displays a different peptide on the surface of a particle
that also contains the DNA encoding the peptide.
Reproduce in E. coli
Amplified phage
with selected
peptide on surface.
elute
M13 genome
Construct phage
recombinants with a library
of all possible hexapeptides
fused to the gene for a viral
structural protein.
Infection of E. coli produces a
huge collection of phage, each
displaying a different peptide on
its surface.
Repeat
selection as
needed
wash
INADEQUACY OF FILAMENTOUS PHAGE DISPLAY FOR VACCINE DEVELOPMENT - The affinity selection capability of
filamentous phage display is really good at epitope identification, but the peptides they display are poorly immunogenic (not dense
repetitive arrays). Further, peptides identified by filamentous phage display frequently lose the desired activity when displayed in
other structural contexts. NEEDED: a vaccine platform that enables affinity selection on a highly immunogenic platform.
Desirable features of a VLP phage display platform.
•
Peptides should be displayed as dense repetitive
arrays, ensuring high immunogenicity.
•
Since VLP-display requires fusion to a viral
structural protein, insertion of the peptide must not
interfere with correct protein folding and VLP
assembly.
•
The VLP should encapsidate the nucleic acid that
encodes its synthesis, thus enabling the recovery of
affinity-selected sequences from complex random
sequence or antigen fragment libraries.
The MS2 VLP platform satisfies these requirements.
The goal of this work is to create an analogue of phage
display that can be conducted on MS2 VLPs, thus creating a
single, integrated platform for epitope identification and
immunogen presentation.
This would enable identification and production of vaccine
candidates by the following approaches:
1. Rational Design - engineered display of peptide epitopes
already identified by other means.
2. Irrational Design - selection from highly complex random
sequence peptide libraries of peptides having affinity for any
selecting agent (e.g. a mAb).
3. Semi-rational Design - optimization of the properties of a
peptide epitope by mutation of its amino acid sequence, or by
randomization of its flanking sequences, followed by affinity
selection.
Phage display requires:
1. A site in a viral structural protein that (a) tolerates
insertion of foreign peptides, and (b) displays peptides
in accessible form on the surface of the viral particle.
2. Linkage of phenotype to genotype. The viral (or viruslike) particle must package the nucleic acid sequence
that encodes the viral protein and the foreign peptide it
carries.
An Integrated Platform for Epitope Identification and Immunogenic Display
Bacteriophage MS2
•Consists of an icosahedral capsid containing a short ssRNA genome.
•A single structural protein, coat protein, that forms a homodimer.
•VLPs can be generated by overexpression of coat protein in E. Coli.
•VLPs consist of 90 dimers, arranged with T=3 symmetry.
•Coat Protein dimers also function by interacting with an RNA hairpin structure on the
viral genome, repressing translation of the downstream replicase.
How do we display antigens
on VLPs in a highly
immunogenic format?
The AB surface loops (the gold balls) on the
coat protein dimer represent a good target
for engineering surface display.
B
Display of foreign peptides on
MS2 VLPs by genetic fusion to
coat protein expressed from a
plasmid.
B
insert
coat
plasmid
transcription
& translation
in E. coli.
assembly
VLP with
foreign
peptide on
surface
Assays for functional coat protein:
1)Translational repression
Does recombinant coat protein repress translation of a
reporter (LacZ) cloned downstream from the translational operator?
2)Capsid formation
As assessed by agarose gel electrophoresis
Can we engineer target sequences into the MS2 Coat Protein
Insertion Sequences:
CCR5 ECL2
RSQREGLHYT
HIV V3
IQRGPGRAFV Translational Repression?
VLPs?
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
Coat protein monomer
Coat protein dimer
Epitopes are exposed on the surface of recombinant MS2 VLPs
0.8
1.25
0.75
MS2 VLPs
ECL2 VLPs
0.6
OD405
OD405
1.00
Antibodies bind
recombinant VLPs
0.7
V3-VLP
MS2 VLP
ECL2-VLP
0.50
0.5
0.4
0.3
0.2
0.25
0.1
0.00
103
104
105
0.0
10 1
10 2
10 3
Antibody dilution
Anti-ECL2
IgG Antibody Titer
10 5
105
10 4
104
10 3
103
10 2
102
-V
L2
EC
EC
L2
-V
LP
LP
(N
A)
(C
FA
)
(C
FA
)
VL
P
V3
-
VL
P
S2
M
-V
LP
L2
EC
(C
(C
FA
)
(C
FA
)
-V
LP
V3
V3
-V
LP
(N
A)
FA
)
(C
VL
P
S2
FA
)
10 1
101
M
Anti-V3
IgG Antibody Titer
10 5
Serum Dilution
106
Recombinant
VLPs elicit
peptide-specific
antibodies
10 4
10 6
A list of the recombinant MS2 VLPs that we have
engineered and their immunogenicity.
Geometric mean end-point dilution IgG titers (from groups of 3-6 mice immunized with the
recombinant VLPs) as determined by ELISA. Titers were determined using V3 peptide, ECL2
peptide, full-length human IgE, or full-length anthrax protective antigen as the target antigen.
How tolerant is coat protein of
peptide insertions generally?
(NNY)6
(NNY)8
(NNY)10
2%
nd
nd
96%
94
92
Insertion into “monomer”
Insertion into singlechain dimer
% recombinants functional for translational repression.
NNY encodes 15 amino acids and no stops.
Insertion of random sequences is
compatible with VLP assembly
-+
EthBr
Western blot
EthBr
Western blot
EthBr
Western blot
Phage display requires:
1. A site in a viral structural protein that (a) tolerates
insertion of foreign peptides, and (b) displays peptides
in accessible form on the surface of the viral particle.
2. Linkage of phenotype to genotype. The viral (or viruslike) particle must package the nucleic acid sequence
that encodes the viral protein and the foreign peptide it
carries.
Recombinant VLPs package their RNAs.
MS2 CP Dimer
wt
wt ECL2 V3
MS2 CP Dimer
wt
wt ECL2 V3
MS2
CP
MS2
CP
Qß MS2
Qß MS2
Agarose gel
Northern blot
(MS2 probe)
Display of Random Sequence Peptide Libraries and
Affinity Selection on the MS2 VLP Platform
MS2 coat
RT-PCR
Transform bacteria
with coat-peptide library.
Affinity
selection
transcription
Lyse
cells
translation
Methods for random sequence peptide library construction.
SalI
1. Cloning of a PCR fragment in pDSP1.
PT7
BamHI
Produce a SalI - BamHI fragment with a
primer that inserts a random peptide
sequence and clone it between SalI and
BamHI of pDSP1.
coat
coat
pDSP1
ColE1 ori, KanR
2. Site-directed mutagenesis by primer extension on a ssDNA template.
U
U
U
U
mutant
U
U
U
mutagenic primer
annealed to ssDNA
template raised in a
dut- ung- host.
U
U
U
Primer
extension
and ligation
Transformation
of ung+ host
coat
juggled coat
pDSP62
Kanamycin
M13 ori
ColE1 ori
We can now routinely generate libraries with 109 – 1010 recombinants
Wash &
discard
unbound
VLPs
VLP peptide
library
Vaccine
Candidate
Elute bound VLPs
Recover sequence
by RT-PCR
mAb immobilized on a surface.
Unlike filamentous phage display, our method both (1) optimizes epitope structure by
affinity-selection, and (2) then presents it to the immune system in the same structural
context and at high immunogenicity. Therefore the MS2 VLP system could be
especially useful for the production of mimotope vaccines.
Affinity Selection: Proof‐of‐Principle
Flag‐VLPs wt VLPs
RT
+
1. Affinity selection
using an anti‐
Flag mAb
PCR
PCR
KpnI
BamHI
A method for recovering selected sequences. Following selection, VLPs
are eluted, then subjected to RT (using the RT primer, above), followed
by PCR (using the primers shown above). Resulting PCR product can
be visualized by agarose gel electrophoresis (shown below) or recloned
into pDSP1 by digest with the restriction enzymes KpnI and BamHI.
Flag VLPs (ng)
4
0
0.4
2
10
40
WT VLPs (ng) 0
40
40
40 40
40
Flag
VLPs
wt
VLPs
2. Wash off unbound, elute bound VLPs
3. Reverse transcription, PCR
Immobilized
Anti‐FLAG mAb
Selection with anti‐Flag mAb
RT‐PCR controls
H2O
Selection of an anthrax protective antigen
epitope in the MS2 display system.
Consensus
Native PA epitope
Alignment
VIGGTHLD-GIGGSFID-DIPSSFLD--ASGSIYDS-YS-SIYDID
-VDATHYDY-GGSTLYDR-FTASTFDR-GIASVLDV-IASSRFSSVVSSSRFD-IVSDRSFG-TDLASFVG--HYASSYDL-
IgG Ab against Anthrax PA
ASFFD‐MS2
OD405
Clone #
F20A4
F20B12
F20B14*
F20A5*
F20A6*
F20A12
F20B18*
F20B11
F20B3*
F20B9
F20B19*
F20B5*
F20A1*
F20A12b
IVSASSFD-... EVHASFFDIGGS....
VALENCY ISSUES:
These are the results of a single round of selection. Although the consensus sequence matches that of the wildtype epitope, no member of this family is itself a perfect match. We suspect these are relatively low affinity ligands,
isolated as a result of our inability to distinguish intrinsically tight binders from weak binders displayed multivalently.
Solution: We now have a means of controlling the valency of epitope display on MS2 VLPs, and can reduce it to a
few peptides (or fewer) per VLP.
Valency Control and Affinity Maturation
First round selection is conducted using
multivalent display so that a diverse
population of peptides is obtained whose
individual members bind the antibody
over a wide range of intrinsic affinities.
In subsequent rounds, valency is reduced,
thereby increasing the selection stringency
so that only the tightest binders are
recovered.
SalI
PT7
PT7
coat
coat
pDSP1(am)
ColE1 ori, KanR
Ala-inserting
suppressor tRNA
gene
Plac
pNMsupA
P15A ori, CamR
pDSP1(am)-flag / pNMsupA
pDSP1(am) / pNMsupA
pDSP1-flag
UAG
pDSP1-flag
pDSP1
ColE1 ori, KanR
pDSP1
pDSP1
pDSP1(am) / pNMsupA
coat
coat
pDSP1(am)-flag / pNMsupA
BamHI
Why MS2-VLP display for vaccine discovery?
The single-chain dimer of coat protein is compatible with the display of
diverse peptide sequences at high density on the VLP surface.
The MS2 platform combines epitope identification and/or affinity
optimization with epitope presentation in a single structural platform, thus
preserving structural context from epitope identification through
immunization.
Preservation of structural context should increase the likelihood of selecting
accurate molecular mimics, thus yielding vaccines able to elicit antibodies
with the same activity as the selecting antibody.