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.