Who am I? – Vet, Virologist and Mountaineer

Who am I?•Doctor of Veterinary Medicine (DVM) – 1990

Member WVA - 1990•PhD, Veterinary Virology – 1998, from The University of Tehran and Trained at EMBL, Germany in the field of molecular virology and bioinformatics.

Member of EVVA.•Associate Professor, Faculty of Veterinary Medicine, The University of Tehran 1998-2005•Post Doctoral research fellow in the field of molecular biology and mutagenesis of Mycoplasma. University of Melbourne, School of Vet. Science. 2005.•Post Doctoral research fellow in the field of Molecular biology and evolution of Avian Influenza virus. University of Melbourne, School of Vet. Science.2007.•Senior lecturer in Virology, SAVS, The Uni Adelaide, since 2009.•Virology, Immunology, Microbiology and public health coordinator since 2012

Phage Display System and Recombinant Antibodies

Dr Farhid HemmatzadehSenior Lecturer. VIMPH coordinator

The University of AdelaideSchool of Animal and Veterinary Sciences

Bacteriophage display technologyA powerful tool in molecular diagnostic

tests

The enemy of my enemy

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What is phage display?

In-vitro selection of genetically fused peptide or antibody to the coat protein of a bacteriophage

http://www.bio.davidson.edu/people/dawessner/micro/images/bacteriophage.gif

• Phage display is a term describing display of foreign (poly)peptides on the surface of phage particle. This is achieved by splicing a gene encoding such a peptide into a gene encoding a capsid structural protein.

• Phage Display was originally invented by George P. Smith in 1985

Phage Display Libraries

• A diverse population of recombinant bacteriophages displaying different peptides.

• Sensitive E. coli cells can quickly create large libraries of phage clones.

http://ifa.hawaii.edu/~jrich/oldstuff/tens/bacteriophage2.jpg

Amplification

• Recombinant bacteriophages can be selected using specific antigens.

• Eluted phages showing specificity are used to infect new host cells for amplification.

• Cycle repeated 2-3 times for stepwise selection of best binding sequence.

http://www.washington.edu/alumni/p

artnerships/biology/200710/images/ker

r_ecoli2.jpg

Analyze

• Final phages can be propagated then characterized with DNA sequencing.

• A panel of reactive bacteriophages can be used for mapping the antigens.

• Common motifs involved with binding may emerge for further study.

Advantages of Phage Display

• Easy to screen large number of clones >109

• Easy to amplify selected phages in E. coli

• Selection process easy and already in use in various forms.

• Can create Phage library variation by inducing mutations, using error prone PCR, etc.

• Genetically modified bacteriophages can be used as source of rABs or peptides or as a specific tool for phage therapy.

Disadvantages

• Might not have long enough peptide insert so critical folding can be disrupted.

• Very short and very long peptides are not display able in bacteriophages.(15-40kd)

• Could lose phage variations if first bind/wash step too stringent.

• Affinities or binding that results during selection might not work in vivo.

Bacteriophages

Filamentous M13 phage, EM image.T. Samoylova

7 nm

900 nm

pIII pVI pVIII pVII + pIX

M13 phage genome structure

Inoviridae

Why does it needed

Bacterial Pili and F protein

F- Bacterial chromosome

Bacterial chromosome

F episome F piliF+

Bacterial chromosome

Integrated F episome F piliF’

Bacterial chromosome

Polarized F episome F piliHRF

E.coli XL Blue MRF’

• Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)

173 endA1 supE44 thi-1 recA1

gyrA96 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]

• Used in lambda phage infection, amplification, expression

• Tetracyline resistant

• Specific tRNA for reading phagemid amber codon (TAG) as glutamine (GAG).

Bacteriophage cell interactions

• Lytic

– Bacteriophages kill the target cells

• Lysogenic

– Bacteriophages infect the cell but don’t lyse them

Lytic VS Lysogenic

lysogenic conversionand

bacteriophage transduction

Corynebacterium diphtheriae

lysogenic conversiontransduction

Significance of bacteriophages:• Transduction is responsible for transfer of drug resistance, especially

in Staphylococci

• Lysogenic conversion is responsible for acquisition of new characteristics

• Random insertion into bacterial chromosome can induce insertional mutation

• Epidemiological typing of bacteria (phage typing)

• Lambda phage is a model system for the study of latent infection of mammalian cells by retroviruses

• Phages are used extensively in genetic engineering where they serve as cloning vectors.

• They are responsible for natural removal of bacteria from water bodies

• Libraries of genes and monoclonal antibodies are maintained in phages

Significant of phage display

• Large numbers of virions just occupy a small volume (> 10 12 particle /ml)

• It is possible to construct foreign polypeptide libraries of the required diversity using recombinant DNA technology.

• In such libraries each phage display a unique random peptide.

• filamentous phage is predominant phage display vector

Phagemid VS PlasmidPhagemids combine features of a plasmids with features of a ssDNAphage vectors.

Phagemids contain an origin of replication (ori) for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles.

A phagemid can be replicated as a plasmid, and also be packaged as single stranded DNA in viral particles.

Bacterial host containing a phagemidwith a 'helper' phage, for example VCSM13 or M13K07 produces recombinant phage.

31

Monoclonal

• Expression of just a single clone of B cells

Normal protein serum B cell myeloma

Monoclonal antibodies

• Hybridoma system (Myeloma + B cell)

Phage Display system

• Displaying antibodies on the surface of Bacteriophages

Immunoglobulin genes

• Assembly and rearrangement

Kappa and lambda light chains mu, delta, gamma, epsilon and alpha heavy chains

VJCClusters of immunoglobulin genes

• 300 Variable regions, 5 junction region and 1 constant regions of immunoglobulin genes

• With 150 V (light) genes, 5 J and 10 possible VJ joining possibility for Light variation is 7500

• With 80 V (heavy) gene, 50 D gene, 6 J genes and up to 2.4 million possible can happening

• 7500 ×2,4 million give 18 billion possible clone

Mu, delta, gamma, epsilon and alpha heavy chains

Structure of Immunoglobulins

PHAGE DISPLAY FORMATS

• It is not possible to display larger polypeptides or proteins without affecting the function of the bacteriophage proteins.

• Six residue insertions are usually tolerated, but, when the insert is

increased to eight residues,

only 40 % of phage are

infectious and, at 16 residues,

this number drops to less than 1%.

helper phage

• Phagemids are engineered to express recombinant p3 fusions under controlled conditions, but do not encode any viral structural or replication proteins.

• Only upon superinfection of a bacterial host by a helper phagewhich contributes the missing genes can the phagemid ssDNA replication and packing take place.

• Helper phage are defective in their origin of replication or packaging signal thereby ensuring preferential packing of phagemids.

mRNA

library consists of billions of phage clones – billions opportunities to select phage clones specific to desired targets

A4- Primer list and PCR.docx PFU DNA Polymerase

E-tag

Kinds of Recombinant Immunoglobulin

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E-tag

Trypsin site

Purification of displayed antibodies

Phage display vectors

P8 P3

Phage rescue

Role of antigen for screening

• Size

• Purity

• Antigenicity

• Domains

• Complexity

• Glycosylation

Increasing the antigenicity by polymerisation

Antigenic mapping

Antigenic mapping for M2e protein of avian influenza protein.

Invited speaker for world influenza congress, London

Structure of rAb

• Sequencing

• Rare codons

• Hydrophobicity

• Linkers

• Tags

• Further purification

10 20 30 40 50 60

....|....|....|....|....|....|....|....|....|....|....|....|

F12HL24r_B08 IFAIPLVVPFYAAQPAVTLDESGGGLQTPGGALSLVCKASGFTFSDRGMGWVRQAPGKGL

F12HL30r_B10 IFAIPLVVPFYAAQPAVTLDESGGGLQTPGGTLSLVCKASGFTFSSFNMGWVRQAPGKGL

F12HL20r_B06 IFAIPLVVPFYAAQPAVTLDESGGGLQTPGGTLSLVCKASGFTFSSYGMVWVRQAPGKGL

70 80 90 100 110 120

....|....|....|....|....|....|....|....|....|....|....|....|

F12HL24r_B08 EWIAGIDDDGGSTYYAPAVKGRAIISRDNGQSTVRLQLNNLRAEDTATYYCAKGAYNCGN

F12HL30r_B10 EFVALINAAGSYTNYGAAVKGRATISRDDGQSTVRLQLNNLRAEDTGIYFCTKGSGGPYG

F12HL20r_B06 EYVAQISNDGGSTTWYATAVKGRATISRDDGQSTVRLQLNNLRAEDTGTYYCAKGAWSGC

130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|

F12HL24r_B08 CGAEIDAWGHGTEVIVSSGGGGSGGGGSGGGGSALTQPSSVSANPGETVEITCSGGYSDY

F12HL30r_B10 WCSAGSIDAWGHGTEVIVSSGGGGSGGGGSGGGGSALTQPSSVSANLGETVKITCSGGSG

F12HL20r_B06 ISWCWDAAGVIDAWGHGTEVIVSSGGGGSGGGGSGGGGSALTQPASVSANLGGTVKITCS

190 200 210 220 230 240

....|....|....|....|....|....|....|....|....|....|....|....|

F12HL24r_B08 GWFQQKSPGSAPVTVIYYNDKRPSDIPSRFSGSKSGSTATLTITGVQAEDEAVYYCGSAD

F12HL30r_B10 SWYGWYQQKAPGSAPVTVIYDNTNRPSDIPSRFSGSKSGSTATLTITGVQAEDEALYFCG

F12HL20r_B06 GGGSSYGSYYYGWYQQKSPGSAPVTVIYNNNNRPSDIPSRFSGSKSGSTATLTITGVQAD

250 260 270 280 290

....|....|....|....|....|....|....|....|....|....|....|....

F12HL24r_B08 SNYAGIFGTGTTLTVLGAAAGAPVPYPDPLEPRAA*TVESCLAKPHT------------

F12HL30r_B10 GYDSSSGAIFGAGTTLTVLGAAAGAPVPYPDPLEPRAA*TVESCLAKPHT---------

F12HL20r_B06 DEAVYFCGSYDSSNTAGIFGAGTTLTVLGAAAGAPVPYPDPLEPRAA*TVESCLAKPHT

SFi

Linker

E tag

No gaps

Variable VH

Variable VL

E tag

Linker

Applications

• Peptide mapping (proteomics)

• Immunogens (antibodies, therapy)

• Random peptide library

• Peptide ligands

• Drugs

• High definition antibodies

Applications

• Epitope mapping and mimicking

• Identification of new receptors & ligands

• Drug discovery

• Epitope discovery – new vaccines

• Creation of antibody libraries

• Organ targeting

Phage Nanobiotechnology Tatiana Samoylova

What we are doing with phage nanomaterials

(current focus)• whole phage particles as vaccines for contraception of animals phage particles that

display peptides with contraceptive properties

- GnRH peptide, zona-pellucida binding peptides

- phage body – carrier protein, peptides – antigens

• phage is effective immunogen (nanosize, architecture), low cost, stable, safe

• shown below – sperm-oocyte interaction in in vitro fertilization experiment in the

presence of (A) normal serum or (B) serum from immunized animal

Normal porcine serum Porcine anti-serum, contains anti-sperm antibodies

A B

Evaluation of the test on field serum samples

Improve purity of antigen

MBP

MBP M2e

M2e His/ Streptavidin tag

Double purification

MBP M2e

Protease digestion&

Double purification

TEV Thrombin

Ramin’s project

Multimeric expression of M2e antigen

Improve antigenicity

Improve purity by protease digestion

MBP M2e

M2e

Development a competitive ELISA to improve the specificity and sensitivity of the DIVA ELISA test

Rabbit Phage display system

cDNA libraryRNA isolation PCR

Cloning of Immunoglobulin library into Phagemid vector

Honours project

Further plans

• GFP conjugated rAbs

• Heterologous multimeric antogens