Mouse skin 4 days after inoculation

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Advanced Aspects of Genetic Immunization
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Genomics - Proteomics Gap
Genomic-based technology is much faster than
proteomic-based technology
Genomics
Throughput
Proteomics
Time
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Number of Proteins

• 30,000 human genes
• gt1million human proteins?

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Proteomics-Scale Antibody Production
GCGTTAGGCTCCTAGGATGATCGATGATGCTTATATCGCGGCTGCTGATG
CTAGTCGATTATGCTCTCGCTAATTAAGCGCTAGCTGCTAGCTGACTGCA
AGCACGCAAAGGTTTCTGGGATGATTTCAGCCAGACCTCTGGTGACAAGG
GCCGCGTGGAGCAGTGACACCAGCAAAAGATGGCACGCGAACCAGCCAAA
AGCTAGCTAGCATCGATAGCATCAGCATCAGCATCAGCACATTATATCAG
CGATAGACTAGTAGCATGAGTTATGAGCGCGCGCATTAGCATATTAGCGA
TAGCGCGGCGCATTATATAGCATCATCGACTAGTCGATCGATAGTAGATG
ACTACTAGCTAGATGCAGCATTATAGCATTATATATGCATGAGGCGCGGG
CCGATACGATATTATAGCTCTATCACACAACGTTGTAAACGTATATACGG
CAGTCAGAGATCAGCGCGCGCTATT
Genome Sequence
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High Throughput Measurement of Proteins
Example antibody microarray
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Protein Problem
GCGTTAGGCTCCTAGGATGATCGATGATGCTTATATCGCGGCTGCTGATG
CTAGTCGATTATGCTCTCGCTACTTAAGCGCTAGCTGCTAGCTGACTGCA
AGCACGTAAAGGTTTCTGGGTATTTAGCGACGAGCGTGCTAGTGTTCATT
Most ligand selection methods require at least
?g quantities of soluble purified target protein
Overexpress Purify
Obtaining the target protein is a major
bottleneck
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Protein Purification Problems
Labor intensive
Technically difficult
Contamination
Denaturation
Not easily automated
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Polyclonal Antibodies
Protein immunization
Johnston et al. (1992) Nature 356 p152-154
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Advantages of Producing Antibodies by Genetic
Immunization
Native recognition
No contaminants
Faster/cheaper/easier
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Immunization Regime
Antibody levels
3 weeks
3 weeks
On average most mice produce high titer responses
after two immunizations
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Antibody Responses
100
Anti-AAT Equivalents (mg/ml)
80
60
40
20
0
1
2
3
4
5
Ave.
Individual Mice
Typical titer 20,000
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Reliability
gt90 success rate
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Detecting the Natural Antigen
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Antibodies to Difficult Antigens
Ubiquitin
INPUT
IP
GI
Comm.
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Mouse Antibodies to Mouse proteins
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Antibodies to Peptide Antigens
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Antibodies to Splice Variants
ELISA
Exon1
Exon3
IEERLGS VSGGELF
500ng
100ng
20ng
4ng
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Larger Animals
Control
Antigen
Control
Antigen
Rabbit anti-ABCG5
Chicken anti-HDAC5
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The Two Arms of the Immune System
Immune system
Antibodies
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Antigen Localization
DC
MHCII
MHCI
B cell
MHCII
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CTL Vector Design
Val
Ubiquitin
Antigen
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Antigen targeting -CTL
Endoplasmic reticulum
MHCI
Antigen
Proteasome
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Antigen targeting-Ab
Antigen
Secretion leader peptide
A leader sequence is fused to antigens to allow
secretion
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Expression of Antigen
In general the more antigen produced the better
the antibody response
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DNA Dose
Dose is limited to about 2.5mg per shot
The limitation is due to the capacity of the
gold for binding DNA and the amount of gold that
can be delivered into tissue without causing
damage.
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DNA Dose - Antibodies
10ng
1000ng
1ng
100ng
Dose immunized into mice
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Transcriptional Promoters
Immediate early enhancer/promoter from human
cytomegalovirus with an intron from b-globin
(hCMVieI) 1150bp
Synthetic enhancer/promoter designed from
transcription factor consensus binding sites and
assembled from oligonucleotides (SP72) 600bp
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Transcriptional Terminators
Rabbit beta globin terminator 500bp
RGT
Human growth hormone terminator 700bp
HGH
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Optimizing Translation
Translation can be enhanced by recoding genes
with codons that are decoded by high abundance
tRNAs
GCT GCC GCA GCG
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Codon Optimization Examples
Antigen
Control
Control
Antigen
Antigen
Control
Control
Antigen
Wild Type
Codon Optimized
Wild Type
Codon Optimized
Bacterial example
Mammalian example
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Codon Optimization
ATGTGGGCCGCCGACTCGGACAGGGAGGACCTGACCATGATGATCGACGA
CCTGGACCGCAGAGGCTTCAGATCCATCTACCGGCTGCTGGCCAGCCGCT
GCAAGCTGGCCGACGCGTTCCAGAATTGGGCGCGCTCCCGGCTCGCCCGC
TACATCATCCTGTCGGCCGCGATCGTCATCTACTACGTGCTCTCGTCGAT
CCTGTCCCTGTCGATCTCGATCTACCGGGCCTCCTCGATGTG
ATGTGCGCTGCTGATTCTGATAGGGAGGATCTTACTATGATGATAGATGA
TCTAGATCGTAGAGGATTTAGATCTATATACCGGCTATTGGCTAGTCGAT
GCAAGCTAGCTGATGCTTTTCAAAATTGGGCGCGCTCTCGGCTCGCTCGC
TATATTATTTTATCGGCTGCTATCGTCATCTACTATGTTCTCTCGTCGAT
ATTATCTCTATCTATTTCTATCTATCGGGCCTCTTCGATGTG
Assemble gene from oligonucleotides
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Gene Synthesis
Pool of Oligos
Assembly (PCR)
etc
Amplify (PCR)
PCR assembly method (Gene v164 p49 1995)
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DNA Builder Software
Designs oligos for gene synthesis
Available free at pga.swmed.edu
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Gene Synthesis Example
Assembly
PCR
Gene 3
Markers
Gene 1
Gene 2
Gene 3
Gene 1
Gene 2
No natural DNA template required
1500-
1000-
500-
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Oligo Quality
Fraction of correct genes
(Midland)
0.1 deletions
0.3 deletions
(IDT, Sigma)
Length of Gene
1 bp deletions are the most common mutations
found in synthetic oligos
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Secretion of Antigen
Nucleus
Golgi
E.R.
Folding/Solubility
Translation
Transcription
Processing
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Antigen Selection -antibodies
Small regions (20-100aa) are selected to allow
high levels of expression and secretion and is a
convenient size for gene synthesis.
Surface regions are selected to ensure efficient
expression and that the resulting antibody will
recognize the native antigen.
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Antigenic Index
Select for immunization
Jameson Wolf algorithm combines
hydrophilicity, surface probability, flexibility
and secondary structure prediction scores.
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Passing Quality Control
Nucleus
Golgi
E.R.
Folding/Solubility
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Enhancing Secretion
Antigen
Secretion leader peptide
A small highly soluble and stably folded protein
is fused to the antigen to enhance secretion
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Post-translational modifications
Antigen
Glycosylation
NX
S
T
Furin proteases
R
RX R
K
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Immune Response Problems
Antigen uptake
B cell stimulation
B cell epitopes
DC
DC stimulation
T cell stimulation
Antigen uptake
T cell epitopes
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Enhancing Antigen Uptake
DC
B cell
An oligomerization domain is fused to the antigen
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T cell epitopes
Universal P2 and P30 epitopes from tetanus toxin
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Internal Control Tag
T cell epitopes
Antigen
SE/oligo
Leader
Tag
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Genetic Adjuvants - Antibodies
GMCSF/Flt3L
Antibodies
GMCSF
None
Time (weeks)
GMCSF and Flt3L are DC growth factors
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Genetic-Based Antibody System
PCR Product
Overexpression Vector
Immunization Vector
Overexpress in E. coli
Immunoassay
Genetic Immunization
Chambers Johnston (2003) Nat. Biotech. 21
p1088-1092
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Linear Expression Elements
Antigen
Control
Antigen
Promoter
Terminator
Terminator
Antigen
Promoter
Sykes Johnston (1999) Nat. Biotech. 17 p355-359
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Assembly of LEEs by Overlap PCR
Antigen
P
T
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LEEs Encoding Peptides
Antigen
Control
T
Ag
T
GDETRQAGKNEEFGGEDTSS
TYGRIRRVNNQAEEDQASAS
WGDTWTQKKSVCIKTEEDQQ
DGGSAASITRKIRQSDEDSR
Select peptides
2 weeks after 2nd immunization
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In Vitro Transcription and Translation (IVT)
RTS continuous-exchange cell-freetechnology
(CECF) boosts yields

• Continuous exchange boosts protein yields by
• Replenishing energy sources
• Removing inhibitory reaction by-products
• Extending translation period

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LEE-Based IVT
T7T
T7P
DNA
Markers
GST-----------TEV-Flag-----Gene-----SII
-DNA
Yield 10-100 ng/ul
GST Fusions
PDGFb
PDGFa
TNFb
TNNCI
TNFa
TGFb
Vegf
SOD
a-SII Probe
Western
Coomassie
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HTP Antibody System
Oligonucleotides
Overexpression LEE
Immunization LEE
In vitro Expression
Immunoassay
Genetic Immunization
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Antibody Production -Step 1
ATGTTAGGCTCCTAGGATGATCGATGATGCTTATATCGCGGCTGCTGATG
CTAGTCGATTATGCTCTCGCTACTTAAGCGCTAGCTGCAGCGTGCTAGTG
TTCGCGGTAG
Oligo-1ATGTTAGGCTCCTAGGATGAT Oligo-2GTATGCTGATCG
TAGCTAGCT Oligo-3GATCTAGATTACGTAGTCGTC
Oligo-4GCGCTAGTCGATGCTAGATAG Oligo-5TCTCGATAGATC
GTAGATATA Oligo-6GCTCGTAACACACGTCGCCAA
Oligo-7GATAGTCGCTCGCTATTACGC etc
ATGTTAGGCTCCTAGGATGATCGATGATGCTTATATCGCGGCTGCTGATG
CTAGTCGATTATGCTCTCGCTACTTAAGCGCTAGCTGCAGCGTGCTAGTG
TTCGCGGTAG
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Assembly of LEEs - Step 2

Mix a set of 16 Oligos (40mers)
Assembly (PCR1)
Oligos
Add overlap flanks (PCR3)
Mix PCR fragments
Amplification (PCR2)

Assembly (PCR4)
Amplification (PCR5)
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Robotic Assembly of LEEs
Robot can assemble 192 LEEs/day
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Immunization Protein Production
4-8 weeks
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Antibody Production Service
http//pga.swmed.edu
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Ordering Pre-made Antibodies
803 Antibodies made or in production
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Your name
Your institution
Enter requested antibodies
MTA Order Form
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MTA Order Form
Either sign pdf version electronically or print
out html version and FAX it to us
Shipping address and shipping account number
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Acknowledgements
Stephen Johnston
Joey Nguyen
Tom Kodadek
Vickie Seward
Dave Fancy
Theresa Chou
Xiang Chen
Alice Shaw
Xiao-hua Li
Greg Urquhart
Jerry Lin
Eileen Ward
Rosemary Wilson
Steve Liu
Southwestern PGA
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Order Pre-made antibodies
Request Antibodies for production
http//pga.swmed.edu
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Workshop -Thursday
Gene building part 2
LEE construction part 2
Assessing immunizations (Luc assays)
Analysis of GB, LEEs and bullets
DNA Builder software
Discussion of days results open question time