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Exploring the potential of novel multivalent DNA-based vaccines [Elektronische Ressource] / Nicolas Miguel Fissolo

104 pages
Universität Ulm Institut für Medizinische Mikrobiologie und Immunologie Sektion für Molekulare Infektionsimmunologie Ärztlicher Direktor: Prof. Dr. med. R. Marre Exploring the potential of novel multivalent DNA-based vaccines Dissertation zur Erlangung des Doktorgrades der Humanbiologie der Fakultät für Medizin der Universität Ulm vorgelegt von Nicolas Miguel Fissolo aus Argentinien, Cordoba 2004 Amtierender Dekan: Prof. Dr. med. R. Marre 1. Gutachter: Prof. Dr. R. Schirmbeck 2. Gutachter: PD R. U. Knippschild Tag der Promotion: 18-02-2005 A mis viejos For my Parents 1TABLE OF CONTENTS Abbreviations……………………………………………………………………….. 4 1. Introduction………………………….. 7 1.1. DNA vaccination................................................................................................. 7 1.1.1 Basics of DNA vaccination………….......................................................... 7 1.1.2. Vector design............................................... 7 1.1.3. Immunization strategies using DNA-based vaccines……………………... 9 1.2. Priming immune responses by DNA-based vectors ………….………..…….… 9 1.
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Universität Ulm
Institut für Medizinische Mikrobiologie und Immunologie
Sektion für Molekulare Infektionsimmunologie
Ärztlicher Direktor: Prof. Dr. med. R. Marre





Exploring the potential of novel multivalent DNA-
based vaccines





Dissertation
zur Erlangung des Doktorgrades
der Humanbiologie
der Fakultät für Medizin
der Universität Ulm






vorgelegt von
Nicolas Miguel Fissolo
aus Argentinien, Cordoba
2004




























Amtierender Dekan: Prof. Dr. med. R. Marre

1. Gutachter: Prof. Dr. R. Schirmbeck
2. Gutachter: PD R. U. Knippschild

Tag der Promotion: 18-02-2005





























A mis viejos
For my Parents
1
TABLE OF CONTENTS

Abbreviations……………………………………………………………………….. 4
1. Introduction………………………….. 7

1.1. DNA vaccination................................................................................................. 7
1.1.1 Basics of DNA vaccination………….......................................................... 7
1.1.2. Vector design............................................... 7
1.1.3. Immunization strategies using DNA-based vaccines……………………... 9
1.2. Priming immune responses by DNA-based vectors ………….………..…….… 9
1.3 Hsp73-mediated antigen expression facilitate priming of immune responses…. 10
1.3.1. Stress proteins are potent innate adjuvants………………………………. 11
1.3.2. Current experimental approaches for hsp-facilitated vaccine
delivery ………………….………………………………. 11
1.3.3. Usage of a stress protein-mediated expression system for DNA based
vaccines………………………………………………………………….. 12
1.4. Novel DNA-based vaccines…………………….. 14
1.4.1 Design of multivalent DNA-based vaccines …………………………...... 14
1.4.2 Viral overlapping reading frames ………………………………………... 14
1.4.3 Expression of cryptic antigen fragments in alternative reading frames…….16
1.5. Aim of the study……….…………………………………………………........... 17


2. Materials and methods……….……………………………………………………. 18

2.1. Mice……………………………………………………………………………... 18
2.2. Polymerase chain reaction (PCR)….………………………………………......... 18
2.3. Agarose gel electrophoresis...………………………………………………....... 18
2.4. Purification of DNA fragments from agarose gels………………………........... 19
2.5. DNA ligation…………………………………………………………………… 19
2.6. DNA transformation of E-Coli…………………………………………….......... 19
2.7. Plasmid isolation by cleared lysates method…………………………………..... 19
2.8. Restriction digestion……………………………………………………...…….... 20
2.9. Plasmid DNA vectors………………………………………………………........ 20
2.10. Cell cultures……………………….…………………………………………….. 26
2.11. Cell lines………………………………………………………………….……... 26
2.12. Transient transfection …………………………. 27
2.13. Immunoprecipitation…………………….………………………………………. 27
2.14. Protein analysis by SDS-PAGE…………………………………….................... 28
2.15. Western Blot……………………………………………………………………. 28
2.16. DNA vaccination……………………………………………………………….. 29
2.16.1. Intramuscular vaccination…………….… 29
2.16.2. Intradermal vaccination………………….. ………………….………… 29
2.17. Determination of antibody response………………..…………………………... 30
2.18. ELISA…………………………………………………………… 30
2.19. Determination of CTL response………………… 31
2.19.1. Preparation of splenocytes……………………………………..…………... 31 2
2.19.2.Determination of splenic CTL frequencies..................................................... 31
2.20. Statistical analysis……………………………………………………………….. 32


3. Results……………………………………………………………………………....... 33

3.1 Experimental design……………..………………………………………………… 33
3.1.1. Generation of expression vectors for DNA based vaccines……………….. 33
3.1.2. DNA vaccination…………………………………………………………... 33
3.2. Characterization of polytope vaccines………………………………………….… 35
+ 3.2.1. Priming multispecific, murine CD8 T cell responses by polytope DNA
vaccines……………………………………………………………………. 36
d +3.2.2. L -restricted CTL down modulate CD8 T cell priming to other epitopes
presented by different MHC class I molecules…………………………….. 36
d + d3.2.3. L -restricted CD8 T cell responses not L surface expression, down
+ modulate co priming of CD8 T cells restricted by unrelated MHC class I
molecules ………………………………………………………………….. 38
3.2.4. Construction of a hsp73-associated polytope DNA vaccine………………. 39
3.2.5. Hsp-associated expression of polytope vaccines facilitates co priming of
+ multispecific CD8 T cell responses in F1 (B6 x Balb/c)…………………. 41
3.2.6. Immunization with the hsp73-associated T -pt10 vaccine overrides the 77
d + suppressive effects of co primed L -restricted CD8 T cell responses…...... 42
3.2.7. Enhanced CTL response priming by hsp-associated T -pt10 with gene 77
gun DNA delivery………………………..………………………………... 43
3.3. Expression and immunogenicity of DNA vaccines encoding single- or multi-
copies of antigen domains ……………..…………………………………………. 44
3.4. Characterization of DNA vaccines encoding two overlapping reading frames…... 46
3.4.1. The HBV pol and surface antigens are expressed from two overlapping
ORFs of the same sequence……………………………...………………… 46
3.4.2. Immunogenicity of HBsAg and Pol expressed from alternative reading
frames of a DNA vaccine………………………………………………..... 48
d 3.4.3. The immunodominant L -restricted T cell response to HBsAg down
modulates T cell responses to other HBsAg, but not Pol epitopes………… 51
3.4.4. Promoter-dependence of HBsAg expression for HBV-pol encoding
vectors……………………..……………………………………………….. 53
3.4.5. Characterization of HBsAg expression by Pol-encoding vectors………….. 53
3.4.6. Priming of Pol and HBsAg-specific CD8+ T cell responses in mice
vaccinated with the different HBV-Pol encoding vectors…………………. 54
3.4.7. Construction of hsp-binding HBV Pol- of Pol fragment- encoding vectors. 55
3.4.8. Induction of Pol specific CD8+ T cell responses………………………….. 59
3.5. Translation of cryptic reading frames of DNA vaccines generates an extended
repertoire of immunogenic, MHC class I-restricted epitopes…………………….. 60
3.5.1. Expression of viral antigens from alternative reading frames of a DNA
vaccine……………………………..…………………………………......... 60
3.5.2. An immunogenic Pol epitope is processed from a peptide translated from
an out-of-frame reading frame of a HBsAg- encoding expression plasmid. 61
+3.5.3. Different HBsAg-encoding expression constructs that support CD8 T cell
priming to the Pol 3 epitope………………………..……………….......… 64
+3.5.4. Induction of ´out-of-frame´ OVA-specific CD8 T cell responses………... 66 3
4. Discussion……………………………………………………………………………. 70

4.1. Detection of immunodominance hierarchies with a polytope DNA vaccine ……. 70
d 4.2. Overcoming the suppressive effect of L -dependent immunodominance by
hsp73-associated polytope DNA vaccine ……………………………………….. 71
+ 4.3. Anti-viral CD8 T cell immunity to antigens derived from two overlapping ORFs
can be primed by DNA vaccines……………….…………………………………. 72
4.4. Cryptic reading frames of DNA vaccines generates an extended repertoire of
immunogenic, MHC class I-restricted epitopes …………………..……………. 74


5. SUMMARY……………………………………………………………………………76


6. REFERENCES……………………………………………………………………….. 78


7. APPENDIX…………………………………………………………………………….98
7.1. List of publications………………….98
7.2. Curriculum Vitae……………………………………………………99
7.3. Acknowledgements………………..100



























4
Abbreviations

α anti
aa mino acid
ab antibody
ADP adenosine diphosphate
Ag antigen
APC antigen presenting cell
ATP adenosine triphosphate
bp base pair
β2m beta-2-microglobulin
BFA brefeldin A
BSA bovine serum albumin
cat. no. catalogue number
°C degree Celsius
CD cluster of differentiation
CMV cyto-megallo virus
CpG Cytosine-phosphate-guanine
cT truncated T-Ag
CTL cytotoxic T lymphocyte
DC dendritic cell
DMEM dulbecco’s modified eagle medium
DNA deoxyribonucleic acid
Dna-J J domain that recruits cellular chaperones
E.Coli Escherichia coli
ELISA enzyme-linked immunosorbent assay
Enh enhancer
ER endoplasmatic reticulum
EtBr ethydium bromide
FACS Fluorescence activated cell sorted
Fc fragment crystalline
FCS fetal calf serum
FITC fluoresceine isothiocyanate 5
H-2 destination for major histocompatibility complex in mice
HBcAg hepatitis B core antigen
HBsAg hepatitis B surface antigen
HBV hepatitis B virus
HBV X hepatitis B x antigen
HC heavy chain
hCMV human cytomegalovirus
HEPES N-(2-hydroxyethyl)-piperazine-N’-2 ethane
HLA man leukocyte antigen
HEK humaembryonic kidney cells
HRP horseradish peroxidase
hsp heat shock protein
i.d. intradermal
i.m intramuscular
IFN interferon
Ig immunoglobulin
IL interleukin
IRES internal ribosomal entry sequence
Kb kilo base pair
kDa kilodalton
LC ligth chain
LB Luria-Bertani media
LMH chicken hepatoma cell line
LS large surface antigen
µ micro
M molar
mAbs onoclonal antibodies
ME mercapto-ethanol
MHC Major Histocompatibility Complex
min minutes
ml mili-liter
mlU li-units
mRNA messenger ribonucleic acid 6
MS middle surface antigen
MT etallothionin
nm nanometer
ORF open reading frame
OVA ovoalbumin
p plasmid
PAGE polyacrylamide gel electrophoresis
PAS protein A sepharose
PB phosphate-buffered saline
PCR polymerase chain reaction
PE phycoerythrine
poly A polyadenylation site
PPO 2,5 diphenyloxazole
psi pound per square inch
pt10 lytope vaccine
rpm round per minute
RT om temperature
RNA ribonucleic acid
SDS sodium dodecyl sulfate
SV40 papova virus 40
T-Ag large tumor antigen of SV40
TAE TRIS-acetate-EDTA buffer
TAP transporter associated with antigen-processing
TEMED N,N, N, N’, -tetramethylethylenediamine
Th T helper cell
TNF tumor necrosis factor
TRIS 2-amino-2-hydroxymethyl-1,3-propanediol







7
1 Introduction

1.1 DNA vaccination

1.1.1 Basics of DNA vaccination
DNA vaccination, involves the introduction into tissues of a DNA plasmid
containing antigen-encoding sequences. The plasmid DNA is inoculated into the host by
different techniques. The antigen is expressed from in vivo transfected cells, processed and
subsequently recognized by the immune system. DNA based vaccines induced humoral
and cellular immune responses against a wide spectrum of viral bacterial parasitic and
tumor antigens in different animal species (e.g., rat, woodchuck, mice, pig, rabbit, chicken,
etc) and in man. A key feature of DNA-based vaccination is the expression of
immunogenic proteins with correct posttranslational modifications, three-dimensional
conformations or oligomerizations. The integrity of conformational epitopes that stimulate
neutralizing antibody (B cell) responses is thereby maintained. Furthermore, DNA
immunization is exceptionally potent in stimulating T cell responses. Peptides can be
generated from intracellular or extracellular protein antigens (expressed after transient in
vivo transfection) in both endogenous and exogenous processing pathways that efficiently
stimulate cellular (T cell) and humoral (B cell) immune responses. The efficient and
specific stimulation of T cell responses is a priority in current vaccine research, this makes
genetic vaccination an attractive candidate for prophylactic or therapeutic immunization
approaches against intracellular (viral, bacterial or parasitic) pathogens and cancer
[30;45;61].

1.1.2 Vector design
The expression vectors are engineered for optimal expression in eukaryotic cells
(Fig 1). The essential elements required are: i) the plasmid backbone necessary for
production in bacterial systems that contains the origin of replication and an antibiotic
resistance gene for prokaryotic selection, and ii) an expression unit containing viral
promoter/enhancer sequences, the gene of interest and regulatory elements for mRNA
editing (e.g. intron sequences, polyadenylation sites) (Fig. 1).
Plasmids are closed circular, double-stranded DNA. Supercoiled plasmid DNA
seems to ensure the most efficient, transient transfection in vivo into immunologically

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