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Rational design of DNA epitope vaccines

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Rational design of DNA epitope vaccines

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Content RATIONAL DESIGN OF DNA EPITOPE VACCINES
by
Dimitrios Nikolaos Vatakis
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILISOPHY
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
August 2003
Copyright 2003 Dimitrios Nikolaos Vatakis
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UMI Number: 3119083
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U N IV E R SIT Y O F SO U TH ER N CA LIFO RN IA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES, CALIFORNIA 90089-1695
This dissertation, written by
Dimitrios Nikolaos Vatakis
under the direction o f h IS dissertation committee, and
approved by all its members, has been presented to and
accepted by the Director o f Graduate and Professional
Programs, in partial fulfillment o f the requirements for the
degree of
DOCTOR OF PHILOSOPHY
Director
Date A u gu st 1 2 . 2003
Dissertation Committee ^
- ' t ' i
Chair
/n x c u > (.
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TABLE OF CONTENTS
L ist o f T ables..............................................................................................................................................iii
List of Figures............................................... iv
A bstract....................................................................................................................................... viii
Chapter 1: Introduction........................................................................................................... 1
Chapter 2: The Influence of the Signal Peptide Sequence on the Immune
Response Elicited by DNA Epitope Vaccines......................................................................13
Chapter 3: Helper Epitope Influences the CTL Response Elicited by DNA
Epitope Vaccines........................................................................................................................44
Chapter 4: The Influence of Chemokines on the Immunogenicity of DNA
Epitope Vaccines........................................................................................................................63
Chapter 5: Materials and M ethods................................................................................... 81
References...................................................................................................................................109
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Table 2-1:
Table 2-2:
Table 2-3:
Table 3-1:
Table 5-1:
Table 5-2:
ill
LIST OF TABLES
The num bers o f m ice used, the vaccine received and the
immunization regim ent.................................................................................21
List of the (3-galactosidase transfectants used in this study..................30
A list o f the transfectants used to determ ine the effect o f leader
sequence on the epitope presentation........................................................ 37
The am ino acid sequences o f the w ild type OVA epitope and its
variants...............................................................................................................46
List of synthetic oligonucleotides used for PCR analysis, sequencing,
and gene assembly..........................................................................................82
Restrictrion endonucleases........................................................................... 87
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LIST OF FIGURES
Figure 1-1: An overview of the immune response..........................................................3
Figure 1-2: The mechanism by which DNA vaccines induce immunity................... 7
Figure 1-3: A schematic diagram of a DNA epitope vaccine....................................... 8
Figure 1-4: The classical M HC I (Class I) and M HC II (Class II) antigen
presentation pathways..................................................................................... 9
Figure 2-1: The amino acid sequences o f the murine Ld leader and the rat KC
chemokine leader.............................................................................................14
Figure 2-2: The schematic diagram s o f the vectors used in this chapter. Their
exact composition is outlined in Materials and Methods section 15
Figure 2-3: Representative 5 1 Cr release assays............................................................... 18
Figure 2-4: Summary plots of two experiments, using a total of 4 mice per group,
showing specific lysis at 200:1 effector to target ratio...........................19
Figure 2-5: A summary plot of the two experim ents showing maximum specific
lysis by effector cells..................................................................................... 22
Figure 2-6: A representative proliferation assay........................................................... 24
Figure 2-7: Mice injected with the vector expressing the new leader (RAOLL)
showed higher proliferation after in vitro stimulation with OVA (323-
339).....................................................................................................................25
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V
Figure 2-8: A representative ELISA assay from two experim ents m easuring the
amount of IFN-y release............................................................................... 27
Figure 2-9: ICCS measuring the amount of TH or CTL IFN-y secreting.cells...... 28
Figure 2-10: The rat chem okine KC and the mouse H-2Ld leaders were inserted
into the pLacz expression vector upstream of the LacZ gene 31
Figure 2-11: A schematic diagram o f the vectors used to measure g p l2 0 secretion
by the two leaders........................................................................................... 34
Figure 2-12: An ELISA assay measuring the amount of g p l2 0 secreted by the cells
transfected with the SA6H and RA6H expression vectors................... 35
Figure 2-13: An ELISA measuring secreted levels of g p l2 0 ........................................ 36
Figure 2-14: A representative CTL assay from two experiments................................38
Figure 3-1: Schematic diagrams of the vectors used in this portion of the study..47
Figure 3-2: A representative 5 1 Cr release assay..............................................................49
Figure 3-3: A summary plot of two experiments, showing maximum specific lysis
by effector cells................................................................................................50
Figure 3-4: The average values o f the proliferation responses from two
experiments.......................................................................................................52
Figure 3-5: An ELISA assay detecting IFN-y levels...................................................54
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Figure 3-6: ICCS measuring the number of TH IFN-y secreting cells.......................55
Figure 3-7: ICCS measuring the number o f CTL IFN-y secreting cells after in
vitro stimulation with gpl20 (318-327).....................................................57
Figure 3-8: An ELISA assay detecting IL-2 release.......................................................58
Figure 4-1: Schem atic diagram s o f the vectors encoding the chem okine
sequences, the epitopes and the targeting sequences............................ 65
Figure 4-2: The amino acid sequence of the chemokines used in this chapter 66
Figure 4-3: A summary plot of two experiments, showing specific lysis at 100:1
effectontarget ratio.........................................................................................68
Figure 4-4: A representative proliferation assay........................................................... 70
Figure 4-5: An ELISA assay detecting IFN-y levels...................................................71
Figure 4-6: ICCS measuring the amount of TH IFN-y secreting cells......................73
Figure 4-7: ICCS measuring the number of CTLs secreting IFN-y after in vitro
stimulation with g p l2 0 (318-327).............................................................74
Figure 5-1: Construction of RAOYZLL...........................................................................89
Figure 5-2: Construction of RAOLL.................................................................................91
Figure 5-3: Construction of RAOYTLL...........................................................................93
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v ii
Figure 5-4: Construction of pSLacZ and pRLacZ.........................................................95
Figure 5-5: Construction of pKC(A), pKC(B), pK C (C )............................................97
Figure 5-6: Construction of pK C (A B C ).........................................................................98
Figure 5-7: Construction of RFLKC, RKC4, and RK C8.......................... 99
Figure 5-8: Construction of RA6FI and SA6H.............................................................101
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ABSTRACT
viii
The em ergence of new viruses and antibiotic-resistant b acteria has
dem onstrated the need for new vaccines. DNA vectors, when injected in mice, can
elict strong cytotoxic T cell (CTL) responses when compared to peptide vaccines. I
have constructed plasm ids that express the H -2Dd-restricted CTL epitope HIV
g p l2 0 (3 18-327) and the H-2Ad -restricted TH epitope ovalbumin OVA (323-339). In
addition, they encode sorting sequences that direct these epitopes into the class I or
class II antigen presentation pathways.
The goal of this dissertation was to enhance the gp 120-specific CTL response
elcited by the DNA plasm ids, by exam ining three aspects affecting the CTL
response. First, I wanted to determine whether the identity of the signal sequence can
alter the cell-m ediated response induced by a DNA epitope vaccine. Therefore, I
replced the existing leader derived from the H-2Ld molecule with a leader from the
rat KC chemokine, and showed this plasmid elicits an enhanced immune response.
Second, I replaced the OVA (323-339) epitope with variants of the same
epitope that have different bidning affintites for the Ad molecule in order to assess
the effect on the magnitude of the gp 120-specific CTL response. In one RAOM LL, I
replaced the wild type with a variant in which the anchor residue valine 327 has been
substituted by an arginine, which decreases the binding to Ad. In the RAOYTLL, I
used a high affinity variant in which the flanking residues, 323-326 and 333-336, on
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ix
either side of the core region, have been replaced with the sequences YTYT. The
presence of the high affinity helper epitope enahcned the CTL response.
Finally, I introduced chemokine sequences to assess their effect on the CTL
responses generated by the DNA epitope vaccines. M ore specifically, I used the
m ouse KC chem okine, known to attract neutrophils, and a series o f truncated
variants and exam ined whether their use can im prove the gp 120-specific CTL
response. The presence of these sequences did enhance the im munogenicity o f the
DNA vaccines.
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CHAPTER 1: INTRODUCTION
1
V accination has been extensively used over the years to protect from
infectious agents (Ada 2001). The vaccines currently used are live attenuated or dead
viruses, subunit, and conjugate vaccines (Ada 2001). The em ergence, however, of
antibiotic - resistant bacteria (e.g., tuberculosis) (Flynn and Chan 2001) as well as
new pathogens (e.g., Human Imm unodeficiency Virus (HIV) and Ebola virus) has
underlined the need for alternative forms of vaccines.
Recent studies have shown that the injection of “naked” plasm id DNA can
induce a strong cellular and humoral immune response (W olff et al. 1990; Yang et
al. 1990; Tang et al. 1992; Fynan et al. 1993; W ang et al. 1993; Raz et al. 1994;
Boyle et al. 1997; Gurunathan et al. 2000). These findings have the potential to lead
to the developm ent o f a new generation of vaccines, which may be effective in
dealing with the new challenges posed to the world. DNA vaccines are attractive
because they are inexpensive to produce in large amounts and they are heat - stable.
This will allow massive vaccinations in Third W orld countries where medical care is
very expensive, the infrastructure and good environm ental conditions are non
existent.
Antigen processing and presentation
V iruses are m icroorganism s that exploit the nucleic acid and protein
expression machinery of the host to replicate. The majority of the viruses infect host
cells by binding to normally expressed surface receptors, many of them important for
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2
the functions of the host. One example is HIV, which infects cells by binding to the
CD4 molecule on the surface of human T cells. After infection, viruses replicate in
the host cell producing viral proteins, which are degraded in cytosol by proteasom es
(Poignard et al. 2001; Douek et al. 2003). Some of the peptides generated by this
process are the part of the antigen that is specifically recognized by the T cells and
are called epitopes. The epitopes are, then, transported to the endoplasmic reticulum
(ER) and associate with major histocompatibility complex class I m olecules (MHC
I). The M HC I-antigen complexes are, then, presented on the cell surface (Germain
and M argulies 1993; Elliott et al. 1995; York and Rock 1996; Pamer and Cresswell
1998; Rock and Goldberg 1999). This is regarded as the conventional route o f MHC
I presentation. Some of the viral products will be secreted by the infected cells. The
secreted antigens will be internalized by the antigen presenting cells (APC) and can
be directed to the MHC I presentation pathway. This is called cross-priming (Bevan
1976; Carbone and Bevan 1990; Pfeifer et al. 1993; W atts 1997; Grom m e and
Neefjes 2002). In addition, the secreted antigens are endocytosed and degraded in the
endosom e. M HC class II m olecules (M HC II) are synthesized in the ER and
associate with an invariant chain (Ii) to prevent binding of other peptides. The MHC
Il-Ii com plexes move to the endosom e where the Ii is cleaved and the antigenic
peptides are loaded on the MHC II. Once loaded, the antigen is represented on the
cell surface associated with MHC II (Germain and Margulies 1993; Cresswell 1994;
W atts 1997; Chapman 1998) (Figure 1.1).
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CD4 Th cell
MHC II
peptide
complex
E
APC
CDS Tc cell
MHC I-peptide complex
Virus
Infected
cell
Viral Antigen
Figure 1-1: An overview of the immune response. Infected cells present epitopes from endogenously made antigens on
MHC I to CTLs. In addition, antigen is secreted and internalized by local APCs that will process and present associated
with MHC II to the TH cells. Upon engagem ent of the TCR w ith the M H C -peptide com plex and with the help of
costimulatory molecules, TH cells will be activated and secrete IFN-y and IL-2 effector cytokines that provide the second
signal needed to activate CTLs to kill the infected cell (indicated by the botton the figure).
4
The induction o f an immune response
T cells express a diverse array of receptors. T cell receptors (TCR) have been
positively and negatively selected in the thymus to distinguish “self “ from “non
s e lf ’. T cells that recognize specific M H C -peptide com plexes proliferate and
differentiate, mediating a specific immune response. Activated lymphocytes will also
generate memory cells. M emory cells ensure a rapid immune response when the host
is re-infected with the same or a structurally related pathogen. The generation of
memory cells is the most important feature of a successful vaccine since it confers
long lasting protective immunity. This process is facilitated by the secretion of
cytokines and chemokines. Cytokines are proteins secreted by lymphocytes and other
cell types and influence the activation state as well as the effector functions of
lymphocytes (Hunter and Reiner 2000). Chemokines are small polypeptides secreted
by a variety o f cells and influence the migration and activation of lym phocytes
(Rollins 1997; Baggiolini 1998; W ard et al. 1998; Zlotnik et al. 1999; Rossi and
Zlotnik 2000; Yoneyama et al. 2000).
There are two types of T cells, CD4+ T helper cells (TH ) and CD8+ T cells
(cytotoxic T lymphocytes or CTL). T H cells recognize antigens associated with MHC
II, which is expressed on the surface of APCs (M urray, 1998). TH cells will become
activated and will then specialize into Tm and T H 2 cells. Tm cells elicit a cell-
m ediated im m une response and release IL-2, IFN-y, and TNF-(3 cytokines.
Furthermore, TH cells play a pivotal role in the activation and generation of memory
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5
CTLs. Th2 cells elicit a humoral immune response and release IL-4, -5, -6, -10, -13,
w hich stim ulate B cells to produce antibodies (Thom pson 1995; C onstant and
Bottomly 1997; Bonecchi et al. 1998; M urray 1998).
CTLs recognize peptide associated with M HC I. Once activated, they elicit a
cytotoxic response and kill infected cells. Killing is mediated via Fas - Fas L ligand
interactions, perforin, and granzym e release. Furtherm ore, activated CTLs release
cytokines such as IL - 2 and IFN - y, thus, enhancing the immune response (Barry
and Bleackley 2002) (Figure 1.1).
Epitope DNA Vaccines
The research discussed in this thesis, concentrates on the design and
optim ization o f DNA epitope vaccines. DNA epitope vaccines utilize the specific
antigenic epitopes of a protein instead of the entire polypeptide, thereby, rem oving
any sequences that may adversely affect the efficacy of the vaccine. Also, I can use
different epitopes from different proteins. M ore specifically, there are epitopes that
are recognized by CTLs and others by TH cells. The use of both types o f epitopes
enables the design of a vaccine that elicits a com plete immune response. The use of
epitopes, however, may pose a problem. Due to major histocompatibility com plex
(M HC) polym orphism, each individual responds to a different epitope. This issue
can be resolved by adding different epitopes of the same protein in a DNA vaccine.
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6
To further enhance the effectiveness of DNA vaccines, I can introduce other
sequences from chem okines and cytokines that will improve the induced im mune
response. Also, I can add the appropriate targeting sequences that w ill direct the
expressed epitopes to the appropriate antigen presentation pathways.
Mode o f Action and Features o f DNA Vaccines
The mechanisms by which DNA vaccines elicit immune responses have not
been fully elucidated. Based on studies thus far, there are three m echanism s by
which the encoded antigen can be processed and presented (Figure 1-2): (a) direct
transfection of the local antigen presenting cells (Casares et al. 1997; Akbari et al.
1999; G urunathan et al. 2000), (b) transfection of local som atic cells and
presentation of antigen via class I to CTLs (Fu et al. 1997; Gurunathan et al. 2000),
and (c) cross-priming where transfected local somatic cells or APCs secrete antigen
which in turn is taken up by other APCs (Figure 1.2) (Casares et al. 1997; Fu et al.
1997; Akbari et al. 1999; Gurunathan et al. 2000).
A DNA plasmid vaccine consists of a strong mammalian prom oter such as
cytom egalovirus (CM V), the gene(s) o f interest, as well as a polyadenylation-
transcription term ination site (Figure 1-3). The genes of interest consist o f the
epitopes that I want to express, any immune enhancing sequences such as cytokines
and chemokines, as well as targeting motifs (Gurunathan et al. 2000). These protein
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Somatic Cells
CD4 Thoell
P la sm a
DNA
Plasmid
DNA
Secreted Ag
CD8 Tc cell
CD8 Tc cell
Figure 1-2: The m echanisms by w hich D NA vaccines induce immunity. Plasm id D N A is taken up by the local
APCs that express, process and present the encoded antigens to TH cells and CTLs. Local somatic cells can take up
the plasmid and secrete the encoded antigens which will be then in turn endocytosed and processed by APCs. DNA
can be also internalized by local somatic cells that will express and present the antigens to CTLs.
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Gene Insert
DNA vaccine
Th EPITOPE Tc EPITOPE
EHDOSOME
TARGET
LEADER
TRANSMEMBRANE
DOMAIN
Figure 1-3: A schematic diagram of a DNA epitope vaccine.
00
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Virus
MHC I-peptide complex
it
MHC H-peptide complex
Proteasome
ENDOSOME
Class I Pathway
Class II Pathway
Figure 1-4: The classical MHC I (Class I) and M HC II (Class II) antigen presentation pathways.
10
targeting sequences will direct the epitopes to the appropriate cell com partm ents,
directing them to the class I or class II presentation pathway (Figure 1-4). M ore
specifically, all vaccine constructs contain a hydrophobic leader sequence, which
destines the polypeptide to be synthesized in the ER. There, it will be degraded to the
peptides that will associate with M HC I. In addition, there is an endosome targeting
sequence that will direct the remaining epitope to the class II presentation pathway
(Figure 1-4).
Project goals and overview
The primary goal of this project is to optimize the CTL responses generated
by a DNA epitope vaccine. This is achieved by replacing or introducing com ponents
crucial to the development o f an immune response.
For my thesis, I have used two epitopes in most of the vector vaccines. I have
constructed a series of plasm ids that encode a CTL epitope, HIV g p l2 0 IIIB (318-
327) targeted by a hydrophobic leader to the ER and a T H epitope (M ichalek et al.
1992), ovalbumin OVA (323-339) targeted by and endosom al/lysosom al targeting
m otif (Schmid 1997; Geuze 1998).
In Chapter 2, I replaced the existing leader derived from the murine H-2Ld
molecule with a leader from the rat KC chemokine (Huang et al. 1992) and measured
the immune response elicited by both plasmids. The rationale was that a leader,
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11
derived from a secreted molecule, might be more effective than one derived from a
transm em brane molecule at generating protein in the constitutive secretory pathway
(M ucke L., personal communication). As a consequence, the antigen concentration
would increase, and hence boost the observed immune response. Based on the data,
the vectors expressing the rat KC leader elicit a stronger immune response com pared
to the H-2Ld-derived one. Furthermore, expression vectors encoding protein secreted
by the two leaders were transfected in cell lines. The presence of the rat KC leader
resulted in higher protein secretion. In addition, I transfected P815 cells with the
vectors used in our immunizations and generated stable transfectants that were used
as targets in a CTL assay. Cells transfected with the vector encoding the new leader
were killed more efficiently by effector lymphocytes.
In Chapter 3 , 1 address the question of affinity of helper epitope for the class
II molecule and its effect on the immune response. T H cells have been shown to play
a crucial role in the generation of effective and protective CTL responses. The
rationale in this chapter is that altering the affinity of the T H epitope will affect its
ability to stimulate helper T cells. Thus, any changes in TH activation will affect the
strength of the CTL response against the encoded CTL epitope. More specifically, I
used well-characterized variants of the OVA (323-339) epitope. These variants have
higher or lower affinities to the MHC II molecule Ad (Lamont et al. 1990; Sette et al.
1990). M ice were injected with vectors encoding the variant sequences and used in
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12
experim ents to analyze the immune response. The data prove that m anipulating the
affinity to the M HC II affects the immune response generated by a DNA epitope
vaccine (M aecker et al. 1998; M urray 1998; Slansky et al. 2000; Ahlers et al. 2001).
Finally in Chapter 4 , 1 examined the effect o f a chem okine sequence on the
CTL response elicited by the DNA epitope vaccines. More specifically, I used the
KC chem okine, a neutrophil (PMN) chemoattract and truncated variants o f it. PMNs
have been recently shown to express high levels of M HC I on their surface and
present epitopes to CTLs. Thus, increased attraction o f PM Ns to the site o f
im m unization can potentially enhance the CTL response due to increased antigen
presentation. To address this, I inserted the full-length gene o f the m ouse KC
chem okine as well as truncated versions of it (Bozic et al. 1995; Loetscher et al.
1998; King et al. 2000). I im m unized mice with these vectors, and studied the
elicited im m une response. M ice im m unized with vectors encoding chem okine
sequences showed improved CTL responses and higher numbers of epitope specific
CD8+ T cells. These data suggest that the introduction of a chem okine sequence can
influence the immunogenicity of a DNA epitope vaccine.
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CHAPTER 2: THE INFLUENCE OF THE SIGNAL PEPTIDE
SEQUENCE ON THE IMMUNE RESPONSE ELICITED BY DNA
EPITOPE VACCINES
Introduction:
Endogenously synthesized antigens are degraded by the proteasom e in the
cytoplasm and translocated into the endoplasmic reticulum (ER) by the transporter
associated with Ag presentation (TAP) where they are loaded onto the M HC I
molecules followed by presentation on the cell surface (M ichalek et al. 1993; Neefjes
et al. 1993; Rock et al. 1994; York and Rock 1996; Pamer and Cresswell 1998; Rock
and G oldberg 1999). This is the classical pathway of class I epitope presentation.
H ow ever, studies have shown that there are alternative pathw ays for M H C I
presentation. It has been shown that secreted antigen can be presented on the surface
o f APCs via crossprim ing (Bevan 1976; M oore et al. 1988; Carbone and Bevan
1990; Rock et al. 1990; Pfeifer et al. 1993; Huang et al. 1994; K ovacsovics-
Bankow ski and Rock 1995; W atts 1997; Carbone et al. 1998; Grom m e and Neefjes
2002). In addition, a number of peptides have been shown to be presented on class I
m olecules in T A P-deficient m ice (H enderson et al. 1992; E lliott et al. 1995;
Ham m ond et al. 1995; Snyder et al. 1997). In these studies, antigens w ere directly
transported to the ER using a signal peptide, and epitopes are loaded on the class I
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M G A M A P R T L L L L L A A A L A P T Q T R A
M V S A T R S L L C A A L P V L A T S R Q A T G
Figure 2-1: The am ino acid sequences o f the murine
chem okine leader.
14
Murine H-2Ld
Rat KC chemokine
L d leader and the rat KC
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SOYZLL
SAOYZLL
RAOYZLL
SOLL
SAOLL
RAOLL
hydrophobic leader
from H2-L4
ova (323-339) transmembrane
YZLL
H i epitope domain to endosome
hydrophobic leader gpl20 (315-329) ova (323-339) transmembrane YZLL
from H2-L* Tc epitope Tit epitope domain to endosome
hydrophobic leader gpl20 (315-329) ova (323-339) transmembrane YZLL
from KC chemokine Tc epitope H i epitope domain to endosome
hydrophobic leader
from H2-L*
O
ova (323-339)
H i epitope
transmembrane
domain
K >
LL
endosome
hydrophobic leader
from H2-L1
gpl20 (315-329)
Tc epitope
ova (323-339)
H i epitope
transmembrane
domain
o
LL
endosome
hydrophobic leader gpl20 (315-329) ova (323-339) transmembrane LL
from KC chemokine Tc epitope H i epitope domain endosome
Figure 2-2: The schem atic diagrams of the vectors used in this chapter. Their exact com position is outlined in
Materials and M ethods section.
16
molecule in a TAP-independent fashion. Leader sequences have been used in DNA
vaccine constructs with m ixed results. However, in these studies the mode o f
im m unization, the type of epitopes used, and the construct design were different,
thus the im portance of the signal peptide cannot be fully assessed. Based on these
studies, it is inferred that the leader sequence may be needed but is not crucial to
influence the magnitude of a CTL response (Ciernik et al. 1996; Haddad et al. 1997;
Haddad et al. 1998; Iwasaki et al. 1999; Ji et al. 1999; Drew et al. 2000).
The leader sequences of a wide variety of eukaryotic species have been
identified. W hile a consensus sequence is absent, there are several common features
am ong leaders sequences. Their length ranges from 13 to 36 amino acid residues,
there is at least one positively charged amino acid residue, there is a hydrophobic
stretch of about 10 to 15 amino acids long in the center o f the signal peptide and a
signal peptidase cleavage site (von Heijne 1990).
In this chapter of my thesis, the goal is to determ ine whether the identity of
the signal sequence can alter the CTL response induced by a DNA epitope vaccine.
A leader, derived from a secreted molecule, m ight be more effective than one
derived from a transm embrane molecule at generating protein in the constitutive
secretory pathway (Figure 2-1). As a consequence, the antigen concentration would
increase, and hence boost the observed immune response. I have constructed a series
of plasm ids that encode a CTL epitope, HIV gpl20 IIIB (318-327) (A) targeted by a
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17
hydrophobic leader derived from the murine H-2Ld molecule (S) and a Th epitope,
ovalbum in OVA (323-339) (O) targeted by the dileucine lysosomal targeting m otif
(YZLL or LL) (Figure 2-1). I replaced the murine H-2Ld leader sequence o f these
plasm ids w ith one derived from the rat KC chem okine(R) (Huang et al. 1992)
(Figure 2-1) and measured the immune response elicited by vaccine vectors encoding
the two different signal peptides. The CTL response was evaluated by a CTL assay,
the proliferative responses by a proliferation assay, and the cytokine profiles of the
im m unized mice were examined by an ELISA and intracellular cytokine staining
(ICCS). I also performed a series of in vitro assays, which confirmed the suggested
role of the leader sequence. More specifically, I measured the amount of protein
secreted by the two leader sequences. Also, I transfected P815 cells with the vectors
used in our immunizations and used as targets in a CTL assay.
Results:
CTL responses elicited by DNA immunized mice
The vectors shown in Figure 2-2 were constructed and used in mouse
im m unizations to determine the role of the leader sequence on the immunogenicity
o f the DNA epitope vaccines. In preliminary experiments, I injected small groups of
BALB/c-H-2d m 2 (dm2) mice (n=4) with the plasm ids shown in Figure 2-2 and
measured immune responses. Splenocytes were in vitro stimulated with gp 120(318-
327) for 5 days and used as effectors in 5lCr release assays to lyse gpl20-pulsed
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18
■ R AO YZLL
• SAOYZLL
—h— SOYZLL
- B - NEGATIVE CONTROL
-G - NEGATIVE CONTROL
— A- NEGATIVE CONTROL
200:1 100:1 50:1 25:1 12.5:1 6.25:1
E:T RATIO
RAOLL
SAOLL
-hr- SOLL
-0— NEGATIVE CONTROL
-©- NEGATIVE CONTROL
NEGATIVE CONTROL
200:1 100:1 50:1 25:1 12.5:1
E:T RATIO
6.0:1
Figure 2-3: Representative 5lCr release assays. In the top plot, mice were injected
with SOYZLL, SAOYZLL, and RAO YZLL vectors while in the bottom mice were
im m unized with SOLL, SAOLL, and RAOLL vectors. P815 target cells were
incubated w ith either gp 120(318-327) peptide or with the Dd -binding MPI peptide
(negative control).
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19
100
90-
SOYZLL SAOYZLL RAO YZLL
100- |
90-
£ 80-
70-
u 6 0 -
50-
8 « -
a 3° -
^ 2 0 -
10-
SAOLL RAOLL SOLL
Figure 2-4: Summary plots of two experiments, using a total o f 4 mice per group,
showing specific lysis at 200:1 effector to target ratio. In the top, dm2 mice were
injected with vectors SOYZLL, SAOYZLL, and RAOYZLL while in the bottom plot
mice were immunized with SOLL, SAOLL, and RAOLL.
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20
P815 target cells. M ice injected with the plasm ids encoding the KC chem okine
leader (RAOYZLL or RAOLL) showed significantly stronger CTL responses when
com pared to the mice immunized w ith the vectors expressing the m urine H -2Ld
leader (SAOYZLL or SAOLL)(Figures 2-3 and 2-4). As expected, mice immunized
with SOYZLL or SOLL vectors showed no CTL responses since they do not express
the g p l2 0 epitope indicating that the response is epitope-specific.
In repeat experiments, I im m unized 2 groups of mice with the SAOLL or
RA OLL vectors using two different im m unization schedules (Table 2-1). The
numbers of mice used in this study are shown in Table 2-1. The first immunization
schedule com prised of an injection followed by a single boost, while the second
included an additional boost. I w anted to exam ine w hether an additional boost
enhances the immune responses. Regardless of the im munization schedule, in both
experiments the mice immunized with RAOLL showed stronger gpl20 specific CTL
responses confirm ing the results we obtained from earlier assays (Figure 2-5). The
introduction of a second boost in the immunization regim ent did have an overall
im provem ent in the reactivity of effector cells (approxim ately 10% increase). In
addition, the CTL responses of the immunized mice within each immunization group
is more consistent.
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21
GROUP Numbers of Immunized
Mice
Immunization Schedule
A 3 Naive, 4 SAOLL, 4 RAOLL Inject Boost -> Sacrifice
B 3 Naive, 6 SAOLL, 6 RAOLL Inject -> Boost Boost Sacrifice
Table 2-1: The numbers of mice used, the vaccine received and the im m unization
regiment.
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22
GROUP A
6 0 -
ys
“ 50-
u
U 4 0 -
b
U 3 0 -
&.
M 2 0 -
10-
SAOLL RAOLL NAIVE
GROUP B
J l
fa
10-
NAIVE SAOLL RAOLL
Figure 2-5: A summary plot o f the two experiments showing maximum specific lysis
by effector cells. Balb/cJ mice were injected with vectors NAIVE (control), SAOLL
(old leader), and RAOLL (new leader). The p-values are: for control p<0.01, old
leader p<0.01, new leader p<0.01. Comparing all three groups, p<0.01.
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23
Proliferative responses o f DNA immunized mice
In addition to the 5lCr release assay, I also w anted to exam ine the
proliferation responses of the immunized mice. I wished to determine w hether the
nature o f the leader sequence influenced the TH cell responses. In prelim inary
experiments, splenocytes from dm2 immunized mice (n=4) were in vitro stimulated
with peptide (ovalbum in(323-339) and M BP(89-101)) for 3 days and used in a
proliferation assay (Figure 2.6). CD4+ T cells from mice injected with the plasmids
encoding the KC chem okine leader (RAOYZLL or RA OLL) show a higher
proliferation response to ovalbum in com pared to the group im m unized with the
plasm ids encoding the m urine H -2Ld leader (SA O Y ZLL or SA O LL). The
experiments were repeated using larger groups of mice. Splenocytes from Group A
and Group B (Table 2-1) were used in a proliferation assay. In Group A, due to
technical problems, I was unable to detect significant differences between the mice.
However, the magnitude of improvement seen in the CTL assays was not replicated
in the proliferation assay. Thus, the presence of the leader sequence influences more
the class I rather than the class II presentation pathway.
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24
SOYZLL SAOYZLL RAOYZLL
15000
13000 OVA(323-339)
11000-
9000-
MBP(89-101)
7000-
^ 5000 -
1000-
■ 1000-
-3000
■5000
SOLL SAOLL RAOLL
11000
9000-
j S 5000 -
3000-
■ 1000-
| OVA(323-339)
0 MBP(89-101)
■3000
Figure 2-6: A representative proliferation assay. Mice injected with the vector
expressing the KC chemokine leader (RAOYZLL, top, and RAOLL bottom) showed
higher proliferation after in vitro stimulation with OVA(323-339). M BP(89-101) is a
class II epitope used as a control. SOYZLL and SOLL are the control vectors.
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NAIVE SAOLL RAOLL
MBP ® 9- 101)
-300
Figure 2-7: Mice injected with the vector expressing the new leader (RAOLL) showed higher proliferation after in
vitro stimulation with OVA(323-339). M BP(89-101) is a class II epitope used as a control.
2 6
Cytokine profiles o f mice immunized with DNA epitope vaccines.
DNA vaccines are known to induce T hl type immune responses (Gurunathan
et al. 2000). Thus, I would predict that mice immunized with the plasm id vaccines
w ould show elevated levels of IFN-y. In order to exam ine the cytokine release
patterns, splenocytes from dm2 m ice (n=4) were in vitro stim ulated with peptide
(ovalbum in(323-339) and M BP(89-101)) for 3 days to detect IFN-y. T H cells from
mice injected with the vectors encoding the KC chemokine leader secreted higher
levels of cytokine release. In repeat experiments, mice im munized with the RAOLL
vector showed higher IFN-y release than the mice immunized with SAOLL (Figure
2-8).
In the repeat experiments, I used another assay to assess the efficacy of the
DNA plasm ids. In order to quantitate the numbers of epitope-specific T H or CTL
cells, splenocytes from the above experiments were used in a intracellular cytokine
staining assay (ICCS). This assay enabled me to examine OVA-specfic TH cells and
gp l2 0 specific CTLs secreting IFN-y (Figure 2-9). Mice immunized with the vectors
encoding the new leader (RAOYZLL or RAOLL) possessed higher numbers of
epitope specific T ceils. These data correlate with the im proved CTL and
proliferative responses.
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NAIVE SAOLL RAOLL
MBP (89-101)
j q J __________________________ \ __________________________ |__________________________ |
Figure 2-8: A representative ELISA assay from tw o experim ents m easuring the am ount o f IFN-y release. M ice were
immunized with SAOLL or RAOLL. Naive m ice are the control group. These data w ere obtained from G roup B of
immunizations. Splenocytes were pooled from each group of mice.
N >
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NAIVE SAOLL RAOLL
I j
8.
1
$ 2 A
'S
£
s u
Tjj/IFN-g
CTL/IFN-g
Figure 2-9: ICCS m easuring the am ount o f TH or CTL IFN-y secreting cells. These data w ere obtained from
G roup A of im munizations. Splenocytes were pooled from each group of mice. M ice w ere immunized with
SAOLL or RAOLL. Naive mice were the control group.
to
o o
29
Secretion levels o f the fi-galactosida.se by the two leaders
Based on the results obtained from the previous experim ents, the use o f a
m ore effective leader can im prove the immune response. This im provem ent is
possible by two different pathways. An effective leader targets antigen more
efficiently to the ER leading to increased antigen secretion w hich can be
crosspresented by APCs. Furtherm ore, increased targeting w ill enhance class I
presentation of antigen. To measure the amount of protein secreted by the two leader
sequences, I generated two expression vectors. In one, pSLacZ encodes the (3-
galactosidase secreted by the murine H-2Ld leader and the other pRLacZ expresses
the (3-galactosidase secreted by the KC chemokine leader. As a positive control, I
generated the pLacZ plasmid that encodes the [3-galactosidase in the cytosol. I
transfected two cell lines with the above vectors. One was A20, a B cell lymphoma,
and the other was P815, a mouse m astocytom a cell line. The identity o f the
transfectants is shown in Table 2-2. After I established stable cell lines, I collected
supernatants and used them in a chemiluminescent detection assay. Thus, the amount
o f enzym e secreted will be m easured as a function of its activity. Based on our
results (Figure 2-10), there is no difference between the two groups. To ensure that
the the protein was not in the cytosol, I lysed the transfectants and tested the lysates
along with the supernatants. They did not show any (3-galactosidase activity. Thus,
the protein was targeted to the ER. The pLacZ transfectants were also lysed and were
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3 0
Name of transfectants Description
DV1A A20 transfected with pRLacZ
DV1B A20 transfected with pSLacZ
DV1C P815 transfected with pRLacZ
DV1D P815 transfected with pSLacZ
DV1E A20 transfected with pLacZ
DV1F P815 transfected with pLacZ
Table 2-2: List of the transfectants used in this study. pR LacZ is the vector
expressing the LacZ gene under the control of the rat KC chem okine leader, pSLacZ
expresses LacZ under the murine H-2Ld leader, and pLacZ expresses LacZ in the
absence of any leader (positive control).
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10
0-1
8
8 ^
I 6_
I 5 "
4-
3-
2 -
1
0
-1
DV1A DVTB DV1C DV1D
n rri in n nTHTrn nTrrTprnw1
&
>
O
3500
-1500
-1000
DV1A:A20-KC
DV1B :A20-LD
DV1G:P815-KC
DV1D-.P815-LD
DVIE:A20-LacZ
DVlF:P815-LacZ
Figure 2-10: The rat chemokine KC and the mouse H-2Ld leaders were inserted into the pLacz expression vector upstream
of the LacZ gene. The cloned vectors were transfected into P815 and A 20 cells. The supernatants (solid bars) were
positive for lacZ secretion while the pellets (dashed bars) were not. There were no significant differences between the two
leaders. Untransfected A20 and P815 cell lysates were the negative controls. Both positive controls showed high levels of
ennzym atic activity (D V lE=40pg, D V lF=3000pg).
32
tested positive for [3-galactosidase activity (Figure 2-10). This was expected since the
[3-galactosidase was not targeted to any vesicular com partm ents. Therefore, the
transfections were successful. The lack of enzym atic activity in the transfectants
encoding secreted (3-galactosidase could be due to the fact that it is a cytosolic
protein that was either incorrectly folded or degraded in the ER.
Secretion o f gp 120 mediated by the murine Lf and rat KC chemokine leaders
Based on the results above, I decided to use the secreted protein g p l2 0 to
evaluate the effectiveness of the two leader sequences. g p l2 0 , an HIV surface
glycoprotein, must be secreted and postranslationally modified before it reaches the
cell surface. In this experiment, I used the syngpl20 gene. This gene encodes the
gpl20M N strain. Its codon usage has been modified from a viral to a mammalian
one and it has been shown that this change results in an increased production of
g p l2 0 by mammalian cell lines (Haas et al. 1996). I constructed two expression
vectors that encoded the murine Ld (SA6H) or the rat KC leader (RA6H), g p l2 0 and
the 6xHis tag at the carboxy terminus of the gp l2 0 (Figure 2-11). These vectors were
transfected using Ca3 (P 0 4)2 into P815 m ouse m astocytom a, BclO M E m ouse
fibroblast, 293T human em bryonic kidney, and S I 94 m yelom a cell lines.
Supernatants were collected in 36 hours and 4 days after transfection and used in an
indirect ELISA (Figure 2-12). The levels of g p l2 0 secreted by the 293T cells
transfected by the RA6H were slightly higher than the SA6H transfectants on both
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33
36-hour and 4-day supernatants. BclOM E transfected with RA6H showed high levels
o f g p l2 0 secretion in the 36-hour supernatants and higher levels in the 4-day
supernatants while transfection with the SA6H yielded no or lower levels of protein.
P815 and S I94 transfectants did not show any secretion. Thus, the presence of the rat
KC leader does indeed increase the secretion of the gpl20 protein.
Following the transient transfection data, I generated stable transfectants to
obtain high-expressing clones. Transfectants w ere cloned twice and used in an
indirect ELISA to detect gp l2 0 secretion. The cells were grown in serum-free media
to minimize non-specific background. Nine clones were tested from each transfectant
group. As with the transient transfections, I did not observe higher secretion of
protein in the S194 clones. On the other hand, 293T and BclOM E clones derived
from cells transfected with the RA6H plasmid secreted higher levels o f gpl20. In
addition, the amounts of protein secreted by the 293T cells was low er than the
transient transfections. Their morphology as well as their growth patterns seemed to
be adversely affected by culturing them in serum-free media. On the other hand,
BclO M E clones secreted com parable levels o f protein when com pared to the
transient transfectants. From these experiments, I conclude that the rat KC leader
was secreting higher levels o f protein. The experim ents dem onstrate that
immunogenicity of DNA vaccines was enhanced by increasing the amount of antigen
secreted by cells internalizing the plasmid.
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X
C D
<
C /3
O
C M
C l
OJ
c
&
C D
<
os
o
C M
ro
c
&
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Figure 2-11: A schematic diagram o f th e vectors used t o measure gpl20 secretion b y th e tw o leaders.
35
293T
g ] BclOME
SA6H RA6H
293T
BclOME
■ ■ P 30
m m is8 a sss_ /_ 7 7 7 i
SA6H RAdH
Figure 2-12: An ELISA assay measuring the am ount of gpl20 secreted by the cells
transfected w ith the SA6H and RA6H expression vectors. In the top graph,
supernatants were collected 36 hours post-transfection, and in the bottom 4 days
post-transfection.
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36
2SA
45-
40-
2RA ■
35-
30-
25-
60'
10-
BSA
45-
40-
BRA ■
35-
15-
10-
Figure 2-13: An ELISA m easuring secreted levels of gpl20. The above data are a
representative of two experim ents. 2SA clones were derived from 293T cells
transfected with SA6H. 2RA clones were derived from 293T cells transfected with
RA6H. BSA clones were derived fromBclOM E cells transfected with SA6H. BRA
clones were derived from BclOME cells transfected with RA6H.
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37
Tranfectant Clones Description
DV2A D V 2A 1.1-1.6 P815 cells transfected with
RAOLL(New Leader)
DV2B D V 2B1.1-1.6 P815 cells transfected with
SA 0LL(01d Leader)
Table 2-3: A list of the transfectants used to determine the effect of leader sequence
on the epitope presentation.
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38
60-
^ 50 -
4 0 -
30-
10-
P 815/HIV P815/MPI DV2A DV2B
Figure 2-14: A representative CTL assay from two experiments. A sum m ary plot
showing specific lysis at 50:1 E:T Ratio. P815 cells were transfected with RAOLL
(DV2A) or SAOLL (DV2B) and used as targets in a CTL assay. The values are
statistically significant (pcO.OOl).
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39
Presentation o f gpl20 epitope by P815 cells transfected with RAOLL
A nother m echanism a leader sequence can influence the im munogenicity of
D NA vaccines is by efficiently targeting antigen to the ER to enhance class I
presentation. To address this issue, I transfected P815 cells w ith the RA OLL
(DV2A) and SAOLL (DV2B) vectors used in mouse vaccinations to m easure the
am ount o f gpl20 (318-327) epitope-specific presentation under the two leaders. The
transfectants were cloned twice(Table 2-3) and used as targets in a chrom ium release
assay. The effector cells were splenocytes from mice immunized with the RAOLL
vector. I expected that targets transfected with RAOLL (DV2A) would have higher
epitope presentation, thus, they would be killed more efficiently by the CTLs. The
targets were treated w ith IFN-y 48 hours before the assay to enhance class I
presentation. DV2A clones were indeed killed more effciently by CTLs than the
DV2B clones (Figure 2-14). Based on this data, the rat KC leader did target the
g p l2 0 epitope more efficiently to the ER thereby improving class I presentation.
D iscussion:
The replacement of a leader sequence from the rat KC chem okine enhanced
the immunogenicity of the DNA epitope vaccines. The hypothesis was that a leader
from a secreted molecule w ill more efficiently target antigen to the ER, thus,
enhance the immunogenicity of DNA epitope vaccines. This increased efficacy can
be achieved by two ways. Effective targeting will result in increased antigen
secretion. The secreted antigen will the be internalized by APC and presented via the
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40
cross priming (Bevan 1976; M oore et al. 1988; Carbone and Bevan 1990; Rock et al.
1990; Pfeifer et al. 1993; Huang et al. 1994; K ovacsovics-Bankow ski and Rock
1995; W atts 1997; Carbone et al. 1998). In addition, the increased targeting to the
ER will enhance antigen loading to the class I molecules resulting in higher numbers
of epitope specific T cells due to increased presentation (H enderson et al. 1992;
Elliott et al. 1995; Hammond et al. 1995; Snyder et al. 1997).
In the mouse experiments, immunizations of mice with vectors encoding the
KC chem okine leader (RAOYZLL or RAOLL) resulted in greater CTL responses
than the murine H-2Ld immunizations (SAOYZLL or SAOLL). W hen examining the
proliferative responses, splenocytes from mice immunized with the RAOLL showed
slightly higher proliferative responses. I would expect that more OVA eptiope will
be secreted thus enhancing TH cell responses. However, since the OVA epitope is
targeted to a different presentation pathway, its levels of presentation may be
influenced more by the lysosomal/endosomal motifs rather than the leader sequence.
Sim ilar patterns were seen when I examined at the levels of IF N -y secretion by an
ELISA. This assay was also limited by the fact that we used mixed lym phocyte
populations rather than exam ining the epitope-specific T cells. To address this issue,
I used ICCS to quantitate the numbers of OVA-specific TH cells secreting IFN -y and
gp 120-specific CTLs secreting IFN-y. Mice immunized with the vector encoding the
new leader possessed higher numbers of epitope-specific TH and CTL cells secreting
cytokine.
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41
The results from the mouse immune responses following plasm id vaccination
suggest that the identity of the leader can improve the immune response by increased
targeting o f antigen to the ER. To address the mechanistic aspects o f this, I created
the P-galactosidase expression vectors to determ ine amounts of P-galactosidase
secreted by the two leaders. The enzymatic activity of the secreted (3-galactosidase
was very low m aking any conclusions im possible. P-galactosidase was being
secreted since there was no activity detected from the cell lysates. Thus, the low
levels of enzym e activity could be attributed to several reasons. First, the protein
may be digested due to targeting to the ER. P-galactosidase is a cytosolic protein,
and it may lack the appropriate signals or sequences that will protect it from
digestion. Second, it may not be correctly folded in the ER thus lacking any activity
when secreted. Finaly protein expression is normal, since cells transfected with
pLacZ, a vector encoding the P-galactosidase protein without a leader, were lysed
and showed high p-galactosidase activity.
I then decided to use a secreted protein to measure the levels of secretion
under the two leaders. The most direct way would have been to exam ine the
secretion levels o f the g p l2 0 epitope expressed in our immunization vectors. The
main lim itation is the lack of affinity purified monoclonal antibodies against the
g p l2 0 epitope that can be used in a sandwich ELISA which can detect picogram
levels of protein. Unfortunately, the above expression systems do not express high
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42
levels of epitope that can be coated directly on plate and used in an indirect ELISA.
In addition, epitopes can be degraded easier in culture by proteases released by dying
cells or present in serum media. This can be rem edied by the use o f serum -free
media. Because of the above limitations, I generated two vectors encoding the g p l2 0
fused w ith a 6xHis tag under the two leaders and transfected them into a series of
cell lines. Supernatants collected at times showed that cells transfected with the
vector encoding the chemokine leader secreted higher levels o f g p l2 0 com pared to
the cells transfectant with the murine Ld. This was further dem onstrated when I
screened a series of clones generated from the transfected cell lines. Thus, the rat KC
leader does indeed secrete more protein.
In addition, I transfected P815 cells with the RAOLL or SAOLL vectors and
extablished stable transfectants. This was done to address the issue o f increased
presentation. The clones were used as targets in a CTL assay. The cell transfected
with the vector encoding the chemokine leader were killed more efficiently by the
CTLs suggesting that they presented higher levels of g p l2 0 epitope. Thus, the
presence o f the chem okine leader did enhance targeting to the ER resulting in
im proved class I presentation.
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43
Conclusion:
The presence of a leader sequence is important in the rational design of DNA
epitope vaccines. Based on the results presented in this chapter, the use of a more
efficient leader from the rat KC chemokine, a secreted molecule, resulted in stronger
im mune responses due to increased targeting of antigen to the ER. These results are
in agreem ent with the lim ited num ber of studies that exam ine the use o f leader
sequences. The use of a leader seems to be crucial for the generation o f strong CTL
as well as antibody responses. However, there have been evidence suggesting that
direct targeting to ER is favored by certain epitopes and not others. These data
though have not been supported by an extensive screening of different leaders. The
amino acid composition of the signal peptidase cleavage site can be a crucial factor.
Therefore, the use of efficient leaders can improve the im m unogenicity of DNA
epitope vaccines.
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44
CHAPTER 3: HELPER EPITOPE INFLUENCES THE CTL
RESPONSE ELICITED BY DNA EPITOPE VACCINES.
Introduction:
The role of TH cells in the generation of effective CTL responses has been an
issue of intense debate. Studies have demonstrated that CTLs can be activated and
becom e effector cells in the absence of TH help (Rahemtulla et al. 1991; W ang et al.
2001; M intern et al. 2002). H ow ever, these responses are short-lived and non-
protective. It has been shown that the presence o f T H help is crucial in order to
generate memory cells that will confer long-lasting protective immunity (Keene and
Form an 1982; von Herrath et al. 1996; Bennett et al. 1997; Bennett et al. 1998;
Ridge et al. 1998; Schoenberger et al. 1998; Marzo et al. 2000; Shedlock and Shen
2003; Sun and Bevan 2003). The mechanisms by which TH cells influence CTL
responses have not yet been found. However, it is hypothesized that T H cells activate
dendritic cells (DCs) through CD40-CD40L interactions. Then, activated DCs can
present class I peptide to the CTLs, activate them, and convert them into effector
cells (Bennett et al. 1998; Ridge et al. 1998; Schoenberger et al. 1998).
Therefore based on the evidence above, the immune response elicited by
DNA epitope vaccines may be affected by manipulation o f T H epitopes. In this
chapter, I replaced the sequence o f the OVA T H epitope in our RAOLL construct
with OVA variants that have different binding affinities to the class II Ad molecule.
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45
Studies by Sette et al. have defined residues on the OVA epitope that associate with
class II Ad molecule (Sette et al. 1987). They synthesized a number of variant OVA
(323-339) peptides and determined their affinity for the class II Ad molecule. In these
studies, I used two of those variant epitopes (Table 3-1) (Lamont et al. 1990; Sette et
al. 1990). In the first one, OVA-YT, residues 323-326 and 333-336 have been
replaced by YTYT sequences and its affinity to the class II Ad molecule is increased
by a factor of 10 (Lamont et al. 1990; Sette et al. 1990). These residues are located in
either side of the core region of the epitope and are called peptide-flanking residues
(PFR). PFRs stabilize epitope binding to the M HC II and have been shown to
interact with the T cell receptor (TCR) (Carson et al. 1997; M oudgil et al. 1998). The
second one, OVA-M , has a m utation at residue 327 where the valine has been
replaced by an arginine decreasing the affinity by a factor of 10. The valine at
position 327 has been shown to be the critical anchor residue for binding to the Ad
(Sette et al. 1987; Lamont et al. 1990; Sette et al. 1990).
I hypothesize that changes in the affinity of OVA to the class II Ad molecule
will affect the im mune response elicited by our DNA epitope vaccines. M ore
specifically, higher affinity class II epitopes will result in increased and longer class
II presentation while a lower affinity class II epitope will have the opposite effect
(Nelson et al. 1997). In these experiments I used the RAOLL vector which expresses
the wild type OVA, and I replaced the OVA epitope of this vector with OVA-YT
and OVA-M to generate RAOYTLL and RAOMLL respectively (Figure 3-1).
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46
PEPTIDES AMINO ACID SEQUENCE
OVA (323-339)
HIGH AFFINITY
M UTANT
OVA-YT
LOW AFFINITY
M UTANT
OVA-M
flanking
residues
I S Q A VHAAHA
flanking
residues
EI NE AGR
no Ad contact
core epitope
Y T Y T V H A A H A Y T Y T A G R
I S Q A R H A A H A E I N E A G R
Table 3-1: The amino acid sequences of the wild type OVA epitope and its variants.
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RAOLL
hydiaphobic leadei gpl.20 (315-339) OVA (323-339) tr ansmembi ane LL
from KC chemokine CTL epitope TB epitope domain endosome
RAOMLL
hydiaphobic leadei gpl20 (315-329) OVA-M (323-339) tr ansmembi ane LL
bom KC chemokine CTL epitope Ta epitope domain endosome
RAOYTLL
hydiaphobic leadei gpl20 (315-329) OVA-YT (323-339) tr ansmembi ane LL
bom KC chemokine CTL epitope Tb epitope domain endosome
Figure 3-1: Schem atic diagram s of the vectors used in this portion of the study. The exact
com position is outlined in the M aterials and M ethods section. LL is the dileucine endosome-
targeting motif.
- 4
48
Results:
A vector expressing the high affinity mutant elicits stronger gp-120 specific CTL
responses
To exam ine the influence o f TH cells on the m agnitude o f g p l2 0 CTL
responses elicted by the DNA epitope vaccines, I constructed the vectors shown in
Figure 3-1 and injected them into BALB/cJ mice as described in the M aterials and
M ethods section. Following immunization and two boosts, the mice were sacrificed
and spleens were extracted. Splenocytes were stimulated in vitro with g p 120(318-
327) for 5 days and used as effectors to lyse gpl20 peptide pulsed P815 target cells
in 5 1 Cr release assays. M ice injected with the RAOYTLL vector, which encodes the
high affinity OVA variant OVA-YT, elicited a stronger gp l2 0 specific CTL response
than the ones injected with the RAOLL plasmid, which expresses the wild type OVA
(Figures 3-2 and 3-3). The mice injected with the RAOMLL, the vector encoding the
low affinity OVA mutant, showed no CTL activity (levels sim ilar to naive mice)
(Figures 3-2 and 3-3). Therefore, increasing the affinity o f the helper epitope
improved the CTL responses generated by the DNA epitope vaccine. Furthermore,
the ablation o f help by using a low affinity mutant resulted to dim inished CTL
responses. Overall, this underlines the importance of TH in the generation of effective
CTL responses.
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c/5 30-
-B-
- e -
RAOYTLL
RAOLL
RAOMLL
NAIVE
NEGATIVE CONTROL
NEGATIVE CONTROL
NEGATIVE CONTROL
NEGATIVE CONTROL
200:1 100:1 12.5:1 6.25:1
Figure 3-2: A representative 5lC r release assay. M ice w ere injected with RA O LL, RA O Y TLL, and
RAOM LL vectors. P815 targets cells w ere incubated with g p l2 0 (318-327) peptide or with the Dd binding
MPI peptide (negative control). The naive mouse is the control.
VO
50
100
C D
u
Ed A n
0* 40-
gq
20 -
NAIVE RAOMLL RAOLL RAOYTLL
Figure 3-3: A summary plot of two experiments, showing m aximum specific lysis
by effector cells. BALB/cJ mice were injected with vectors RAOM LL, RAOLL, and
RAOYTLL. The values are statistically significant (p<0.001).
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51
Th cell proliferation data indicate the generation o f an OVA-YT specific T-helper cell
population.
Due to its high affinity, the OVA-YT epitope should have prolonged class II
presentation as a result of a stable class II-epitope interaction leading to the
stim ulation o f higher numbers o f TH cells (Nelson et al. 1997). Therefore, the
proliferation responses of the RAOYTLL group of mice are predicted to be higher.
Furtherm ore, since OVA-YT has been extensively modified, it is probable that an
O VA -Y T specific TH population may be generated. To exam ine these issues, in
addition to the 5 1 Cr release assays, I performed [3 H] thymidine incorporation assays
to m easure TH cell proliferation. Spleen cells were stim ulated in vitro with OVA
(323-339) and M BP(89-101) peptides. Mice injected with RA O Y TLL proliferated
only to OVA-YT(323-339), while the mice injected with RAOLL responded only to
wild type OVA(323-339) (Figure 3-4). These results indicate the generation of high
affinity mutant, OVAYT, specific TH cells. Mice injected w ith the low affinity
variant did not show any proliferation (Figure 3-4). Unexpectedly, the RAOYTLL
im m unized mice did not proliferate higher than the RAOLL im m unized group. The
am ount of proliferation between the two groups was com parable after repeated
experiments. However, since I was stimulating mixed lymphocyte populations, it is
possible that the differences between the two groups are m asked. A more likely
explanation is the fact that the OVA-YT has been m odified so extensively that
stimulates different T cell populations (Carson et al. 1999).
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NAIVE RAOMLL RAOLL
RAOYTLL
65000
55000 -
45000-
35000 -
& 25000
15000-
5000-
-5000
-15000
1
OVA033-339)
OVA-YT (323-339)
OVAM<323 339)
gpl20 <315-329)
MBP(B9 1G1)
Figure 3-4: The average values of the proliferation responses from two experim ents. The num ber of m ice used are
n=12 for the RA OLL, RA O M LL, and R A O Y TLL groups, and n=6 for the N A IV E. All the differences are
statistically significant(p<0.001).
to
53
Mice immunized with the vector encoding the high affinity TH epitope variant show
epitope-specific cytokine secretion
In addition, IF N -y ELISAs w ere perform ed to m easure the am ount of
cytokine released. Based on the CTL data, I would expect that the secretion of
cytokine would be enhanced in ths RAOYTLL immunized mice. Spleen cells were
stim ulated in vitro with peptide (OVA(323-339), OVA-YT(323-339), OVA-M (323-
339), g p l2 0 (3 15-329), M BP(89-101)) for 3 days. As shown in Figure 3-5, mice
injected with the RAOYTLL vector secreted high levels cytokine after stimulation
with OVA-YT peptide. Mice im munized with the RAOLL vector secreted cytokine
after wild type OVA stimulation while in the low affinity mutant there was no IFN-y
release. Furtherm ore, after in vitro stim ulation with the g p l2 0 (3 15-329) both the
RAOYTLL and RAOLL groups secreted comparable levels of IFN-y. The secretion
is most likely by gp 120-specific CTLs even though the 15-mer has been described as
a class II epitope (Shirai et al. 1992). W hen using gp 120(315-329) in proliferation
assays, I failed to see any proliferation. The g p l2 0 (3 15-329) is most likely processed
by A PCs to generate the gp 120(318-327), 10-mer HIV, and stim ulate the CTLs
(Ochoa-Garay et al. 1997).
In order to further analyze the immune response, I performed intracellular
cytokine staining (ICCS) assays. In these assays, I measured the num ber epitope-
specifc T h cells and CTLs secreting IFN-y. Mice immunized with the RAOLL vector
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NAIVE RAOMLL RAOLL RAOYTLL
250
1
150-
50-
-50-
OVA-YT(323-
OVA-M(323
gpl30(3'15. 329)
H MBP $9-101)
Figure 3-5: An ELISA assay detecting IFN-y levels. M ice injected with the RAOLL vector showed cytokine release
after in vitro stim ulation with O VA (323-339), w hile RA OY TLL im m unized m ice responded to the O VA -Y T
peptide. MBP(89-101) is a class II epitope used as a control. Splenocytes were pooled. The num ber of mice used are
n=6 for the RAOLL, RAOM LL, and RAOYTLL groups, and n=3 for the NAIVE.
L /t
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NAIVE RAOMLL RAOLL RAOYTLL
a
i i
s ®
1
8 -
1 7 -
s
h "
6-
!
5 -
1
V s
4 -
O
3-
1
2-
I
1-
0-
OVA(323-339)
OVA-YT(323-339)
OVA- M (323-339)
Figure 3-6: ICCS m easuring the num ber o f TH IFN-y secreting cells. Splenocytes were pooled from each
group o f mice. The num ber of mice used are n=6 for the RAOLL, RAOM LL, and RA OY TLL groups, and
n=3 for the NAIVE. The vectors are indicated at the top of the graph.
L f\
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NAIVE RAOMLL RAOLL RAOYTLL
CTL/IFN-g
&
$
Figure 3-7: ICCS measuring the num ber o f CTL IFN-y secreting cells after in vitro stim ulation with
gp 120(318-327). Splenocytes were pooled from each group o f mice.The num ber of mice used are n=6
for the RAOLL, RA OM LL, and RA O Y TLL groups, and n=3 for the NAIV E. The vectors are
indicated at the top o f the graph.
O
57
showed higher numbers of cytokine secreting TH cells only when stim ulated with the
OVA(323-339) peptide while RAOYTLL immunized mice responded only to OVA-
YT stim ulation (Figure 3-6). Also, m ice im m unized with the RA O Y TLL vector
showed higher numbers of cytokine-secreting epitope-specific CTLs com pared to the
RAOLL immunized group (Figure 3-7). The last set of results are in agreement with
the CTL assay data.
D 011.10 T cell hybridoma stimulation by A20 cells pulsed with OVA epitope
variants yields distinct responses
Based on the results from the m ouse experim ents, im m unization with a
vector encoding the OVA-YT variant resulted in the generation o f a distinct T H
population. To confirm these observations in vitro, I used an established T cell
hybridom a specific for the OVA epitope used in this thesis project. DO11.10
hybridom as are OVA(323-339) specific and upon peptide stimulation release IL-2
(M arrack et al. 1983).
Irradiated A20 cells were pulsed with various amounts of the OVA peptides
(OVA, OVA-M , OVA-YT) to stim ulate the DO11.10 T cell hybridoma. Cytokine
secretion was measured by a sandwich ELISA. Based on the IL-2 ELISA, there is a
10-fold difference between the O V A and O VA-M peptides while there is no
stim ulation by OVA-YT (Figure 3-8). The differences between the OVA and the
OVA-M were expected since the OVA-M has a 10-fold lower affinity to the class II
Ad when comapred to the wild type epitope. Thus, presentation will be less efficient
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1 9 0 -
OVA-YT
OVA-M < 1 4 0 -
9 0 -
4 0 -
-10
0 0.01 0.1 10 100 1
Peptide concentration (uM)
Figure 3-8: An ELISA assay detecting IL-2 release. DO10.11 hybridom as (7.5xl04) were
incubated with irradiated A20 B lym phoma cells (2 x l0 5 ) and 10-fold dilutions of OVA(323-
339), OVA - YT(323-339), OVA - M(323-339) for 48 hrs.
00
59
compared to the wild type influencing T cell stimulation. The lack of IL-2 secretion
following stim ulation with OVA-YT is due to the fact that residue 333 o f the core
region is critical for the stim ulation o f DO10.11 cells (M cFarland et al. 1999;
Robertson et al. 2000). The replacement o f this residue with any other amino acid
will result in loss of DO10.11 activation (M cFarland et al. 1999; Robertson et al.
2000). In the OVA-YT, this amino acid has been changed to a tyrosine (E->Y ) in
order to yield the high affinity variant (M cFarland et al. 1999; Robertson et al. 2000).
Discussion:
The role of T H cells on the generation of strong and protective CTL responses
has been a subject o f intense debate. W hile CTL responses can be elicited in the
absence of help from TH cells, these responses are neither long lasting nor protective
(Keene and Forman 1982; von Herrath et al. 1996; Bennett et al. 1997; Bennett et al.
1998; Ridge et al. 1998; Schoenberger et al. 1998; Marzo et al. 2000; Shedlock and
Shen 2003; Sun and Bevan 2003). Even though the mechanisms by which TH cells
influence the CTL responses are not yet understood, it is apparent that in order to
rationally design a vaccine, one must include TH epitopes.
The necessity for TH epitope inclusion in DNA epitope vaccines has similarly
raised an intense debate (Ahlers et al. 1997; M aecker et al. 1998; Chan et al. 2001;
Yoshida et al. 2001). In previous groups, the importance of T H help was evaluated by
the ability of DNA epitope vaccines encoding only the class I epitope to elicit strong
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60
CTL repsonses or by the removal of TH cells from mice (Ahlers et al. 1997; M aecker
et al. 1998; Chan et al. 2001; Y oshida et al. 2001). Unlike the above studies, I
decided to use epitopes of varying affinity to the class II molecule to exam ine their
influence on the CTL response. TH help is not completely absent from the vaccine
constructs but com prom ised. Therefore, it can be determined at what threshold T H
help has an impact.
Based on the results presented in this chapter, alterations in the affinity o f the
class II epitope does affect the immune response elicited by DNA epitope vaccines.
The use of a high affinity OVA-YT TH epitope enhanced the elicited g p l2 0 specific
CTL response. Furthermore, decreasing the affinity of the helper epitope resulted in
a dim inished im m une response, suggesting the need of a helper epitope for a
successful CTL response. In support of the CTL data, I observed increased numbers
of gp 120-specific CTLs secreting IFN-y. Therefore, the increased number o f stable
MHC II-peptide complexes stimulate more T H cells (Nelson et al. 1994) resulting in
better CTL responses.
In a peptide vaccine study, the use of a high affinity helper epitope improved
CTL responses (Ahlers et al. 2001). However, this improvement was not only due to
high numbers of T H cells stimulated. The use o f the high affinity TH epitope resulted
in increased levels of CD40L in TH cells. This resulted in more effective conditioning
of DCs which showed increased expression of costimulatory molecules and secretion
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61
of IL-12, a TH 1 polarizing cytokine (Ahlers et al. 2001). Thus, the use of high affinity
T h epitopes can polarize TH cells to enhance CTL responses.
In addition to the stronger CTL response, I observed the generation of OVA-
YT specific Th population. Follow ing in vitro stim ulation w ith peptide, m ice
immunized with RAOYTLL proliferated to the OVA-YT peptide and the RAOLL
group responded to wild type OVA. Sim ilar patterns were seen in the ELISA and
ICCS experiments. These results correlated with the in vitro stimulation o f DO 10.11
cells by peptide pulsed A20 cells. A 10 - fold higher amount of the low affinity
variant, OVA-M, was needed to reach the levels of the wild type OVA. On the other
hand, the T cells were not stim ulated when A20 cells presented the high affinity
variant, OVA-YT. Amino acid residue 333 is a glutamic acid. Studies have shown
that substitution of this residue results in loss of T cell activation. In the OVA-YT
peptide, residue 333 has been replaced by a tyrosine. Several groups have reported
on this T cell receptor (TCR) PFR dependency seen above (Carson et al. 1997;
M oudgil et al. 1998; Carson et al. 1999). The PFRs are involved in epitope stability
and have been shown to interact w ith the TCR and influence VP chain usage
depending on the nature of the amino acid present (Carson et al. 1997; M oudgil et al.
1998; Carson et al. 1999). Also, quantitatively the high affinity TH cells were not
higher than the wild type group (Figures 3-4 and 3-6). Proliferation responses were
com parable, so were the levels of cytokine release and numbers of epitope specific
T h cells secreting IFN-y. The enhancing effect, thus, on the CTL response may have
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62
been due to a qualitative shift in the nature of the immune response rather than a
quantitative. The presence of a high affinity helper epitope enhanced T m type
responses that strengthen the generation of gpl20 specific CTLs.
Conclusion:
The use of high affinity TH epitopes is very important in the generation of
effective vaccines. An increasing number of studies demonstrates how crucial T H
cells are to the orchestration of protective immune responses. In these studies, when I
manipulate help by lowering the affinity of the OVA epitope to the class II molecule,
the CTL killing is ablated. W hen I substitute the wild type helper epitope with a high
affinity variant, CTL responses are significantly improved. These results are in
contrast to the studies, which suggest that TH epitopes are not needed to generate a
CTL response w ith DNA epitope vaccines. This study dem onstrates that helper
epitopes are required to elicit a strong CTL response.
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63
CHAPTER 4: THE INFLUENCE OF CHEMOKINES ON THE
IMMUNOGENICITY OF DNA EPITOPE VACCINES.
Introduction:
Chem okines are a large fam ily o f small proteins that are involved in
leukocyte trafficking and play a crucial role in immune and inflammatory responses
(Baggiolini 1998; Rossi and Zlotnik 2000). Based on a conserved cysteine motif,
they are divided in four fam ilies: C, CC, CX C, and C X 3C (Rollins 1997).
Chem okines interact with G - coupled receptors. The expression of the receptors is
crucial in the action of chemokines. Even though there is considerable redundancy
(M antovani 1999) among the different chem okines within the same family, certain
cells express a given set of chemokine receptors, thus, rendering them responsive to
specific chemokines (Bonecchi et al. 1998).
In this chapter, the goal is to determine w hether the use of chem okines in
DNA epitope vaccines can enhance their immunogenicity. To examine this issue, I
decided to use the mouse KC chemokine in the DNA epitope vaccines (Bozic et al.
1995). KC is a member of the CXC ELR+ family. This family of chem okines is
known to be potent chemoattractants of neutrophils due to the presence of the ELR
m otif (Rollins 1997). It is, along with MIP-2, the major chem okine in inflammatory
states. It is secreted prim arily by endothelia and epithelia cells as well as
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64
lym phocytes. KC binds to the CXCR2 receptor, which is expressed on neutrophils
(Bozic et al. 1995; White et al. 1998).
The role of neutrophils (PM N) was believed to be lim ited to the innate
im m une response. Recent studies, however, have shown that PMNs can play a role
in the adaptive immune response (Potter and Harding 2001). More specifically, they
are capable of presenting class I epitopes to memory CTLs providing them with a
strong stim ulus to lengthen their lifespan. Since they lack class II m olecules, they
cannot directly stimulate T helper cells. H owever, they are capable o f releasing
epitopes by regurgitation to surrounding antigen presenting cells that will in turn
present epitopes to T helper cells (Potter and H arding 2001). Thus, PM Ns can
enhance CTL responses as well as further improve the stimulation of T helper cells
to elicit a stronger CTL response (Potter and Harding 2001).
In this chapter of my thesis, the goal is to determ ine the role of the KC
chem okine on the immunogenicity of our DNA epitope vaccines. Also, I wanted to
determ ine the most minimal seqeunce required to elicit the desired effect. To address
these questions, I designed four vectors shown in Figure 4-1. The first encodes the
full-length chemokine gene (FL-KC). The second is a truncated version of the full-
length chem okine, lacking the first 4 N-terminal amino acids (King et al. 2000). This
is a naturally occurring intermediate (T-KC) and its activity has been shown to be 10
m illion tim es higher than the full length chem okine (King et al. 2000). I also
constructed a vector encoding the first 8 amino acids residues (8-KC) as well as one
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hydrophobic leader
FL-KC
gpl20 (315-329) OVA (323-339) transmembrane LL
from KC cliemokiiie CTL epitope Tm epitope domain endosome
hydrophobic leader
T-KC
gpl20 (315-329) OVA (323-339) transmembrane
LL
from KC chemokine CTL epitope T, epitope domain endosome
hydrophobic leader
8-KC
gpl20 (315-329) OVA (323-339) transmembrane LL
from KC chemokine CTL epitope epitope domain endosome
hydrophobic leader
4-KC
gpl20 (315-329) OVA (323-339) transmembrane
LL
from KC chemokine CTL epitope Tk epitope domain endosome
Figure 4-1: Schem atic diagram s o f the vectors encoding the chem okine sequences, the epitopes and the targeting
sequences. FL-KC is the full-length gene, T-KC is the truncated form that lacks the first 4 N -term inal amino acids,
8-KC is the 8-mer and 4-KC is the 4-mer.
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1 10 20 30 40 50 60 70
Full length KC (FLKC) APIANELRCQCLQTMAGIHLKNIQSLKVLPSGPHCTQTEVIATLKGREACLDPEAPLVQKIVQKMLKGVPK
Truncated KC (TKC) NELRCQCLQTMAGIHLKNIQSLKVLPSGPHCTQTEVIATLKGREACLDPEAPLVQKIVQKMLKGVPK
8 - m e r KC (KC8) APIANELR
4 - m e r KC (KC) NELR
Figure 4-2: The amino acid sequence of the chemokines used in this chapter.
G \
ON
67
encoding only the NELR portion in an attempt to isolate the bioactive portion o f the
chem okine (4-KC) (Loetscher et al. 1998). The ELR m otif is believed to be the main
factor of neutrophil attraction. The amino acid secquences o f the chemokines used in
this study are shown in Figure 4-2.
Results:
The presence o f chemokine sequences influences the gp-120 specific CTL response.
The vectors (Figure 4-1) w ere injected into BALB/cJ mice (n=6 for each
group) as described in the M aterials and M ethods, chapter 5. As in the previous
experiments, after the mice were sacrificed, spleen cells were isolated and stimulated
with gp 120(318-327) peptide for 5 days in vitro. Subsequently, they were used as
effectors in 5lCr release assays to lyse g p l2 0 pulsed P815 target cells. As shown on
Figure 4-3, mice injected with the vector expressing the truncated KC (RTKC) as
well as the 8-mer (RKC8) elicited a stronger immune response than the ones injected
with the non-chemokine expressing vector (RAOLL). The introduction of the 4-mer
(RKC4) and the full length chem okine (RFLKC) did not have an effect. In the
RFLKC im m unized group of mice there was a w ider spread of responses. There
were num bers of mice that were influenced by the presence of the chem okine
sequence and others that were not. These data suggest that the presence of a
chemokine seqeunce can improve the CTL response.
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68
100
90-
70-
60-
50-
40-
£ 30-
^ 20-
10 -
NAIVE RAOLL KKC4 RKC8 RTKC RFLKC
Figure 4-3: A summary plot of two experim ents, showing specific lysis at 100:1
effectontarget ratio. BALB/cJ mice (n=6) were injected with the indicated vectors.
The values are statistically significant (p<0.001).
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69
Th cell proliferation o f mice immunized with the chemokine and control vectors
In addition to the 51C r release assays, I perform ed [3 H] thym idine
incorporation assays to measure TH cell proliferation. Spleen cells w ere in vitro
stim ulated w ith O V A (323-339) and M BP(89-101). There w ere no significant
differences among the five groups o f im munized mice (Figure 4-4). It is possible
that the increased infiltration of PM Ns at the local draining lym ph nodes may
influence the CTLs through the increased M HC I presentation of gpl20(Figure 4-4).
Mice immunized with chemokine encoding plasmids showed increased cytokine
secretion
Furthermore, I examined the cytokine profiles of the immunized mice. The goal
was to determ ine whether the presence of a chem okine sequence influenced the
cytokine profiles of the im m unized mice. I performed ELISAs to measure the
amount of IFN-y released. Spleen cells were stimulated in vitro with peptides (OVA
(323-339), gp 120(315-329), M BP(89-101)) for 3 days. The results are presented in
Figure 4.4. A fter in vitro stim ulation with OVA, cells from m ice injected with
RKC8, RTKC, and RFLKC plasm ids showed higher cytokine secretion than the
RAOLL (Figure 4-5). W hen stim ulated with gpl20, all the chem okine encoding
vectors showed higher levels of cytokine secretion. I did expect differences among
the chemokine groups after g p l2 0 stimulation. The lack of variation could be due to
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NAIVE RAOLL RCK4 RKC8 RTKC RFLKC
34000
24000-1
g 14000
S <
v
4000-
-6000-
16000
*
OVA(323-339)
gpl20(315-329)
MBPC89-101)
Figure 4-4: A representative proliferation assay. There are no significant differences among the five groups of
immunized mice after in vitro stimulation with OVA(323-339) peptide. The num ber of mice used for each group
in this experiment is n=3.
O
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NAIVE RAOLL RKC4 RKC8 RTKC RFLKC
■ OVA (323-339)
mm
M m
MBP $9-101)
Wm
Mm
wM
v l v f f i
WM
m t
MM
- 100-
- 200 -
Figure 4-5: An ELISA assay detecting IFN-y levels. Mice were im m unized with the indicated vectors. M BP(89-
101) is a class II epitope used as a control. M ouse spleen cells were pooled. The number o f mice per group was
n=3.
72
the fact that I am stimulating a mixed splenocyte population in which the differences
between the immunized groups can be masked. To obtain a more clear picture as to
the cytokine profiles of epitope-specific T cells, I performed intracellular cytokine
staining assays (ICCS).
In these assays, I measured the epitope-specific IFN-y secreting T H cells and
CTLs. There were no significant differences in the numbers of cytokine secreting T H
cells when stim ulated with the OVA(323-339) (Figure 4-6), a pattern seen in the
proliferation assay (Figure 4-5). M ice immunized with the RKC8 and RTKC vectors
showed higher numbers of epitope specific CTLs compared to the RAOLL group
(Figure 4-7). On the other hand, immunizations with the RKC4 and RFLKC did not
have a similar effect. These data are in agreement with the CTL assays.
Discussion:
The use o f chem okines in DNA vaccine constructs is a relatively new
strategy to enhance their immunogenicity. The majority of studies have focused on
im m unizing mice w ith two plasm ids, one encoding a chem okine and another
encoding the full length antigen (Loetscher et al. 1998; Xin et al. 1999; Youssef et al.
1999; Kim et al. 2000; Sin et al. 2000; Eo et al. 2001; Eo et al. 2001; W ildbaum et al.
2002). Only, very recently, there have been studies using chem okine sequences
(interferon inducible protein 10 (IP -10) and monocyte chemotactic protein3 (M CP-
3)) fused with the full-length tumor antigens to generate stronger antitum or immune
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NAIVE RAOLL RKC4 RKC8 RTKC RFLKC
T „ /IFN-T
*
Figure 4-6: ICCS measuring the am ount o f TH IFN-y secreting cells. Splenocytes were pooled from
each group of mice and in vitro stim ulated with OVA(323-339). The num bers o f mice im munized
per group is n= 3. The vectors are indicated at the top of the graph.
U J
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NAIVE RAOLL RKC4 RKC8 RTKC RFLKC
| CTUlFN-y
Figure 4-7: ICCS m easuring the num ber o f CTLs secreting IFN-y after in vitro stim ulation with
g p l2 0 (3 18-327). Splenocytes w ere pooled from each group of m ice. T he num ber of m ice
immunized per group is n=3. The vectors are indicated at the top of the graph.
75
responses (Biragyn et al. 1999; Biragyn et al. 2001; Biragyn et al. 2002). DNA
vaccines encoding these fusion proteins were able to generate strong antitum or and
HIV im m une responses. The m echanism suggested in these papers is that the
chem okine segment of the expressed fusion peptide attracts target cells. Then, the
target cells bind and internalize the fusion peptide leading to antigen presentation.
Thus, the chem okine is used as a carrier to target the antigen to APCs (Biragyn et al.
1999; Biragyn et al. 2001; Biragyn et al. 2002). However, it is possible that the
fusion protein may be degraded into the individual proteins after targeting to the ER.
The chem okine will, then, attract more antigen presenting cells to the site of
im m unization thus improving the presentation of a poorly im m unogenic tum or
antigen.
In this study, I examined the effect o f an inflammatory chem okine on DNA
epitope vaccine immunogenicity as well as the most minimal sequence required to
elicit the desired effect. To accom plish this, I generated four vectors encoding
variants o f the KC chemokine and com pared the immune responses elicited to a
plasm id that did not express any chem okine (RAOLL). The CTL responses elicited
by the RKC8 and the RTKC were stronger than the RAOLL while im m unization
with the RFLKC did not show consistent improvement. The lack of a reproducible
effect by the RFLKC can be attributed to the low amounts of chem okine secreted by
the DNA vaccine. Studies have shown low levels of naked plasmid transfection in
vivo and low amounts of expressed protein (Casares et al. 1997; Gurunathan et al.
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76
2000). Furtherm ore, it is possible that the secretion of large am ounts o f bioactive
chem okine may be com prom ised by another factor. The KC m olecule is expressed
w ithin the context o f a string of epitopes. This may affect to a certain degree the
correct folding o f the chem okine, thus, com prom ising the secretion o f bioactive
protein. On the other hand, RTKC can bypass the above limitations because o f its
higher potency as a KC truncated variant (King et al. 2000). The effect by RKC8 is
not unusual. The am ino-term inal portion of chem okines possesses m uch o f the
bioactivity o f the m olecule. This region is about 8-10 amino acid residues long.
Previous groups have generated chemokine anatagonists by truncating the 8-10
residues from the N- term inus of full-length chem okines. In addition, 8-m er
chemokine peptides have been shown to be bioactive (Loetscher et al. 1998). Studies
done with S D F -1 have shown that an 8-mer peptide can induce chem oattraction but
its bioactivity is about 1000 fold lower than the full-length (Loetscher et al. 1998).
Due to its small size, folding is not an issue for generating a bioactive secreted
peptide. However, I am unable at this point to determine whether the positive effect
is due to increased secretion, higher bioactivity or both.
The enhanced im munogenicity was further dem onstrated when I exam ined
the numbers o f IFN-y secreting T cells. RTKC and RKC8 im m unized mice had
higher numbers o f epitope specific CTLs while IFN-y secreting T H cells were
com parable in all groups of immunized mice. Proliferation assays did also show
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77
com parable proliferative responses am ong all im m unization groups further
strengthening the rationale that this is more o f a CTL targeted effect due to the
expression of M HC I molecules on the surface of PMNs.
C onclusion:
The introduction of a chem okine sequence w ithin the context o f DNA
vaccine encoding antigens is a relatively new strategy for generating effective
immune responses. The presence of a chemokine seems to enhance epitope specific
im m une responses. M ore interestingly, I was able to dem onstrate the potential of
using smaller fragments of the chemokine to elicit the same beneficial effects.
The discrepancy between CTL and TH effect on the KC chem okine suggests
that the following may be occurring. Imm unization with the chem okine encoding
vectors leads to the increased attraction of PMNs to the site of immunization as well
as the draining lymph nodes. The increased infiltration of PMNs results in better
presentation of gp l2 0 epitope to CTLs.
The above data are bolstered by recent literature where chem okines have
been fused to tum or antigens to enhance their im m unogenicity. The fusion of
chemokines to the tumor antigen was shown to enhance antitumor resposes (Biragyn
et al. 1999; Biragyn et al. 2001; Biragyn et al. 2002). Even though the mechanisms
have not been fully elucidated, it is clear that a chemokine fused to an antigen can be
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78
expressed, correctly folded, and be bioactive. In my results, the full-length
chem okine does not seem to have a significant effect on the CTL response. Even
though this contradicts the above findings, there is an im portant difference. In the
absence o f the chemokine sequence, the DNA epitope vaccine is already eliciting a
strong response. In Biragyn et. al, immunization with a DNA plasmid encoding only
the tum or antigen did not elicit any immune response (Biragyn et al. 1999; Biragyn
et al. 2001; Biragyn et al. 2002). The implications of these are very important. First,
the need for separate genetic im m unizations, one for the chem okine expressing
plasm id and one for the antigen will be elim inated. Second, sm aller bioactive
fragments can replace large chemokine m olecules allowing flexibility in rationally
designing a DNA epitope vaccine. Lastly, the inclusion of chem okines in DNA
epitope vaccines will further enhance their immunogenicities.
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79
Coda:
Several questions were posed in the Introduction (Chapter I) w hich have
been answered:
(a) Does the replacement o f the leader sequence influence the epitope-specific
CTL response elicted by the DNA epitope vaccines? The use o f a more
efficient leader, rat KC chem okine, did indeed enhance the CTL responses
elicited by the DNA epitope vaccines. Thus, the identity o f the targeting
sequence is very important.
(b) Can changes in the affinity of TH epitopes to MHC II affect CTL responses?
The presence of strong T H epitopes does influence the potency of CTL
responses. I replaced the wild type OVA(323-339) with the high affinity
variant OVA-YT and the low affinity variant, OVA-M. The vector encoding
the high affinity TH epitope did elicit stronger CTL responses than the one
encoding the wild type. In addition, immunization with the plasmid encoding
the low affinity variant showed diminished CTL responses. Therefore, there
is a T h epitope threshold that is needed to be met in order for the response to
be effective.
(c) Can the introduction of a chemokine sequence enhance im m unogenicity of
DNA epitope vaccines? The introduction o f the KC chem okine, a PMN
chem oattractant, did affect the CTL responses. In addition, the use of the
am ino term inal 8-m er chem okine w as sufficient to enhance the
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80
immunogenicity of the DNA epitope vaccine. Thus, like epitopes, it is possible to
use smaller bioactive fragments instead of the entire molecule.
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81
CHAPTER 5: MATERIALS AND METHODS
Oligonucleotides
The oligonucleotides used in these studies were synthesized at the USC
N orris Cancer Center M icrochemical Core Facility and are listed in Table 5-1.
Annealing o f oligonucleotides fo r cloning
200pmol of sense and anti-sense strands of oligonucleotides are added in a
55fil reaction mixture containing l.lfxl of 5M NaCl, and l.ljil Tris HC1, pH = 7.2.
The sam ples were heated in a 9600 therm ocycler (Perkin-Elm er C orporation,
M eriden CT) at 90°C and ramped to 20°C in 3 hrs. Samples w ere run on a 2%
agarose gel, to confirm annealing, and were then ethanol precipitated.
Polymerase chain reaction
All PCR reactions described here used a 9600 therm ocycler (Perkin-Elm er
Corporation, Meridian CT). In most cases standard PCR conditions were used: lOOpI
reaction containing 2mM M gCl2, 50mM KC1, lOmM Tris-HCl, pH8.3, 20pmol of
each prim er, 0.5 units of Taq DNA polymerase (Roche, Indianapolis IN) run at 30
cycles at 96°C for 30 seconds, 65°C for 30 seconds, 72°C for 30 seconds. The amount
of template was never more than lOOng per reaction.
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82
Table 5-1: List of synthetic oligonucleotides used for PCR analysis, sequencing, and
gene assembly.
N um ber Description Sequence 5’— » 3 ’
M C520 KC Leader, first half sense GTGACCCGCCGCACCATGGTC
TCAGCCACCCGCTCGCTTCTCT
GTGCAGCGC
MC521 KC Leader, first half anti-sense GAAAGCGAGCGGGTGGCTGAG
ACCATGGTGGCGGCGG
M C518 KC Leader, second half, sense TGCCTGTGCTGGCCACCTCTAG
ACAAGCCACAGGGGGGCC
M C517 KC Leader, second half, anti
sense
CCCCTGTGGCTTGTCT AG AGGT
GGCCAGCACAGGCAGCGCTGC
ACAGA
MC515 Quick change primer; corrupt
BstEII site, sense
CACTGCAGTGGATCGCGTGAC
CATCGCCGCGG
M C514 Q uick change prim er; corrupt
BstEII site, anti-sense
CCGCGGCGATGGTCACGCGAT
CCACTGCAGTG
MC535 Q uick change prim er; insert
H indlll restriction site, sense
GTGGATCGGAAGCTTATCGCC
GC
M C534 Q uick change prim er, insert
H indlll restriction site, sense
GGCGATAAGCTTCCGATCCAC
TGCA
MC542 OVA mutant, replaces flanking
regions with YTYT, sense,
first half
CCGGT AGCGC AGAGAGCCTG A
AGT ACACCT ACACCGTCCATG
CAGCA
MC546 OVA mutant, replaces flanking
regions w ith Y TY T, a n ti
sense, first half
GGTGT AGGTGACTTCAGGCTC
TCTGCGCTA
MC545 OVA mutant, replaces flanking
regions with Y TY T, sense,
second half
CATGCATACACCTACACCGCA
GGCAGAGAGGTGC
MC541 OVA mutant, replaces flanking
regions with YTYT, antisense,
second half
TTAAGCACCTCTCTGCCTGCGG
T GT AGGTGT AT GCATGTGCTGC
ATGGAC
M C543 Quick change primer, mutates
OVA epitope (V— »R), sense
G AAGAT ATCTC AAGCT AG AC A
TGCAGCACATGCAG
M C544 Quick change primer, mutates
OVA epitope (V — >R), anti
sense
CTGCATGTGCTGCATGTCTAGC
TTGAGATATCTTC
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83
Table 5-1, continued
M C564 Q uick change prim er, insert
A pal restriction site, sense
CT GCAGATCGAAGGGCCCATA
GATCCCGTCG
MC565 Q uick change prim er, insert
A pal restriction site, anti-sense
CG ACGGG AT CT AT GGGCCCTT
CGATCTGCAG
M C569 H indlll insertion primer AAAAAAGCTTGGAGATCTGCC
GCCACCATG
M C570 H indlll insertion primer AAAAAAGCTTCGCCGCCACCA
TGGTCTCAG
M C568 B o v in e g ro w th horm one
priming site primer for pcDNA
3.1/lacZ/c-myc/6-His vector
CTAGAAGGCACAGTCGAGGCT
G
MC573 L acZ p rim er fo r pcD N A
3.1/lacZ/c-myc/6-His vector
CGCT ATT ACGCC AGCT GGCG A
AAGGGG
M C656 pSyngpl20JR -FL sense Apa I
PCR extension primer
AAAAAAGGGCCCGTGGAGAA
GCTGTGGGTG
MC657 p S y n g p l2 0 JR -F L anti-sense
H indlll PCR extension primer
AAAAAAAAGCTTTCAGCCCAC
AGCGCGCTTCTC
M C670 Quick change primer; removes
4 N-terminal amino acids from
KC chemokine; sense
AGACAAGCCACAGGGAATGAG
CTGCGCTGT
MC671 Quick change primer; removes
4 N-terminal amino acids from
KC chemokine; anti-sense
ACAGCGCAGCTCATTCCCTGT
GGCTTGTCT
M C672 8-m er (8KC) KC chemokine,
sense
CT AGACAAGCCACAGGGGCGC
CTATCGCCAATGAGCTGCGCG
GGCC
MC673 8-m er (8KC) KC chemokine,
ant-sense
CGCGCAGCTCATTGGCGATAG
GCGCCCCTGTGGCTTGT
M C674 4-m er (4KC) KC chem okine,
sense
CT AGACAAGCCACAGGGAATG
AGCTGCGCGGGCC
MC675 4-m er (4KC) KC chem okine,
anti-sense
CGCGCAGCTCATTCCCTGTGGC
TTGT
M C676 KC gene (A), sense CT AGACAAGCCACAGGGGCGC
CTATCGCCAATGAGCTGCGCT
GTCAGTGCCTGCAGACCATGG
CTGGGATTCACCTCAA
MC677 KC gene (A), anti-sense CCAGCCATGGTCTGCAGGCAC
TGACAGCGCAGCTCATTGGCG
ATAGGCGCCCCTGTGGCTTGT
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84
Table 5-1, continued
M C695 KC gene (B), sense GAACATTCAGAGCTTGAAAGT
GTTGCCTTCAGGACCTCACTGC
ACTCAAACTGCAGTCATAGCT
ACACTCAAGAATGGTC
M C696 KC gene (B), anti-sense GTGT AGCT ATG ACTT C AGTTT G
AGTGCAGTGAGGTCCTGAAGG
CAACACTTTCAAGCTCTGAAT
GTTCTTGAGGTGAATC
M C694 KC gene (C), sense GCGAGGCTTGCCTTGACCCTG
AAGCTCCCTTGGTTCAGAAAA
TTGT CCA A A AG AT GCT A A AGG
GTGTCCCCAAGGGGCC
M C697 KC gene (C), anti-sense CCTTGGGG AC ACCCTTT AGC AT
CTTTTGGACAATTTTCTGAACC
AAGGGAGCTTCAGGGTCAAGG
CAAGCCTCGCGACCATTCTTG
A
M C726 KC gene (A l), sense CTAGACAAGCCACAGGGGCGC
CTATCGCCAATGAGCTGCGCT
GTCAGTGCCTGCAGACCAT
M C718 KC gene (A l), anti-sense CACTGACAGCGCAGCTCATTG
GCGATAGGCGCCCCTGTGGCT
TGT
MC721 KC gene (A2), sense GGCTGGGATTCACCTCAAGAA
C ATCCAG AGCCTT AAG AAA A A
ACTGCA
M C724 KC gene (A2), anti-sense GTTTTTTCTTAAGGCTCTGGAT
GTTCTTGAGGTGAATCCCAGC
CATGGTCTGCAGG
MC722 KC gene (B l), sense CT AG A A A A A A ACTT A AGGTGT
TGCCTTCAGGACCTCACTGCAC
ACAAACAG
MC715 KC gene (B l), anti-sense AGTGAGGTCCTGAAGGCAACA
CCTT AAGTTTTTTT
M C716 KC gene (B2), anti-sense AAGTCAT AGCT ACACTCAAGA
ACGGCCGAAAAAACTGC
MC719 KC gene (B2), anti-sense GTTTTTTCGGCCGTTCTTGAGT
GTAGCTATGACTTCTGTTTGTG
TGC
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85
Table 5-1, continued
M C720 KC gene (C l), anti-sense CT AGAAAAAAACGGCCGCGAG
GCTTGCCTTGACCCTGAAGCTC
CTTT
M C723 KC gene (C l), anti-sense ATTTTCTGAACCAAAGGAGCT
TCAGGGTCAAGGCAAGCCTCG
GGCCCGTTTTTTT
M C725 KC gene (C2), anti-sense GGTT C AG A A A ATT GTCC A A A A
GAT GCT A A A AGGTGTCCCT A A
GGGGCCCAAAAAACTGCA
M C717 KC gene (C2), anti-sense GTTTTTTGGGCCCCTTAGGGAC
ACCTTTT AGC AT CTTTTGG AC A
M C747 psyngpl20JRFL Age I
insertion primer
AAAAAAACCGGTGCCCACAGC
GCGCTTCTCCCT
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86
Digestion with restriction enzymes
All restriction digests were perform ed according to the m anufacturer’s
instructions. Table 5-2 outlines the restriction enzym es used and their conditions.
The buffers listed are described in the respective com panies’ catalogues.
Ethanol precipitation
DNA was precipitated by adding 1/10 volume of 3M sodium acetate pH 5.2
and 2.5 to 3 volumes of 100% -20°C ethanol to the sample, mixing and incubating on
ice for 30 m inutes. The sam ple was then spun in a B eckm an refrigerated
m icrocentrifuge (Beckm an, Irvine CA) at m axim um speed for 30 m inutes.
Supernatant was removed and the pellet was washed with 70%, -20°C ethanol and
centrifuged for 30 minutes at 4°C. Supernatant was discarded and the pellet was air
dried for 5 minutes. The pellet was resuspended in the appropriate volume in double
distilled sterile water (ddH2 0 ).
DNA ligation
Ligation reactions were carried in 15(il reaction volume. A typical reaction
contains the digested DNA (10-50ng), 1.5(il of lOx ligase buffer, and 1 unit of T4
ligase (New England Biolabs, Beverly MA). The reaction was incubated at 15°C for
at least 16 hours.
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Table 5-2: Restriction endonucleases
87
E N ZY M E B U FFE R T E M PE R A T U R E IN C U B A TIO N
T IM E
SO U R C E
A pal 4 26°C Overnight New England
Biolabs
A flll 2 37°C At least 4 hrs New England
Biolabs
A gel 1 37°C At least 4 hrs New England
Biolabs
BstEII B 55°C 4 hrs Roche
EagI 3 37°C 4 hrs New England
Biolabs
H indlll 3 37°C 4 hrs New England
Biolabs
N otl 3 37°C 4 hrs New England
Biolabs
PstI 3 37°C 4 hrs New England
Biolabs
X bal 3 37°C 4 hrs New England
Biolabs
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Transformation o f Bacterial Cells
The Escherichia coli bacterial strain D H 5a was made com petent using
calcium chloride incubation according to the protocol in C urrent Protocols in
M olecular Biology. The com petent cells were transformed by incubating the ligated
DNA reaction with lOOpl of com petent cells for 30 minutes on ice. The DNA and
cell m ixture was heat-shocked for 90 seconds at 42°C and plated on am picillin
(100p.l/ml) Luria Broth (LB) agar plates at 37°C overnight.
PCR Screening
Colonies that grew on the ampicillin/LB plates were used to inoculate 1ml of
LB broth cultures, and the cultures were incubated for 4 hours on a rotary shaker at
37°C. In order to PCR amplify the insert DNA, 10|il of bacterial culture and 90|ol of
0.1% Tween-20 (Sigma-Aldrich, St Louis M O) was added to a 200|il-thin walled
PCR tube (Axygen, Union City CA). The mixture was then incubated at 100°C for
15 m inutes to lyse the bacteria. 2(0 .1 o f the lysate was used in a standard PCR
reaction.
DNA preparation
Colonies, tested positive in the PCR screening, were grown in 3ml LB media
containing 10% ampicillin. Vector DNA was extracted using mini-prep kits (Qiagen,
Valencia CA). The DNA plasmids were then sequenced at the USC Norris Cancer
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89
Center M icrochem ical Core Facility and they were shown to be base-perfect. DNA
plasmids with the correct sequence were then grown in 2.5L of LB media containing
10% am picillin. DNA was prepared using EndoFree Giga Prep kits (Qiagen). For
transfection experim ents, DNA was prepared using EndoFree M axi Prep kits
(Qiagen) and was sterilized using phenol/chloroform extraction method.
Construction o f rat KC leader epitope vectors
The pR c/C M V vector (Invitrogen, Carlsbad CA) was used in all the
constructs. The vectors possess a hydrophobic leader sequence (S) derived from the
murine H-2Ld m olecule at the N-terminus of the peptide to direct synthesis of the
peptide to the endoplasm ic reticulum (ER). Furtherm ore, they contain a
transm em brane sequence that positions the polypeptide product such that the N-
terminus is initially located in the lumen of the ER for processing and the C-terminus
in the cytosol for targeting. Also, the C-termini contain two endosome localization
signals, YXXZ (YZ) (Bonifacino and D ell'A ngelica 1999) and di-leucine (LL)
(Teasdale and Jackson 1996). The SOYZLL (Figure 5-1) plasmid encodes the class
II ovalbum in (O) epitope (323-339). This is a T H epitope targeted to the class II
pathway. The SAOYZLL (Figure 5-1) plasmid, also has the HIV class I epitope
gpl20 (A) (315-329), in addition to the ovalbumin epitope. The RAOYZLL (Figure
5-1) is the vector with the rat KC leader sequence(R). The RAOYZLL plasmid was
constructed using the following strategy. The H-2Ld leader of the SAOYZLL vector
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90
= X
1 1 1
< s > C O
OVA TM
SAOYZLL
YZLL Amp H-2d leader
L U
1 S
c a
BstEII/Apal digestion
ligation
OVA TM
RAO YZLL
YZLL Amp KC le a d e r
Figure 5-1: Construction of RAOYZLL. The KC chemokine leader was assembled
using synthetic oligonucleotides . SAOYZLL was digested with BstEII and Apal
restriction enzym es. The synthetic KC leader was ligated between the BstEII and
Apa I sites to generate RAOYZLL. The gpl20 minigene is the gpl20 (315-329) CTL
epitope, OVA is the ovalbumin (323-339) TH epitope, and TM is the transmembrane
domain. YZLL is the endosomal targeting motif.
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gp120 OVA YZLL H-2a lea d er
SAOYZLL
O) C Q
OVA TM g pl20
SOLL
H-2a lea d er
PCR £p120 epitope
minigene
betw een Not) and Agel
Not I/Age I di gesti on
ligation
TM OVA
SAOLL
A m p1
BstEllfApal digestion
tigation
TM CP 120 OVA
RAOLL
Amp* — ^ KC le a d e r
Figure 5-2: Construction of RAOLL. The g p l2 0 epitope m inigene was PCR
am plified and digested with Not I and Age I enzymes. The SOLL plasm id was
digested with the same enzymes and subsequently the HIV PCR product was ligated
to generate SAOLL. SAOLL was then digested with BstE II and Apa I enzym es,
followed by ligation of the KC leader to generate RAOLL.
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92
was digested using the BstEII and A pal enzymes. Then, I inserted the annealed rat
KC synthetic oligonucleotides between the BstEII and Apal sites. The fragment was
ligated using T4 ligase. The SAOLL (Figure 5-2) vector was constructed by the
follow ing procedure. The g p l2 0 (A) epitope was PCR am plified from the
SAOYZLL. In order to generate the overhang ends of the oligonucleotide, the PCR
fragment was digested with N otl and Agel. The resulting oligonucleotide was then
gel purified and ligated into SOLL between the N otl and A gel restriction sites.
RAOLL (Figure 5-2) was constructed using the same protocol as was used in the
RA OY ZLL vector. I transform ed the vectors into D H 5 a com petent cells, as
described previously.
Construction o f OVA mutant epitope vectors
The pRc/CM V vector (Invitrogen) was used in all our constructs. The
RAOLL was manipulated to generate vectors expressing the OVA epitope variants.
The RAOYTLL plasm id was constructed by the following procedure (Figure 5-3).
The OVA epitope from the RAOLL vector was digested between the Agel and A flll
sites. Then, I inserted the OVAYT synthetic oligonucleotide between the Agel and
A flll sites. The fragment was ligated using T4 ligase. The RAOM LL vector was
constructed by the following procedure. I mutated the OVA epitope o f the RAOLL
vector at position 327 with the MC543 and MC544 primers (Table 5-1) and using the
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RAOLL
Agel/Aflll d ig e s tio n
ligation
+
gp 1 2 0 OVA-YT TM
RAOYTLL
A m p KC le a d e r
Figure 5-3: Construction of RAOYTLL. RAOLL was digested between Age I and
Afl II sites. The synthetic OVA-YT gene was then ligated into RAOLL to generate
RA OY TLL. O V A-YT is the high affinity variant of ovalbum in (323-339) T H
epitope.
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94
QuickChange M utagenesis kit (Stratagene, La Jolla CA). I transform ed the vectors
into D H 5 a com petent cells.
Construction o f fi-gcilactosida.se expression vectors
The pcDNA 3.1/lacZ/M yc-His (+) (pLacZ) vector (Invitrogen) was used in
all constructs. Into this vector, I inserted a hydrophobic leader derived either from
the m urine H -2Ld m olecule (S) (pSLacZ) or the rat KC leader sequence (R)
(pRLacZ) at the N-terminus of the P-galactosidase protein to direct synthesis to the
endoplasm ic reticulum. The pSLacZ plasmid encodes the P-galactosidase under the
control o f the murine H-2Ld leader and the pRLacZ under the rat KC chem okine
leader. Both vectors were constructed by the following procedure (Figure 5-4). I
inserted the A pal digestion site upstream the lacZ gene with the M C564 and 565
primers (Table 5-1) and using the QuickChange Mutagenesis kit (Stratagene). Also,
by PCR extension, I added the H indlll restriction site on the two leader sequences
using prim ers M C569 (for the murine H -2Ld) and M C570 (for the rat KC
chem okine) (Table 5-1). Each leader was inserted into the pcDNA 3.1/lacZ vector
between the H indlll and Apal sites. I transformed the vectors into D H 5a com petent
cells.
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Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission.
Pm el
pLacZ
*
Hindlll/Apal digestion
ligation
c
i
H-2Ld lead er
Agel Agel
L U —
I z X CD C O
Pmel
H-2Ld le ad er KC le a d e r
\ pRLacZ I / V . I pSLacZ ) /
Figure 5-4: Construction of pSLacZ and pRLacZ. pLacZ was digested with Hind III and Apa I enzymes. The KC
and H-2Ld leaders were PCR amplified from RA OLL and SAOLL vectors respectively. Both were digested with
Hind III and Apa I and, subsequently, were ligated into pLacZ to generate the expression vectors.
V O
L n
96
Construction o f mouse KC chemokine and chemokine variant vectors
The pRc/CM V vector (Invitrogen) was used in all the constructs. I used the
RA OLL to generate vectors expressing the full length KC chem okine and the
truncated variants (see Figure 4-1 in Chapter 4). The RFLKC plasm id encodes the
full-length mouse KC chemokine (Bozic et al. 1995). The RTKC plasm id encodes a
truncated version of the full-length gene. It lacks the first 4 N-terminal amino acids.
The RKC8 encodes the first 8 am ino-term inal am ino acids o f the chem okine
(Loetscher et al. 1998). The RKC4 encodes the NELR m otif known to be responsible
for the attraction o f neutrophils. The RFLKC plasm id was constructed by the
following procedure. The PGEM 3Zf(+) cloning vector (Promega, M adison, WI) was
used in the assembly of the full-length chemokine. The whole gene was divided into
three parts and each portion was cloned into the vector separately. Synthetic
oliogonucleotides M C726, 718, 721, 724 w ere annealed to yield K C(A ),
oliogonucleotides M C722, 715, 716, 719 yielded KC(B), and oliogonucleotides
M C720, 723, 725, 717 segment KC(C). All three fragments were cloned between
X bal and PstI sites generating pKC(A), pKC(B), and pKC(C) vectors (Figure 5-5).
pKC(A) was digested between Xbal and A flll to generate the KC(A) fragment that
was ligated into pKC(B) between the Xbal and A flll sites to construct the pKC(AB)
vector (Figure 5-6). pKC(AB) was digested between Xbal and EagI sites to generate
the KC(AB) fragment that was ligated into pKC(C) between X bal and EagI sites to
construct the pKC(ABC)vector (Figure 5-6). pKC(ABC) was digested with Xbal and
Apal and KC(ABC) was the assembled full length KC. The assembled full length
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission.
T7 SP6
Am pB
X baPP stl d igestion
ligation
r
* S’ M
x
t u f .
ligation
ligation
| K C ( C ] |
. 8 S S . J I
|U J V > £ < W ,5 s
« -R-&1
U i a.
8 1 E _
I ui m £ < “> m * * |
pKCTA) pKCIBl pKC(C)
Figure 5-5: C onstruction of pKC(A), pKC(B), and pKC(C). The pGEM 3zf(+) was digested between X ba I and
Pst I sites. Synthetic oligonucleotides were ligated into the vector to generate three constructs containing parts o f
the full-length KC chem okine.
VO
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T7 SP6
MCS K C (B )
Amp1
pKC(B)
T7 SP6
MCS KC{AJ Amp1
pKC(A)
XbaWtfl II digestion
LU(/> X < W £
SP6
lAmp1 MCS KC(A) K C(B)
pKC(AB)
T7 SP6
EaglfPstl digestion
MCS K C (C )
Amp1
6 ag PPst I di g e s t on
ligation
PKC(C)
Amp* K C(A) KC(B) KC(C)
pKC(ABC)
Figure 5-6: Construction o f pKC(ABC). pKC(A) and pKC(B) were digested between X ba I and Afl II sites. The KC(A)
segment was ligated into pKC(B) to generate pKC(AB). Then, pKC(C) was digested between Eag I and Pst I to isolate the
KC(C) segment. The latter was ligated into pKC(AB) to generate pKC(ABC). v o
Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission.
sre
lA m P“l---- lA m p * KC(B K C(C)
RAOLL
pKC(ABC)
ligation
Xbal/Apal d ig e stio n
lig ation
ligation FLKC
KC4 g p 1 2 0 OVA KC8 Inp120 OVA
RKC4 RKC8
OP 120 I OVA TM
RFLKC
Figure 5-7: Construction of RFLKC, RK C4, and RKC8. pK C(ABC) was digested betw een X bal and A pal to
generate the full length KC, w hich was ligated into RAOLL to generate RFLKC. KC8 and KC4 synthetic
oligonucleotides were ligated between the sam e sites into RAOLL to generate RKC8 and RKC4. V O
100
KC DNA was gel purified and cloned into the RAOLL vector between the Xbal and
A pal sites to generate the RFLKC construct (Figure 5-7). The RKC8 and RKC4
vectors were constructed in a similar manner (Figure 5-7). Oligonucleotides M C674
and 675 were annealed to generate the KC4. Similarly, oligonucleotides MC672 and
673 w ere annealed to generate the KC8. The RTKC was constructed by the
following procedure. I mutated the KC gene of the RFLKC vector to delete the first
4 N - term inal amino acids with the primers M C670 and MC671 (Table 5-1) and
using the QuickChange Mutagenesis kit (Stratagene). I transformed the vectors into
D H 5a competent cells.
Construction o f gpl20-6xHis expression vectors
The pCDNA3.1 vector was used in all the constructs. I generated two vectors,
SA6H and RA6H. SA6H encodes the murine H-2-Ld leader, g p l2 0 and 6xHis tag at
the C-terminus of the gpl20 while the RA6H encodes the fusion protein under the rat
KC chem okine leader. SA6H was constructed as follows. Using PCR extension, I
added the Apa I and Agel sites on the syngpl20 gene (NIH AIDS Reagent Program)
(Haas et al. 1996) using primers MC656 and M C747 respectively. The reaction was
done using the Pfu Turbo DNA polym erase according to the m anufacturer’s
instructions (Stratagene). Briefly, 50ng of pSyngpl20JRFL template was added into
a PCR reaction mixture containing: 5(il lOx cloned Pfu DNA polym erase reaction
buffer, l|il dNTP mix (lOOmM), l|il MC656 primer (100ng/|il), 1 ptl M C747 primer
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission.
Agel
_ o u i _
Pmel Pmel
LacZ LacZ
myc II 6xH
Amp1 Amp1 KC leader
pRLacZ
Agel/Apal digestion
ligation
m
*
a i
i
sy n g p 120
I
PCR product from p S y n g p l20 JRFI-
Age l/Apa I di gesti on
ligation
6xH Amp1 syngp 120
RA6H I / \ SA6H
Figure 5-8: Construction of RA6H and SA6H. syngp 120 was PCR am plified from the pSyngpl20JR FL vector. The
PCR product was digested with the A pa I and Age I enzym es, as were the pRLacZ and pSL acZ vectors. The
syngp 120 was ligated into pRLacZ to generate RA6H and into pSLacZ to construct SA6H.
102
(lOOng/pl), l(il Pfu Turbo (2.5 U), and 40.6|il ddH 2 0 . The reaction was run in a
9600 therm ocycler at 30 cycles at 95°C for 30 seconds, 65°C for 30 seconds, and
72°C for 2 minutes. The PCR product was run on a 2% agarose gel, gene cleaned,
and digested by A pal and A gel enzymes. The insert was ligated into the pSLacZ
betw een the A pal and A gel sites replacing the fi-galactosidase gene and c-myc
sequence (Figure 5-8). The RA6H was generated in the same manner(Figure 5-8).
DNA Immunization
BALB/c-H-2d m 2 (dm2) mice were injected intradermally (i.d.) at the base of
the tail with 200 pg of DNA in 80pl of endotoxin-free PBS (Sigma-Aldrich). They
were boosted usually twice in two-week intervals with the same quantity o f DNA.
Three weeks later they were sacrificed by cervical dislocation. The spleens were
excised for further experiments.
Proliferation Assay
lxlO 6 spleen cells/w ell were plated in 96-w ell plates in HL-1 m edium
(BioW hittaker, Rockland ME) supplem ented with 2mM glutamine (Invitrogen),
penicillin (lOOU/ml) and streptomycin (lOOpg/ml) (M ediatech Inc, Herndon VI).
Subsequently, they were stim ulated w ith 40pM ovalbum in(323-339), 40pM
OVAYT(323-339), 40pM g p l2 0 (3 15-329), and 40pM myelin basic protein (MBP)
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103
(89-101) peptides for 72 hours at 37 °C, 5% C 0 2. All peptides were synthesized at
the USC/Norris Cancer Center M icrochem istry Core Facility. Then, lpC i/w ell of
[3 H] thymidine (ICN, Irvine CA) was added followed by a 24 hour incubation period
at 37 °C, 5% C 0 2. The cultures were then harvested using a M icron96 Harvester
(Skantron Instrum ents, Sterling VA) on filterm ats (Skantron Instrum ents). The
incorporation of [3 H] thym idine was m easured w ith a B eckm an LS6000IC
scintillation counter (Beckman, Irvine CA).
5 1 Cr Release Assay
7 x l0 6 spleen cells/well were plated in 24-well plates in RPM I 1640 medium
supplem ented with 10%(v/v) fetal calf serum (Gemini, W oodland CA), 50(iM 2-
m ercaptoethanol (Bio-Rad, Hercules CA), penicillin (lOOU/ml) and streptomycin
(100|ig/m l) (M ediatech), and 2mM glutamine (Invitrogen). They were stimulated
with l[iM gp l2 0 peptide(318-327) for 5 days. A total of 4.4x106 P815 target cells
were incubated with g p l2 0 (3 18-327) or metalloproteinase inhibitor (MPI) peptides
and pulsed with 400|iCi 5 1 Cr-sodium chromate (Perkin Elmer Life Sciences) for 1.5
hours. Effector lymphocytes and target cells were plated in 96-well plates at different
effector: target ratios (E: T Ratios) and incubated for 4 hours at 37 °C, 5% C 0 2. The
5 1 Cr release was measured using a LKB Wallac 1272 Clinigamma automatic gamma
counter (Perkin Elmer Life Sciences).
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104
Cytokine ELISA Assays
lxlO 6 spleen cells/well were plated in 96-well plates in HL-1 proliferation
m edium (BioW hittaker) supplem ented with 2mM glutamine, penicillin (lOOU/ml)
and streptom ycin (100|J,g/ml). Subsequently, they were stim ulated w ith 40|iM
ovalbumin(323-339), 40|iM OVAYT(323-339), g p l2 0 (3 15-329), and m yelin basic
protein (M BP) (89-101) peptides at 37 °C, 5% C 0 2. For the IFN -y ELISA ,
supernatants were collected 72 hours later. Supernatants were frozen in -8 0 °C. For
the IL-2 ELISA , supernatants w ere collected after 24 hours o f stim ulation.
Supernatants were frozen in -8 0 °C. The samples were quantitated using a sandwich
ELISA assay according to the m anufacturer's instructions (BDPharm ingen, San
Diego CA).
Intracellular Cytokine Staining
lxlO 6 spleen cells/well were plated in 24-w ell plates in HL-1 proliferation
m edium (BioW hittaker) supplem ented with 2mM glutamine, penicillin (lOOU/ml)
and streptomycin (100pg/ml). For IL-2 cytokine staining, cells were stimulated with
40|iM ovalbumin(323-339) or gp 120(318-327) overnight in the presence of brefeldin
A (Sigm a-Aldrich) (lO pg/m l). On the day o f the assay cells were fixed and
perm eabilized using Cytofix/Cytoperm solution (BD Pharmingen), follow ed by
staining with PE-conjugated anti-CD4 or anti-CD 8. Cells were permeabilized with
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105
Perm W ash buffer and stained w ith FITC -conjugated anti-IL-2. Sam ples w ere
analyzed at the USC N orris C ancer C enter FACS Core Facility. For IFN -y
staining,cells were stimulated for 3 days with the ovalbumin or gp l2 0 peptide at the
concentration indicated above. The day o f the assay, cells were stim ulated
non-specifically w ith PM A (Sigm a-A ldrich) (200ng/m l), ionom ycin (Sigm a-
A ldrich) (l|iM ), and brefeldin A (10jig/m l) for 6 hours or specifically with 40|i.M
ovalbum in(323-339) or g p l2 0 (3 18-327) in the presence of brefledin A (lOfig/ml).
Cells were stained with PE-conjugated anti-CD4/8 (BDPharmingen) and FITC-
conjugated anti-IFN-y (BDPharmingen).
Cell lines
A20 is a BALB/c B cell lym phom a line (IgG+, Ia+, Fc+ , IgM \ IgA', and
com plement receptor negative) kindly provided by Dr. H. v. Grafenstein (University
of Southern California) (Kim et al. 1979). P815 is a mouse mastocytom a cell line
(Ralph et al. 1974). Both cell lines were grown in RPMI 95%, fetal calf serum 5%
(Omega, Tarzana CA), penicillin (lOOU/ml), streptomycin ( 100p.g/ml), and 2mM
glutamine, 50pM 2-mercaptoethanol (for A20 only). NIH/3T3 is a murine fibroblast
line. BclOM E is a chemically - transformed mouse fibroblast cell line derived from
the norm al BALB/c em bryonic fibroblast line BALB/c CL.7. DO 10.11 T cell
hybridoma is an OVA-Ad -specific T cell receptor (TCR) hybrid kindly provided by
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106
Dr. J. W oodward (University o f K entucky), (M urphy et al. 1990). 293T cell is a
kidney transformed cell line (a kind gift from Bret Ball, Dr F. A nderson Lab). All
four cell lines were grow n in DM EM supplem ented with fetal calf serum 10%,
penicillin (lOOU/ml), streptomycin (100|ig/m l), and 2mM glutamine. S194 cells are
a m ouse m yelom a grow n in RPM I supplem ented with fetal calf serum 10%,
penicillin (lOOU/ml), streptomycin (lOOpg/ml), and 2mM glutamine.
Transfections
DNA plasm ids w ere sterilized using the following protocol. DNA was
suspended in 60|il and 50(0.1 o f chloroform /isoam yl was added. The solution was
vortexed for 30 seconds and then centrifuged for 2 minutes at 12000xg. The top
aqueous phase was removed to be used for transfection. 2 x l0 7 A20 and P815 cell
lines were transfected with 25pg of sterile plasmid DNA (pLacZ, pSLacZ, pRLacZ)
by electroporation using the BIORAD Gene Pulser II Electroporation System at
960|oF, 300mV. After growing for 48 hours, cells were transferred to 24-well tissue
culture plates, and grown in G418 selection (1200pg/ml) (Invitrogen). 10-12 days
later, colonies were harvested and expanded in 6-well plates. These bulk cultures
were screened for P-galactosidase secretion using the LacZ Chemiluminescence
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107
Assay Kit (Roche). Briefly, bulk cultures were plated at a density of 5 x l0 6 cells per
well, in 24-well plates. Cultures were incubated at 37 °C, 5% C 0 2 until they were
confluent. Then, cells were cloned by limiting dilution. 14 days later, 15 colonies
from each transfectant were selected. Secretion of (3-galactosidase was tested using
the LacZ Chem ilum inescence Assay Kit (Roche). Briefly, 15 clones from each
transfectant was grown to confluency and plated at a density of 5x105 cells per well
in 96-well plates. Cultures were incubated at 37 °C, 5% C 0 2. After 24 and 48 hours
supernatants were collected and assays were performed.
For the g p l2 0 secretion assays, 293T, S I 94, BclOM E, P815 cells w ere
transfected with 25pg of sterile plasmid DNA. P815 and S I 94 were transfected by
electroporation (pulsed at 960|iF, 300mV) while 293T and BclOME by C a3 (P 0 4)2
transfection (Strain and W yllie 1984). Briefly, cells were split 1:5 the day before the
transfection. Next day, sterilized DNA (pSA6H and pRA6H) was precipitated and
added to the cells for 12-16 hours, followed by media change and incubation for the
desired amount of time.
P815 cells transfected with SAOLL or RAOLL were collected 48 hours after
transfections seeded in 24-well plates in RPMI-5 + G418(1200pg/ml). Colonies were
selected 10 days later and screened by a CTL assay. Positive clones were cloned by
limiting dilution and used as targets in a CTL assay.
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108
In vitro stimulation o fD O 1 0 .ll by peptide pulsed A20
Briefly, 2 0 x l0 6 A20 cells were irradiated at 3000R. The cells were then
w ashed and resuspended in HL-1 m edium (B ioW hittaker) at 2 x l0 6 cells/m l.
D O 10.11 were suspended in HL-1 medium (BioW hittaker) at 1.5xl06 cells/ml. Cells
were plated in 96-w ell tissue culture plates (2 x l0 5 A20 cells and 7.5xl04 DO 10.11 in
each well) with peptide at 10-fold dilutions (OVA, OVA-M, OVA-YT) for 48 hrs at
37°C, 5% C 0 2. Supernatants were collected and used in an IL-2 sandwich ELISA
according to the manufacturer’s instructions (BD Pharmingen).
Measurement o f gp 120 secretion
Supernatants from P815, S194, BclOME, 293T cells were collected 36 hours
and 4 days after transfection with SA6H or RA6H. IOOjj.1 of the supernatants were
coated onto M etal Chelate Binding 96-well plates (Pierce, Rockland M D) and
incubated for 1 hour at room temperature. Then, I added 100(0.1 of gpl20M N goat
antisera (1:50 dilution) (NIH AIDS Reagent program) and incubated for 1 hour at
room temperature. Subsequently, I added lOOpl of rabbit anti-goat H+L alkaline
phosphatase-conjugated detection antibody (Pierce) and incubated for 1 hour. P-
nitrophenyl phosphate (PNPP) substrate (Pierce) was used according to the
instructions of the manufacturer at lOOpl/well. Plates were developed 1 hour later and
read at 405nm on an Em ax Precision M icroplate Reader (M olecular D evices,
Sunnyvale CA).
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109
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Creator Vatakis, Dimitrios Nikolaos (author)

Core Title Rational design of DNA epitope vaccines

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