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Engineering viral vectors for T-cell immunotherapy and HIV-1 vaccine
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Engineering viral vectors for T-cell immunotherapy and HIV-1 vaccine
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ENGINEERING VIRAL VECTORS FOR T-CELL IMMUNOTHERAPY AND HIV-1
VACCINE
by
Bingbing Dai
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MATERIALS SCIENCE)
May 2012
Copyright 201 Bingbing Dai
ii
Dedication
This thesis is dedicated to my parents and my sister
for their constant support.
iii
Acknowledgements
I have been fortunate to work with talented people. First and foremost I would
like to thank my advisor Dr. Pin Wang, who has supported and guided me though this
process. He has been always there for me when I had difficulty in research and help me to
reach my full potential. I would also like to thank my dissertation committee Dr. Edward
Goo and Dr. Don Arnold. I had the privilege of both being a student and a teaching
assistant for Dr. Goo, he gave me many advices for my learning career. I was lucky to
have Dr. Don Arnold join my committee as the outside member. He is an expert in
cellular and molecular neurobiology; his comments and suggestions help me to finish this
thesis.
I was also lucky enough to collaborate with Dr. David Baltimore, Dr. Lili Yang,
and Jocelyn Kim from Caltech, their insightful comments and discussion have given me
new insight into my research. It is also my pleasure to work with a great team in RTH-
515 at USC. I would like to show my thanks to Haiguang, Leslie, Alex, Kye-Il for
tutoring me when I joined the lab; Paul, April and Steve for critical reading of the
manuscripts; Liang and Biliang for their help in vaccine projects; Steven for the help in
virus-producing cell line projects; and all the other lab members for their support of my
work.
Many thanks to the professors and staffs in the Materials Science & Chemical
Engineering department. Thanks to Dr. Steven Nutt, he provided me with a lot of
valuable information. Thanks to our academic advisor Dr. Edward Goo, he gave me
guidance on schoolwork.
iv
Last but not least, I'd like to thank my parents Songlin Dai and Guiying Li, my
sister Chunchun Dai, and my boyfriend Zuwei Liu. With their love and support, I'm full
of energy to explore the road ahead of me.
v
Table of Contents
Dedication........................................................................................................................... ii
Acknowledgements............................................................................................................iii
List of Tables ...................................................................................................................viii
List of Figures.................................................................................................................... ix
Abstract.............................................................................................................................. xi
Chapter 1: Introduction....................................................................................................... 1
1.1 Gene Therapy and Gene Delivery Vectors .............................................................. 1
1.1.1 Retroviral vector ............................................................................................ 1
1.1.2 Lentiviral vectors ........................................................................................... 3
1.1.3 Adenoviral vectors......................................................................................... 4
1.1.4 Adeno-associated virus vectors (AAV) ......................................................... 6
1.1.5 Nonviral vectors............................................................................................. 7
1.2 Immunotherapy and Vaccines................................................................................ 10
1.2.1 How vaccine works?.................................................................................... 10
1.2.2 Dendritic cells-based vaccine ...................................................................... 13
1.3 Genetic Therapies Against HIV............................................................................. 16
1.3.1 HIV infection ............................................................................................... 17
1.3.2 Challenges of development of AIDS vaccine.............................................. 19
1.3.3 Current Progress in AIDS Vaccine.............................................................. 21
1.4 Our Method for Targeted Gene Delivery to Dendritic Cells ................................. 25
1.4.1 Targeted Delivery of Recombinant Lentiviral Vectors ............................... 26
1.4.2 Engineering Sindbis Envelope Glycoprotein............................................... 27
1.5 Summary and Thesis Work.................................................................................... 29
Chapter 2: In Vitro Differentiation of Adult Bone Marrow Progenitors into
Antigen-Specific CD4 Helper T Cells Using Engineered Stromal Cells
Expressing a Notch Ligand and a MHC Class II protein...................................... 31
2.1 Introduction............................................................................................................ 32
2.2 Materials and Methods........................................................................................... 35
2.2.1 Mice ............................................................................................................. 35
2.2.2 Construct preparation................................................................................... 36
2.2.3 Cell lines ...................................................................................................... 36
2.2.4 Mouse bone marrow stem cells and OP9 cells coculture ............................ 37
2.2.5 Flow cytometry ............................................................................................ 38
2.2.6 T cell stimulation and functional assays ...................................................... 38
vi
2.2.7 IFN-γ and IL-2 ELISA................................................................................. 39
2.2.8 Statistical analysis........................................................................................ 40
2.3 Results.................................................................................................................... 40
2.3.1 Construction of OP9 cells expressing a MHC class II protein .................... 40
2.3.2 Generation of TCR-positive T cells using adult bone marrow progenitors in
OP9 coculture systems.............................................................................. 42
2.3.3 Generation of TCR-specific CD4 T cells in OP9 coculture systems........... 43
2.3.4 Functional Analysis of TCR-specific CD4 T cells generated by OP9
coculture.................................................................................................... 50
2.4 Discussion .............................................................................................................. 53
2.5 Acknowledgements................................................................................................ 58
Chapter 3: HIV-1 Gag-specific immunity induced by a lentivector-based vaccine
directed to dendritic cells...................................................................................... 59
3.1 Introduction............................................................................................................ 60
3.2 Materials and Methods........................................................................................... 62
3.2.1 Mice and vaccination schedule .................................................................... 62
3.2.2 Plasmid construction and lentivector production......................................... 63
3.2.3 Peptides and peptide pools........................................................................... 63
3.2.4 MHC class I tetramer staining and phenotypic analysis.............................. 64
3.2.5 Intracellular cytokine staining (ICCS) and multicolor ICS ......................... 64
3.2.6 ELISAs......................................................................................................... 65
3.2.7 Gamma interferon (IFN-γ) ELISpot assay................................................... 66
3.3 Results.................................................................................................................... 67
3.3.1 Immune Responses Generated by Various Routes of Vaccine
Administration .......................................................................................... 67
3.3.2 Enhanced Gag-Specific Immunity by Prime/Boost Regimens.................... 69
3.3.3 Comparison of T-Cell Responses Elicited by Lentivector and Adenovector
................................................................................................................... 71
3.3.4 Multifunctional CD4
+
and CD8
+
T-Cell Responses Elicited by Lentivector
................................................................................................................... 73
3.3.5 Breadth of T-Cell Responses Induced by Various Immunization Regimens
................................................................................................................... 76
3.4 Discussion .............................................................................................................. 78
3.5 Acknowledgements................................................................................................ 82
Chapter 4: PD-1/PD-L1 Blockade Can Enhance HIV-1 Gag-specific T Cell
Immunity Elicited by Dendritic Cell-Directed Lentiviral Vaccines..................... 83
4.1 Introduction............................................................................................................ 84
4.2 Materials and Methods........................................................................................... 87
4.2.1 Mice and vaccination procedure .................................................................. 87
4.2.2 Lentiviral vector production ........................................................................ 88
4.2.3 Gag peptide and peptide pools..................................................................... 88
4.2.4 Tetramer staining ......................................................................................... 89
vii
4.2.5 Intracellular cytokine staining...................................................................... 89
4.2.6 Multiparameter intracellular cytokine staining............................................ 90
4.2.7 IFN-γ enzyme-linked immunospot assay..................................................... 91
4.2.8 Vaccinia virus and challenge of mice .......................................................... 91
4.3 Results.................................................................................................................... 92
4.3.1 PD-1 blockade in combination with DCLV immunization increases
frequency of Gag-specific CD8
+
T cells................................................... 92
4.3.2 PD-L1 blockade enhances DCLV-based booster immunization for
generating Gag-specific T cell immunity ................................................. 94
4.3.3 DCLV prime/boost regimen combined with PD-L1 blockade elicits
multifunctional CD8
+
T cell responses ..................................................... 97
4.3.4 DCLV-based prime/boost vaccination combined with PD-L1 blockade
induces long-term immunity..................................................................... 99
4.3.5 DCLV-based prime/boost vaccination combined with PD-L1 blockade
induces broad HIV-1 Gag-specific T cell responses............................... 101
4.3.6 DCLV-based vaccination combined with PD-1 blockade provides better
protection against vaccinia virus challenge ............................................ 103
4.4 Discussion ............................................................................................................ 104
4.5 Acknowledgements.............................................................................................. 109
References....................................................................................................................... 110
viii
List of Tables
Table 1.1: Comparison of properties of gene therapy vectors............................................ 9
Table 1.2: HIV vaccines advancing in human trials......................................................... 25
ix
List of Figures
Figure 1.1: How vaccines work. ....................................................................................... 12
Figure 1.2: The proposed entry mechanism two-component retroviral targeting. ........... 27
Figure 1.3: A schematic representation of the general strategy to engineer a
lentivector system capable of targeting dendritic cells......................................... 29
Figure 2.1: Construction of OP9 stromal cells to express murine MHC class II
protein (I-A
b
) by retroviral transduction............................................................... 41
Figure 2.2: Retroviral transduction of adult bone marrow cells to express OT2
CD4
+
T cell receptor (TCR).................................................................................. 44
Figure 2.3: Coculture of OT2 TCR-transduced bone marrow cells with various
OP9 cells (OP9-MIG, OP9-IA
b
, OP9-DL1, and OP9-DL1-IA
b
).......................... 46
Figure 2.4: Development of CD4
+
T cells from TCR-transduced bone marrow
cells cocultured with either OP9-DL1 or OP9-DL1-IA
b
cells.............................. 49
Figure 2.5: Functional analysis of antigen-specific CD4 T cells generated from
TCR-transduced bone marrow cells cocultured with either OP9-DL1 or
OP9-DL1-IA
b
cells................................................................................................ 52
Figure 3.1: Comparison of immune responses generated from different injection
routes after a single immunization........................................................................ 68
Figure 3.2: DC-directed LV can effectively boost HIV-1 Gag-specific immune
response................................................................................................................. 70
Figure 3.3: Comparison of magnitude, kinetics and memory responses of Gag-
specific CD8
+
T cells after immunization with LV-Gag and rAd5-Gag.. ............ 72
Figure 3.4: Generation of multifunctional CD4
+
and CD8
+
responses by
prime/boost immunization regimens..................................................................... 75
Figure 3.5: Breadth of HIV-1 Gag-specific responses to LV-Gag-based
vaccination. ........................................................................................................... 77
Figure 4.1 In vivo PD-L1 blockade in combination with DCLV immunization
increased frequency of HIV-1 Gag-specific CD8 T cells..................................... 93
x
Figure 4.2: DCLV immunization combined with PD-1 blockade efficiently
boosted primary Gag-specific T cell immunity. ................................................... 96
Figure 4.3: Multifunctionality of CD8
+
T cell immune responses induced by
DCLV prime/DCLV boost in combination with PD-1 blockade.......................... 98
Figure 4.4: DCLV prime/DCLV boost in combination with PD-1 blockade
generated prolonged immunity. .......................................................................... 100
Figure 4.5: DCLV prime/DCLV boost in combination with PD-1 blockade
broadened the profile of vaccine-specific T cell responses. ............................... 102
Figure 4.6: Immunization delivered by DCLV prime/DCLV boost in combination
with PD-1 blockade could protect animals from recombinant vaccinia
virus challenge. ................................................................................................... 104
xi
Abstract
T cell immunotherapy fell into two categories: passive (adoptive) transfer of in
vitro expanded cells, and active expansion of antigen-specific T cells by in vivo
immunization. I present three studies to promote T cell immunotherapy and T cell
vaccine. In my first study, I described a method to generate autologous antigen-specific
CD4
+
helper T cells in vitro from easily accessible bone marrow cells. T lymphocytes are
produced in thymus as the progeny of fetal liver (FL)- and bone marrow (BM)- derived
precursors. A murine stromal cell line (OP9-DL1) expressing a notch ligand, Delta-like-1,
has been shown to partially mimic the function of thymus and to drive the differentiation
of both murine and human hematopoietic progenitors into T cells in vitro. Next, I attempt
to develop a specific T-cell vaccine by in vivo gene delivery Human immunodeficiency
virus-1 (HIV-1) is one of the most catastrophic pandemics confronted by mankind with
33 million infections, and there is an urgent need for an effective vaccine. I choose
lentiviral vector as the vaccine carrier as it is among the most efficient gene delivery
machinery, and can infect both dividing and nondividing cells. In my second study, I
evaluate in mice a dendritic cell (DC)-directed LV system encoding the Gag protein of
human immunodeficiency virus (LV-Gag) as a potential vaccine for inducing an anti-
HIV immune response. The DC-directed specificity is achieved through pseudotyping the
vector with an engineered Sindbis virus glycoprotein capable of selectively binding to the
DC-SIGN protein. To further optimize this T-cell vaccine system to achieve an increased
immune response, in my third study, I attempt to break down the suppressive signaling
pathways involved in T cell function. It was found that exhaustion of CD8
+
T cells and
xii
upregulation of programmed death 1 (PD-1), a negative regulator of T cell activation, is a
characteristic feature of individuals chronically infected with HIV-1. In this project, I
demonstrate that blocking of the PD-1/PD-L1 inhibitory signal via an anti-PD-L1
antibody (αPD-L1) generated an enhanced HIV-1 Gag-specific CD8+ immune response
following a both a single round of DC-targeting LV immunization and a homologous
prime/boost regimen.
1
Chapter 1: Introduction
1.1 Gene Therapy and Gene Delivery Vectors
The concept of gene therapy is to introduce a corrective genetic material into
cells, the product of which alleviates or slows down the progression of disease (Verma
and Somia 1997). The first clinical trial of gene therapy to combat disease was carried out
in 1990, and since then more than hundreds of clinical trials are underway worldwide, the
success of gene therapy is largely dependent on the development of gene delivery vectors
(Verma and Somia 1997). An ideal gene delivery carrier should comprise a few features:
(i) easy of production; (ii) high and sustained transduction; (iii) ability to target specific
tissues or cell types; (iv) large size capacity which wouldn’t limit the genetic materials to
be delivered; (v) immunologically inert so that it won’t induce immune response against
vector components; (vi) ability of site-specific integration or to be maintained as a stable
episome (Verma and Somia 1997; Somia and Verma 2000). There are two broad
categories of vectors available to date: viral vectors and non-viral vectors.
1.1.1 Retroviral vector
Retrovirus is among the first viral vectors used in a gene therapy trial. In 1990,
Anderson used a retroviral vector to ex vivo transfer adenosine deaminase (ADA) gene to
T lymphocytes of patient with severe combined immunodeficiency (SCID) (Anderson
and Hope 2005). Retroviruses are enveloped virus containing two identical strands of
RNA, and have a transgene capacity of 7-7.5 kb. The genome consists of three essential
2
genes: gag encodes viral structure proteins (matrix, capsid and nucleocapsid protein), pol
contains enzymatic proteins (reverse transcriptase and integrase), and env stands for viral
envelope glycoprotein. The RNA genome is flanked by long terminal repeats (LTRs),
which define the beginning and end of the viral genome, regulate integration into host
genome, and control viral genes expression (Somia and Verma 2000; Sinn, Sauter et al.
2005). Retroviruses infect targeted cells by first binding themselves to the host cells,
followed by either an endocytosis with a conformation change of the endosome, or a
direct membrane fusion to allow for capsid release (Anderson and Hope 2005). The viral
RNA is then reverse transcribed into double-stranded DNA, and forms a pre-integration
complex with integrase, which was then transported into nucleus. Newly synthesized
DNA randomly integrates into the host cell genome. For better utilization of retroviral
vectors, the env protein can be replaced for modified target specificity (Anderson and
Hope 2005; Sinn, Sauter et al. 2005). Tissue-specific and inducible promoters are
employed for enhanced transduction efficiency.
The main limitation of retroviral vectors is their inability to infect non-dividing
cells, such as those that make up muscle, brain, eye, and lung tissues (Verma and Somia
1997). The other problem is that random integration may activate some oncogenes, or
inactivate tumor suppressive genes. Despite that, retroviral vectors remain one of the
most promising tools for introducing genes into dividing cells, such as tumor cells, and
hematopoietic cells.
3
1.1.2 Lentiviral vectors
Lentivirus belongs to the family of retroviridae, which is a class of single-
stranded RNA (ssRNA) viruses. They replicate through a double-strand DNA
intermediate, and integrate transgene into host cell genome. Some valuable features of
lentiviral vectors include their large transgene capacity (up to ~8kb), ability to transduce
both dividing and non-dividing cells, efficient and sustained gene delivery, easy to be
pseudotyped and lack of pre-existing anti-vector immunity (Verma and Somia 1997;
Cronin, Zhang et al. 2005). The most commonly used lentiviral vector for gene therapy is
derived from HIV-1 backbone, therefore some safety concerns arise, such as potential
generation of replication competent vectors due to recombination with wide-type HIV
virus or during viral production.
To eliminate safety problems, some accessory virulent proteins (vif, vpr, vpu, nef)
are deleted due to their cytotoxic or cytostatic activities. Further improvement in safety of
the vector is achieved by splitting the package components, where rev gene is expressed
in trans from separate plasmids (Sinn, Sauter et al. 2005). The transfer plasmids for
lentivirus production consist of four components: (1) an expression cassette containing
the gene of interest, (2) the HIV cis-acting factors essential for packaging, reverse
transcription, and integration, (3) rev protein that reacts with the rev responsive elements
(RRE) to help the nuclear export of unspliced gag-pol mRNAs (Delenda 2004), and (4) a
heterologous envelope (in most case, the glycoprotein from other viruses, such as
VSVG). Another benefit of the third-generation lentiviral vector is the development of
self-inactivating (SIN) vector (Sinn, Sauter et al. 2005; Breckpot, Aerts et al. 2007). The
4
transcriptional activation unit of the U3 region of the 3’-LTR has been deleted to
eliminate promoter interference with targeted cells, risk of emergence of replication
competent lentivirus (RCL), and potential oncogenic derivations. In addition, some other
modifications have been made to optimize the lentiviral vector to provide higher vector
production titer, and better transgene expression. Engagement of central polypurine tract
(cPPT) and its central termination sequence represents enhanced transduction efficiency
by improving nucleus import of pre-integration complex (Sinn, Sauter et al. 2005).
Addition of post-transcriptional regulatory element of woodchuck hepatitis B (WPRE)
cis-acting sequence improves gene expression probably by regulating polyadenylation,
nuclear export or translation (Delenda 2004; Sinn, Sauter et al. 2005). The shortcomings
of lentiviral vectors are the instability of viral particles, low virus titer, and difficulty in
scale-up production. Given all of the advantages, lentiviral vector plays a vital role in gene delivery
and immunotherapy. It is a promising vaccine vector for infectious disease due to the
strong Cytotoxic T Lymphocytes (CTL) response, and allows repeated administration.
Direct delivery of lentiviral vector encoding tumor-associated antigens (TAA) to
dendritic cells is capable of generating antitumor response. 1.1.3 Adenoviral vectors
Adenoviruses (Ad) are non-enveloped double-stranded DNA virus with a genome
size of ~30-40 kb. The viral genome flanked by inverted terminal repeats (ITR,
responsible for DNA replication), and contains over a dozen units for RNA polymerase
5
II-mediated transcription (Sinn, Sauter et al. 2005). Most vectors used as vaccine carriers
are deleted in E1, which renders the vector replication-defective, as well as prompts
antigen presentation. Further deletions (E2A, E3, E4) are made to increase the permitted
size of inserted gene (Tatsis and Ertl 2004). Nowadays, “gutless” adenoviral vectors, in
which all of the viral genes are removed, have been developed. HEK 293 cells, which
supply the deleted transcription units in trans, are most commonly used for propagation
of adenoviral vector (Tatsis and Ertl 2004; Lasaro and Ertl 2009). The adenoviral capsid is composed of the hexon, penton base, and fibre proteins.
Most human serotype adenovirus binds its fibre to the coxsackie virus B adenovirus
receptor (CAR) (Meier and Greber 2004), which is expressed on a variety of cell types,
including respiratory endothelial cells, epithelial cells, hepatocytes, myoblasts, and heart
muscle cells. However, DC infection with adenovirus is suggested to be mediate through
a region of fibre shaft that recognizes a heparin-sensitive receptor on DC (Tatsis and Ertl
2004). Adenovirus has a broad tropism, and can efficiently transduce both dividing and
non-dividing cells (Lasaro and Ertl 2009). Engineering the fibre knob and utilizing
bispecific bridge are ways to alter the tropism. There are a few limitations of adenoviral vectors. Preexisting immunity and high
titer of neutralizing antibody caused by natural infections is the main hurdle for effective
usage of adenovirus as vaccine vector (Tatsis and Ertl 2004). Most of the neutralizing
antibodies to Ad are directed against the hexon loops, which is serotype-specific. This
may be overcome by increasing the dose of the vector, or combining with other serotypes
of adenovirus (or other types of vectors) in prime-boost regimens (Tatsis and Ertl 2004).
6
Besides that, the duration of transgene expression is usually short (~5-20 days post
infection). However, adenoviral vectors are still the most widely used for clinical trials.
Most vaccine carriers are based on AdHu5 virus. The Merck “STEP” HIV vaccine trial
involved a rAd5 vector carrying gag, pol, and nef genes (Tatsis and Ertl 2004). Although
showed potent immunity in preclinical models, the “STEP” phase II trial was stopped due
to lack of efficacy in human. Moreover, adenoviral vectors have been developed for
cancers and degenerative diseases, such as influenza and hepatitis viruses.
1.1.4 Adeno-associated virus vectors (AAV)
Adeno-associated virus (AAV) is a member of the Parvoviridae family, which is
a small, non-pathogenic, single-stranded DNA virus (Somia and Verma 2000). AAV
genome is composed of two large open reading frames (ORF) flanked by inverted
terminal repeats (Somia and Verma 2000). It contains only two essential genes: cap that
encodes viral structure proteins; and rep that is involved in replication and integration.
AAV needs additional genes provided by a helper virus (such as adenovirus or herpes
simplex virus) for productive infection and replication. AAV has a broad tropism, they
infect most cell types including muscle, liver, lung, retina, and brain cells. AAV can
integrate its viral DNA into a site-specific location on human chromosome 19 by virtue
of rep protein (Campbell and Hope 2005). However, recombinant AAV (rAAV) are
generated by replace the rep and cap gene with therapeutic genes, and they are usually
integration deficient.
7
The disadvantages of AAV vectors are their small coding capacity (~ 4.5kb), and
the laborious scale-up procedure for vector production. The rep gene and some helper
proteins are cytotoxic to packaging cell lines, and vector preparation requires removal of
contaminating helper viruses (Campbell and Hope 2005). The packaging capacity of
AAV can be extended by co-delivering two separate rAAV vectors to form head-to-tail
concatemers (Verma and Somia 1997; Somia and Verma 2000). Notwithstanding the
drawbacks, AAVs maintain long-term gene expression, and are relatively stable against
heat and low PH. AAV vector is among the most frequently used viral vectors for gene
therapy, over 13 protocols have been approved for human clinical trials. The most
commonly used serotype is AAV2. AAV has shown promising results in curing
hemophilia B and Parkinson’s disease.
1.1.5 Nonviral vectors
Nonviral vectors are attractive gene delivery carriers due to their simplicity and
safety to use. Low toxicity, lack of pathogenicity, and ease of pharmacologic production
are advantages of nonviral vectors over viral vectors (Verma and Somia 1997; Niidome
and Huang 2002). However, nonviral vectors continue to suffer from the low efficiency
of gene transfer (Medina-Kauwe, Xie et al. 2005). To overcome the barriers, efforts have
been made to improve cell surface binding, plasma membrane traversing, endosomal
escaping, and nuclear entry. Naked DNA Direct injection of naked plasmid DNA into the tissue is the simplest and
safest way for gene delivery. Direct injections have been tested in skeletal muscle, liver,
8
heart muscle, skin and tumor (Somia and Verma 2000). However, direct injection gives
to low gene expression level due to DNA degradation by nucleases in the serum, and
clearance by the mononuclear phagocyte system. To address the problem, physical
manipulations, such as electroporation, gene gun, ultrasound, hydrodynamics, have been
employed to improve the efficiency (Niidome and Huang 2002). DNA vaccination has
been used in many occasions, including hepatitis B and HIV. It usually requires repeated
administrations.
Lipoplexes Lipoplexes refer to cationic lipid-DNA complex. Cationic liposomes are
microscopic bubbles of amphiphilic lipid that form an overall positively charged complex
with negatively charged DNA (Medina-Kauwe, Xie et al. 2005). This positively charged
complexes overcome electrostatic barrier faced by naked DNA to enter negatively
charged biological cells surface and get endocytosed (Niidome and Huang 2002). The
cellular uptake process is believed to involve both endocytosis and membrane fusion
mechanisms. Endosomal escape is facilitated with the help of pH-sensitive fusiogenic
lipid (such as dioleoylphosphotidylethanolamine, DOPE), which undergoes a
conformation transition at an acid pH 5 to 6 to destabilize the endosomal membrane, and
promotes the release of its content (Khalil, Kogure et al. 2006). Nuclear envelope is
believed to be a significant barrier for nuclear delivery of DNA, and cell division is an
important factor for transgene translocation.
Polyplexes Cationic polymer-DNA complexes are named polyplexes. Biodegradable
polymers with high biocompatibility are of great interest. The most commonly used
polymer is named polyethylenimine (PEI) and poly L-lysine (PLL). PEI has been used to
9
deliver oligonucleotides, plasmid DNA, RNA, and intact robozymes (Verma and Somia
1997). Receptor-mediated endocytosis of polyplexes can be accomplished by labeling the
complex with targeting moieties. The liberation of polyplexes from endosomes is thought
to take place through membrane disruption and a “proton sponge” effect, in which
imidazole protonation happens during endosome acidification (Khalil, Kogure et al.
2006). Thermosensitive polymers, which undergo solution-to-gel transition in response to
temperature, and ensure sustaining release of encapsulated DNA, have been developed
recently. A hydrophilic copolymer poly (D, L-lactic acid-co-glycolic acid) (PLGA)-PEG
transforms from aqueous phase (4-20°C) to gel phase (body temperature) after injection,
and shows slow release and prolonged transfection (Niidome and Huang 2002).
Table 1.1: Comparison of properties of gene therapy vectors (Nature 389: 239-242, 1997)
10
1.2 Immunotherapy and Vaccines
The ultimate goal of a vaccine is to develop long-lived memory response, which
will provide immunological protection to the host to prevent infection or greatly reduce
the severity of disease. Designing of a vaccine is mainly based on the biology of the
pathogen, the nature of the disease and the cell population it targets (Berzofsky, Ahlers et
al. 2001). An ideal vaccine for optimal control of pathogens should be able to induce both
the humoral and cellular immune response. There are a few factors need to be taken into
consideration for the development of a vaccine: (i) Choice of antigen; (ii) T helper subset
induced by the vaccine; (iii) Whether or not to induce the CTL; (iv) Which kind of
memory subset is needed? (v) Avoidance of downregulatory signaling events (such as
Treg cells); (vi) Selection of adjuvants to induce best vaccine-specific response; (vii)
Choice of vector systems and routes of vaccination for vaccine delivery; (viii)
Optimization of conditions for clinical trials (safety, doses, etc.) (Berzofsky, Ahlers et al.
2001; Hoft, Brusic et al. 2011). Nowadays, the better understanding of viral biology
facilitates the development in use of viral vectors as vaccine carriers.
1.2.1 How vaccine works?
Vaccine modalities generate specific memory immune response, which is capable
of expanding. The two important types of immune cells induced by memory are B cells
and T cells. B cells are capable to produce neutralizing antibody, which is the main
gatekeeper of adaptive immune. The neutralizing antibodies bind to free viral particles to
11
prevent their entry into host cells, or to eliminate infected cells via host effector system
(Robinson 2007). It takes up to 4 months for naïve B cells to undergo expansion and
differentiation to become memory. Upon stimulation with vaccine vectors, B cells
actively mutated the Ig receptors to compete for antigen recognition and survival in the
germinal center of lymph nodes (Walker and Burton 2008). After maturation, they obtain
the ability to produce high-affinity antibodies, to develop memory cells that traffic
through lymphoid tissues, and to become plasma cells which head to bone marrow or
mucosal surface for long-term antibodies production.
T cells build the second line of defense, cellular immunity. Vaccine strategies
generated a population of virus-specific memory T cells, which undergoes rapid
expansion within the first few days encountering reinfection. CD8
+
T cells, which are
also termed “CTLs”, recognize viral proteins displayed on surface of infected cells via
major histocompatibility complex (MHC) molecules (in mice) or human leukocyte
antigen (HLA) class I molecules (in human), and kill the infected cells through cytolytic
or apoptosis mechanisms mediated by lysis and antiviral cytokines. CD4
+
T cells provide
growth and differentiations factors for CD8
+
T cells and B cells, and are thus called
“helper T cells” (Walker and Burton 2008). Vaccines promote clonal expansion and
differentiation of naive cells that recognize the epitope in the antigens, and generate
antigen-specific CTLs. It takes around 6 weeks to generate a memory T cell response
(Robinson 2007). The memory response usually lasts for years due to its homeostatic
proliferation potential, and the establishment of antigen depots in lymph nodes (Robinson
2007). There are two types of memory T cells. Effector memory T cells patrol the tissues
12
and are responsible for immediate responses to clear targeted infection, while central
memory T cells are sent to reserves (Kaech, Wherry et al. 2001). The ultimate of a “T-
cell” vaccine is to present the antigen to the immune system in a way that can generate
long-lived effector and central memory responses (Hoft, Brusic et al. 2011).
The licensed vaccines for use in humans include live-attenuated viruses (such as
vaccines for yellow fever, measles, mumps, varicella), whole inactivated viruses (such as
the Salk polio vaccine influenza vaccine, and hepatitis A vaccine), and recombinant
subunit proteins (hepatitis B and papilloma vaccines).
Figure 1.1: How vaccines work. The schematic shows the expansion and contraction of
responding immune cells to a vaccine (green shaded area) and then to a challenge
infection (red shaded area). The primary response to the vaccine requires several weeks,
whereas the anamnestic response to the challenge occurs within days. Both the vaccine
and challenge responses contract when the vaccinating antigens or challenge microbes
are cleared by the immune response. Memory responses can last for years due to their
potential for homeostatic proliferation. (Nature 8: 686-693, 2007)
13
1.2.2 Dendritic cells-based vaccine
Dendritic cells (DC) are potent antigen presenting cells that initiate and modulate
adaptive immune response against microbial, tumor, and self-antigens. They play a
pivotal role in mounting T-cell immunity, and are crucial for the therapeutic treatment of
cancer, autoimmunity, and chronic viral infections (Gilboa 2007; Palucka, Ueno et al.
2007). A desirable DC vaccine is expected to generate large number of high-avidity CTL,
and to suppress regulatory T cells.
DCs reside in peripheral tissues and lymphoid organs where they identify and
capture antigens via different receptors [receptors for immunoglobulin (FcR), DEC-205,
DC specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)]
(Tacken, Vries et al. 2007). After antigen uptake, DCs migrate to draining lymph nodes
and undergo maturation. There are two pathways that have been well studied for antigen
presentation. Endogenous antigens (protein from virus or self antigens synthesized within
the cytosol of APCs) are broken down into peptides by proteasome, and are transported
to the endoplasmic reticulum (ER), where they are loaded onto major histocompatibility
complex (MHC) class I molecules. The loaded MHC class I molecules will be
transported to cell surface and presented to CD8
+
T cells (Gilboa 2007; Tacken, Vries et
al. 2007). The other mechanism is named MHC class II pathway in which the exogenous
antigens remain in the endosome and fuse with protease-containing lysosomes. The
antigens are degraded into smaller peptides, followed by fusing with MHC class II
molecules, and are presented to CD4
+
T cells (Bhardwaj 2001). Maturation of DCs
requires appropriate costimulatory signals, such as microbe, bacteria, virus, inflammatory
14
cytokines and T-cell derived signals (CD40L), and cell products (tumor cell lysates)
(Tacken, Vries et al. 2007). DC activation upregulate MHC and costimulatory molecules
(CD86, CD40), express migration chemokine receptors (CCR7), and synthesize essential
cytokines (IL12, IL15 and IL18), which results in development and proliferation of
helper and effector T cells (Bhardwaj 2001). On the other hand, DC maturation is also
accompanied by development of resistance to immunosuppressive factors (IL10), and
generation of regulatory T cells (Tregs).
Delivering vaccines to DCs can be achieved by ex vivo loading or in vivo
targeting. Ex vivo generation of DCs involves culturing CD34
+
hematopoietic
progenitors, or peripheral blood-derived monocytes (PBMC) primed with granulocyte
macrophage colony stimulating factor (GM-CSF) and IL4 (Figdor, Vries et al. 2004;
Tacken, Vries et al. 2007). Although the early studies involved immature DCs,
maturation and activation stimuli is thought to be essential to promote their migration to
nodal T-cell area, and to avoid induction of tolerance instead of immunity (Gilboa 2007).
The signals that can activate DCs include (i) Toll like receptor (TLR) agonists (LPS,
polyinosinic-polycytidylic acid [polyI:C], oligodeoxynucleotide [CpG]); (ii) pro-
inflammatory cytokines (TNF, IL-1β, IL-6); (iii) costimulatory molecules (CD40L,
OX40, 4-1BB) that are cross-linked ligands on DCs to regulate T cells survival and
proliferation (Figdor, Vries et al. 2004). In vivo targeting of DCs represents an ideal
scenario in which the patient’s own DCs can be loaded with antigens and activated. The
receptors that are frequently used for DC targeting include the mannose receptor, CD205,
and DC-SIGN (Tacken, Vries et al. 2007). Studies in which DC-specific antibodies are
15
fused with tumor-derived antigens and are delivered in conjunction with chosen
adjuvants that activate particular TLRs, appealed potential for design of anticancer
vaccines. The advantages of ex vivo methods are that the maturation and activation status
can be well controlled, and only the ex vivo generated DCs will be delivered. However,
this protocol requires labor-intensive procedure, has limitation in DC subsets in regarding
to isolation and culture condition, and arises poor distribution of DCs. In comparison, the
in vivo approach involves single product, which is easy to produce in bulk quantities. The
antigen is delivered in natural environment to multiple DC subsets, and at multiple sites.
However, there is difficulty in achieving high specificity because most receptors are not
single cell type specific.
Numerous approaches have been developed for DC-based vaccination. The
“classic” vaccines involve antigens with or without adjuvants, viral vectors and DNA
vaccines. They target DCs randomly, and might bring some “unwanted” types of immune
response. The ex vivo generated DCs can be loaded with tumor antigens, educated to
generate appropriate cytotoxic or helper T cells, and be injected back into patients
(Palucka, Ueno et al. 2007). One can also generate targeting vaccines directed to specific
DC subsets by fusing anti-DC antibodies with DC activators. As DCs express multiple
molecules from the B7 family, including both stimulatory and inhibitory signals, the next
generation of DC vaccines dedicate to block the regulatory (Treg cells) and suppressive
pathways (CTLA4, PD1), and to breakdown the tumor environment (IL-10, IL-13, TGF-
β) (Palucka, Banchereau et al. 2010).
16
To establish a more potent DC based vaccination protocol, there are a few
parameters need to be taken into consideration. Targeting distinct DC subsets may lead
to improved vaccine (Palucka, Banchereau et al. 2010). It is postulated that humoral
immunity is managed by CD14
+
dermal DCs, while cellular immunity is preferentially
regulated by epidermal Langerhans cells (LCs). Another concern is the forms of antigens
that are loaded onto DCs. The antigens can be provided in exogenous form, such as
peptides, while proteins, tumor lysate, and apoptotic debris; or they can be synthesized
endogenously by transfecting DCs with mRNA or cDNA. The choice of antigens is more
challenge for cancer therapy, with mutated antigens and self antigens inducing immunity
and autoimmunity (breakdown tolerance) respectively (Palucka, Banchereau et al. 2010).
The choice of antigens depends on the types of immune response we want to induce.
Moreover, defining appropriate DC activation signals, and administering them at the right
time, frequency and route are also important as they lead to differentiating DCs with
different functions.
1.3 Genetic Therapies Against HIV Human immunodeficiency virus-1 (HIV-1) is one of the most catastrophic
pandemics confronted by mankind with more than 6500 new infections daily. The highest
prevalence of infection worldwide is found in South Africa, Switzerland, and Asia
(Fauci, Johnston et al. 2008). Since the discovery and characterization of HIV-1 as a
virus that attacks the immune system in the early 1980s, some successes have been
gained for the treatment of HIV-1 infection. However, there is still no safe and effective
17
vaccine that provides sterilizing immunity. Antiretroviral therapies (ARTs) and highly
active antiretroviral therapies (HAARTs) are the main drug treatments of disease control
during last 15 years (Munier, Andersen et al. 2011). However, the high expense and
limited availability restrict only 28% being able to receive drugs. Gene therapy strategies
against HIV are expanding to interfere with viral replication by targeting HIV genes and
their products, or to provide T-cell based therapies by adoptive transferring of gene-
modified T cells, engineering T cell receptors (TCR) and lentiviral gene delivery
(Munier, Andersen et al. 2011). To better control virus spread worldwide, there is a
pressing need for the development of an effective vaccine. 1.3.1 HIV infection
Since the identification of HIV-1 as the causative agent of the acquired
immunodeficiency syndrome (AIDS) in 1983, over 33 million have been living with HIV
and 25 million deaths have been reported worldwide. HIV transmits though genital tract
or the rectal mucosa via sexual activity or exposure to contaminated blood, infected
women can transmit virus to their offspring (Rossi, June et al. 2007). HIV penetration
occurs across the vaginal, ectocervical and endocervical mucosa, with endocervical more
vulnerable, as it is composed of an intact single layer of columnar epithelium. The
mechanisms by which HIV transverse the intact barrier is unclear yet. It is suggested that
transcytosis by a vesicular pathway, direct contact with dendrites of DCs, or direct
infection of intraepithelial lymphocytes are some possibilities (Gamble and Matthews
18
2011). Once crossing the epithelial barrier, HIV-1 mainly targets the CD4
+
T cells, DCs
and macrophages in the underlying mucosal tissue. DCs expressing CD4, CCR5, DC-
SIGN and other C-type lectin receptors (CLRs) facilitate viral capture and cell entry.
Once captured by DCs, the virus can be internalized and then transmitted to nearby CD4
+
T cells (Gamble and Matthews 2011). This “infectious synapse” through DC-T
conjugates rapidly spread infection to more CD4
+
T cells, especially the memory subset,
which is the main virus-producer in vivo (Munier, Andersen et al. 2011). Two to three
weeks following mucosal transmission, the viruses are well established in the lymphatic
tissue reservoirs (Pope and Haase 2003). From the acute stage of HIV infection, billions
of virions are produced daily, and around 10-100 million CD4
+
T cells die every day in a
dynamic process.
Env is the virus surface protein, which mediates virus entry into target cells. There
are two co-receptors involved in HIV infection, CCR5 and CXCR4 with CCR5 more
preferentially used (Haut and Ertl 2009). The Env glycoprotein spikes are comprised of
three identical gp160 molecules, and form trimeric structures. Each gp160 molecule
includes a surface gp120 molecule and a transmembrane gp41 protein. Following
attachment of its conserved CD4 binding sites to the targeted cells, the gp120 molecule
undergoes conformation change, which results in the exposure of the high affinity co-
receptor binding site within the V3 loop and the release of gp41 (Munier, Andersen et al.
2011). The gp41 molecule rearranges its structure within the trimer and activates the
fusogenic machinery, which allows the viral and host cell membrane fuse together, and
facilitates viral entry (Dimitrov 2004).
19
1.3.2 Challenges of development of AIDS vaccine
The main problem of developing AIDS vaccine is the high variability of HIV
pandemic. HIV has a potent capacity for mutation and adaptation due to the error-prone
nature of reverse transcriptation process, the high propensity of recombination, and its
rapid turnover in vivo (Richman, Margolis et al. 2009). This leads to generation of
mutants that will escape the capture of neutralizing antibodies and virus-specific CTLs.
The spikes, which is composed of the gp41 transmembrane protein and the heavily
glycosylated gp120 surface protein (Env), are the sole target for antibodies (Robinson
2007). However, the Env protein is extraordinary variable, and undergoes structure
change upon receptor binding (Fauci, Johnston et al. 2008). Moreover, the conserved
targets are masked by loops of variable sequences and sugars to avoid access of
antibodies. The spike is an unstable structure, and is hardly generated in recombinant
form (Richman, Margolis et al. 2009). Therefore, there are tremendous difficulties in
developing immunogens to induce broadly neutralizing antibodies against various HIV
isolates.
Latency is a characterize feature of HIV. Immediately following the infection,
HIV establishes a latent reservoir of infected lymphocytes that produce small amount of
viral RNA sporadically, yet do not display activation markers (Richman, Margolis et al.
2009). This is an irreversible process, and the long-lived lymphocytes may be activated at
a later time and generates a livelong threaten. Clinical data shows that 1 in 10
6
CD4
+
T
cells undergoes latent infection from HIV. Several factors have been proposed related
with the mechanism that drives latency. Histone deacetylases (HDACs) are recruited to
20
the HIV-1 LTR promotor, and make modification within chromatin of provial promotor,
which limits the ability of RNA polymerase to initiate transcription (Richman, Margolis
et al. 2009). Inadequate nuclear levels of nuclear factor κB (NF-κB) and nuclear factor of
activated T cells (NFAT) in resting CD4
+
T cells impede robust transcription (Richman,
Margolis et al. 2009). In addition, inefficient HIV mRNA export (Tat) due to low levels
of polypyrimidine tract-binding protein (PTB), and impediment in HIV mRNA
translation resulted from cellular microRNAs (miRNAs) contribute to the establishment
of latency (Richman, Margolis et al. 2009). HIV latency imposes a tremendous challenge
for effective therapy.
Unlike other viruses, HIV directly infects immune cells, such as CD4
+
cells and
dendritic cells. The loss of CD4
+
T cells in the gut-associated lymphoid tissues within the
first few days of exposure leads to severe impairment of immune system. The break of
the mucosa barrier results in translocation of bacterial, which facilitates CD4
+
T cells
activation and virus replication (Walker and Burton 2008). Another hurdle is that HIV
has evolved several ways to escape from the capture of immune response. Nef protein,
which is an accessory protein of HIV, downregulates MHC molecules of infected cells to
avert the recognition by CTLs (Haut and Ertl 2009).
The lack of an appropriate and validated animal model is another obstacle in
developing efficacious AIDS vaccine (Fauci, Johnston et al. 2008). Only a few
nonhuman primates are susceptible to HIV-1 infection, nevertheless they do not progress
to AIDS. Macaques can be infected with SIV or SHIV, but it’s still elusive if the
infections are representative of HIV-1 infections in human (Haut and Ertl 2009).
21
However, extensive preclinical trials of HIV vaccines are conducted on macaques to
assess their protection against SIV or SHIV challenge. Although early challenge
experiments are conducted though i.v. injection of challenge virus, it is now suggested
challenge through mucosal routes can be more predicative, which mimics the HIV
transmission (Munier, Andersen et al. 2011). Strain of the challenge virus, as well as the
route and dose of challenge need to be taken into careful consideration to predict the
outcome of vaccination in humans. BLT (bone marrow-liver-thymus) mice, which are
immuno-deficient mice transplanted with human thymus and liver tissues and infused
with hematopoietic stem cells, provide an alternative (Haut and Ertl 2009). More efforts
are need to search for an animal models that more reflect the pathology seen in human
infections.
1.3.3 Current Progress in AIDS Vaccine
Highly active antiretroviral therapy (HAART) was introduced in 1986, and
remains the most important accomplishment in HIV/AIDS medicine (Robinson 2007).
Although HAART expand many life spans, it is not optimal due to the high cost, side
effects, and complex regimens. Many drug involved in HAART regulate lipid and
glucose metabolism, decades of treatment will lead to accumulated toxicity. Side effects,
such as heart disease, diabetes, liver disease, and cancers have been increasingly noticed
in people continuously receiving HAART treatment (Rossi, June et al. 2007). Moreover,
the drug couldn’t eliminate the virus, but only reduce the viral loads.
22
An ideal vaccine against HIV could elicit both humoral (antibodies) and cellular
(T cells) with sufficient potency and quality to recognize diverse stains of HIV, and to
reach the site of virus entry during the early stage of infection before the irreversible
latent reservoir established (Rossi, June et al. 2007). Vaccine vectors that encoding
recombinant env glycoprotein (gp120) are explored for the purpose to generate
neutralizing antibodies (Nabel 2001). However, difficulty relies in the inability of these
vectors to induce neutralizing Abs recognizing the conserved CD4 binding region of
gp120 with high titers (Nabel 2001). VRC01 is the most potent cross-neutralizing
antibody that have been reported till now, which can neutralize 90% of the currently
circulating HIV-1 stains, efforts are underway to identify immunogens that can efficiently
elicit VRC01 antibodies (Rossi, June et al. 2007).
Although T cells couldn’t protect against viral infection, they are able to decrease
the set-point level of viremia, which predicts disease progression as well as further
transmission (Munier, Andersen et al. 2011). Most T cell vaccines are designed to direct
immune response against conserved internal proteins of HIV. A number of vaccine
platforms have been studied during the past few decades, including delivering adjuvant
protein and peptides, plasmid vectors, recombinant viral vectors (MVA, AAV, VSV,
ADV, and vaccinia virus), bacterial vectors, and virus like particles (VPL) (Robinson
2007). Prime-boost regimens are most commonly used to further elevate the magnitude
of HIV-specific immune response, as well as to reduce the dosage of individual
manipulation for alleviating dose-related toxicity (Rossi, June et al. 2007). Heterologous
23
prime/boost regimens, which combines two or more vaccine modalities, are extraordinary
popular because there is limited preexisting antibodies block the function of booster.
Table 1.2 presents a few representative ongoing HIV/AIDS vaccines in human
trials (Robinson 2007). All of these trials use replication-defective viral vectors to deliver
multiple HIV proteins in order to elicit broad T-cell responses, most of them include Env
as an immunogen to induce anti-Env antibodies as well. The STEP trial, which is a phase
2b test-of-concept trial, is the first attempt involving a “T-cell only” vaccine. It is based
on recombinant adenoviral vectors expressing HIV Gag, Pol, and Nef proteins. This trial
enrolled men and women at high risk of HIV acquisition, in regardless of the titers of
neutralizing antibodies against AdHu5 virus in participated individuals. However, the
STEP trial was stopped and unblended due to lack of efficacy (Munier, Andersen et al.
2011). The data accumulated in 2008 showed that 33/922 men in the placebo group got
infected, while 49/914 men in the vaccinated group became infected (Wijesundara,
Jackson et al. 2011). The failure of STEP trail is presumably due to several reasons. It
was found that Ad vectors persist at low levels in a transcriptional active form in T cells,
which may result in high percentage of activated T cells in lymphatic tissues. This raises
the risk of infection as HIV-1 preferentially infects activated CD4
+
T cells (Walker and
Burton 2008). The breath of the CTL response is not adequate; data shows that the
vaccine only induced T cells responding to two or three epitopes on average. The
challenge model (hybrid SIV-HIV virus) and the MHC alleles chosen for the monkey
experiment are not well representative of human infection. Besides that, it is proposed
that the antiviral cytotoxic activity and avidity of T cells, rather than IFN-γ Elispot
24
measurement should be evaluated to assess the efficacy of the trial in NHP (Walker and
Burton 2008).
The phase III efficacy trial in Thailand provide some protective effect. The
participants received prime injections with ALVAC-HIV (Aventis recombinant
canarypox) and boost immunization with AIDSVAX B/E (bivalent VaxGen gp120
protein) (Munier, Andersen et al. 2011). This trail revealed 31.2% vaccine efficacy in the
modified intention-to-treat analysis. It showed modestly reduced risk of HIV infection,
whereas did not affect viral load or CD4
+
T cell counts in subsequently infected
individuals (Wijesundara, Jackson et al. 2011).
For the future development of an optimized T-cell vaccine for AIDS, researchers
believed that besides the magnitude of antigen-specific CD8+ T cells response, a number
of other factors need to be carefully evaluated, such as CTL functionality, differentiation
status, rates of clonal turnover, migration patterns, and proliferation potency
(Wijesundara, Jackson et al. 2011). T-cell avidity is thought to be an important parameter
that may predict viral control. It refers to the ability of T cells to respond to the peptide-
MHC complex, which is correlated with routes of vaccine delivery, immunodominance
and antigen density, as well as the cytokine milieu (IFN-γ, IL-2, TNF-α). Moreover, to
manipulate the anatomical distribution of vaccine-induced immune response is of great
importance as most of the HIV infection takes place at mucosal level (Wijesundara,
Jackson et al. 2011).
25
Table 1.2: HIV vaccines advancing in human trials. (Nature 8: 686-693, 2007)
1.4 Our Method for Targeted Gene Delivery to Dendritic Cells
Engineering lentiviral vectors by substitution of its native envelope protein with a
variety of different envelop pseudotypes is a commonly used means to achieve desired
cell tropism and increase gene transduction efficiency (Delenda 2004). We decorated the
lentiviral vectors with an engineered glycoprotein from Sindbis virus, and realized
targeted gene delivery to dendritic cells.
26
1.4.1 Targeted Delivery of Recombinant Lentiviral Vectors
Initially, our lab developed a two-component targeting system for targeted gene
delivery to specific cell types using retrovirus. The targeting viral particles incorporated
two separate glycoprotein on the viral surface, one is responsible for binding to cell
surface and the other performs the viral-cell membrane fusion function (Yang, Bailey et
al. 2006). When the viruses bud out from the producer cells, they take the cells’
membrane that expresses the binding and fusion glycoproteins, and thus they incorporate
the two proteins into the viral particles. By using this two-component system, we can
engineer the viral vectors with different binding glycoproteins, which can be an antibody,
an antigen or a ligand to allow for diverse repertoire of targeting (Cronin, Zhang et al.
2005). When the viral vectors bind to their targeted cells, the binding of glycoproteins to
the receptors induces endocytosis. The fusogenic molecule responds to the low pH
environment in endosome, and triggers membrane fusion, which allows the release of the
virus core into the cytosol (Dimitrov 2004). Following reverse transcription and transport
of the product into nucleus, the genome of the viral vector is able to integrate into host
cell genome, and incorporate its transgene into inheritance of targeted cells (Fig 1.2).
In my study, I utilize the viral glycoproteins from Sindbis virus, which is a class II
fusogen that triggers fusion with β barrels (Dimitrov 2004). As Sindbis virus naturally
infects dendritic cells, we engineered the viral glycoprotein to avert its non-specific
tropism and achieved specific targeting to dendritic cells. The Sindbis virus glycoprotein
performs binding and fusion simultaneously.
27
Figure 1.2: The proposed entry mechanism two-component retroviral targeting.
1.4.2 Engineering Sindbis Envelope Glycoprotein Sindbis virus, which belongs to the Alphavirus family, is reported to be able to
pseudotype both oncotrtroviruses and lentiviruses (Morizono, Xie et al. 2005). This
provides a means to develop cell-targeted lentiviral vectors. Sindbis virus envelope
contains two integral membrane glycoproteins: E2 binds to targeted cells, and E1
mediates fusion between the viral envelope and targeted cell membrane in a low pH-
dependent fashion (Morizono, Xie et al. 2005; Yang, Bailey et al. 2006). E1 and E2 form
a trimer of the heterodimer, and function as a unit.
28
In 2005, Morizono et al. modified the native Sindbis virus glycoprotein by
inserting the IgG Fc binding region of protein A (ZZ domain) into the receptor-binding
region (Morizono, Xie et al. 2005). The ZZ domain can bind to the Fc region of
monoclonal antibodies, so that the virus can be conjugated with specific antibodies to
recognize desired cell surface antigens to achieve target specificity. However, the wide
distribution and highly conserved nature of high-affinity laminin receptor and heparin
sulfate receptor of Sindbis virus raise the problem of non-specific tropism (Morizono,
Xie et al. 2005). The team made several additional mutations to in-activate the receptor
binding sites, so that they made a binding-deficient and fusion-competent vector (SIN
vector). In 2006, Yang et al. collaborated with our lab and developed a two-component
targeting system, in which two distinct proteins provide the cell recognition function and
the fusion function separately (Yang, Bailey et al. 2006). In this study, we replaced the
ZZ domain of the SIN vector with a 10-residue tag sequence, for which there exists a
monoclonal antibody that allows monitoring of SIN expression.
It is found that Sindbis virus can bind to dendritic cells by recognizing a surface
protein DC-SIGN, which is a C-type lectin-like receptor capable of rapid binding and
endocytosis (Geijtenbeek, Torensma et al. 2000). Therefore, in 2008 Yang and our lab
designed a lentiviral vector that can be targeted to dendritic cells (Yang, Yang et al.
2008). The canonical heparin sulfate binding site and the DC-SIGN binding site on
Sindbis virus envelope glycoprotein are physically separated, which allows us to
introduce mutations that blind the vector to its canonical binding site, while maintain its
29
ability to interact with DC-SIGN. The mutated Sindbis virus glycoprotein can be utilized
to decorate lentiviral vector to direct it towards dendritic cells.
Figure 1.3: A schematic representation of the general strategy to engineer a lentivector
system capable of targeting dendritic cells (Nat Biotechnol 26: 326-334, 2008)
1.5 Summary and Thesis Work
The ultimate goal of gene therapy for human diseases is to engineer the gene
delivery vectors in a way that they only deliver the therapeutic genes to a selected subset
of cells (Somia and Verma 2000). My thesis projects were to engineer retroviral and
lentiviral vectors to achieve prophylactic or therapeutic goals. Chapter 2 is published
(Dai and Wang 2009), and is a completed study describing a method to generate
autologous antigen-specific CD4
+
helper T cells in vitro from easily accessible bone
marrow cells. This provides a means to generate large number of antigen-specific T cells
for adoptive cell transfer (ACT) therapies, which may alleviate patients suffering from
cancer and AIDS. The generation of antigen-specific T cells is fulfilled by transducing
hematopoietic progenitors with a retroviral vector to express a human CD8
+
T cell
receptor (TCR), followed by coculturing the engineered progenitor cells with an OP9-
30
DL1 monolayer to facilitate T-cell commitment. Chapter 3 is also a published study in
which we utilize an engineered lentiviral vector to serve as a promising DC-targeted
vaccine carrier for the treatment of HIV (Dai, Yang et al. 2009). This lentiviral vector is
incorporated with HIV-1 gag protein, and pseudotyped with a mutated Sindbis virus
glycoprotein, which can bind to the dendritic cell surface protein DC-SIGN. It is
demonstrated that this vector, either being manipulated by itself or combined with other
vectors to form prime/boost regimens, could induce robust HIV-specific immune
response. In Chapter 4, which is completed and submitted for publication, we further
optimized the DC-directed lentiviral vaccine by blocking PD1/PD-L1 inhibitory pathway
via an anti-PD-L1 antibody. Programmed death 1 (PD-1) is a negative regulator of T cell
activation. By combining the lentiviral vector prime/boost regimen with PDL1 blockade,
we noticed robust, multifunctional, broad and prolonged HIV-specific CD8
+
T cell
response.
31
Chapter 2: In Vitro Differentiation of Adult Bone Marrow
Progenitors into Antigen-Specific CD4 Helper T Cells Using
Engineered Stromal Cells Expressing a Notch Ligand and a MHC
Class II protein
Portions of this Chapter are adapted from:
Bingbing Dai, and Pin Wang Stem. Cells. Dev. (2009) 18: 235–245
A murine stromal cell line (OP9-DL1) expressing a notch ligand, Delta-like-1, has
been shown to be able to drive the differentiation of both murine and human
hematopoietic progenitors into T cells in vitro. Further studies showed that hematopoietic
progenitors transduced by a retroviral vector to express a human CD8
+
T cell receptor
(TCR) followed by an OP9-DL1 monolayer coculture could generate antigen-specific
cytotoxic T lymphocytes (CTLs) in vitro. It remains unknown if a similar method could
be applied to produce CD4
+
helper T cells. In this report, we show that murine adult bone
marrow cells transduced with an OT2 CD4
+
TCR and cocultured with OP9 stromal cells
expressing Delta-like-1 can differentiate into antigen-specific CD4
+
T cells in vitro.
These cells are capable of inducing the expression of T cell activation markers and
producing cytokines upon stimulation. We have also constructed a new stromal cell line
(OP9-DL1-IA
b
) ectopically expressing a murine major histocompatibility complex
32
(MHC) class II protein, I-A
b
, in OP9-DL1 cells. This new line could accelerate the
development of TCR-transduced bone marrow cells into CD4
+
T cells, resulting in cells
with an improved capacity to respond to T cell stimulation to secrete cytokines. Taken
together, we demonstrate a general and potentially useful method to generate autologous
antigen-specific CD4
+
helper T cells in vitro from easily accessible bone marrow cells.
2.1 Introduction
Although the immune system can deal with most pathogens well, certain
microbial infections, such as HIV (Douek, Picker et al. 2003), can directly target immune
cells, preventing the host from generating an effective pathogen-specific immunity.
Similarly, the immune system can often fail to suppress the growth of tumors, largely due
to a low number of tumor-reactive T cells and/or an anergy of T cells that is induced by
tumor cells (Dudley and Rosenberg 2003). Thus, it is conceivable that immunotherapy by
direct provision of a large quantity of functional T cells through adoptive transfer (termed
passive T cell immunotherapy or adoptive T cell therapy) can be a viable approach to
treat certain infectious diseases and cancers (Dudley and Rosenberg 2003; Gattinoni,
Powell et al. 2006). To carry out such a therapy, we need a sufficient amount of antigen-
reactive T cells for transfer. One approach is to use allogeneic effector T cells from
appropriate donors (Kolb and Holler 1997). Adoptive transfer of these donor T cells has
been shown to be able to treat some leukemia-like diseases (Rooney, Smith et al. 1998;
Khanna, Bell et al. 1999; Bollard, Aguilar et al. 2004). However, the low availability of
33
these donor cells limits the widespread application of this type of treatment. Moreover,
patients usually suffer from severe toxicity due to graft-versus-host disease (GVHD). An
improved approach for obtaining T cells is through the in vitro expansion of patients’
own lymphocytes (Dudley and Rosenberg 2003). One example is the autologous transfer
of in vitro expanded tumor-infiltrating lymphocytes (TILs) for the treatment of solid
melanoma tumors (Dudley and Rosenberg 2003). Due to patient-to-patient variation, the
major limitation of this approach is that isolation and selection of good quality T cells
from patients is not always successful; therefore, the treatment will be limited to a small
number of patients. Thus, it is of great interest to develop methods to reliably generate
large numbers of antigen-specific T cells in vitro, which could address many of these
described limitations.
One of the popular methods for developing T cells from hematopoietic
progenitors in vitro is the use of fetal thymic organ culture (Ceredig, Jenkinson et al.
1982). But this method is expensive and difficult to scale up, making it impractical for
generating a sufficient amount of T cells for adoptive transfer. Recently, an OP9-based
coculture system capable of supporting the hematopoietic differentiation of progenitor
cells into functional T cells has been reported (Schmitt and Zuniga-Pflucker 2002;
Zuniga-Pflucker 2004; de Pooter and Zuniga-Pflucker 2007). OP9 is a stromal cell line
derived from the macrophage CSF-deficient osteopetrotic mouse (Wiktor-Jedrzejczak,
Bartocci et al. 1990; Yoshida, Hayashi et al. 1990). It was found that over-expression of a
notch ligand, Delta-like-1, in OP9 cells resulted in the generation of a new cell line (OP9-
DL1) that could drive the differentiation of both murine and human hematopoietic
34
progenitors into T cells in vitro (Schmitt and Zuniga-Pflucker 2002). Thus far, murine
progenitors isolated from fetal liver (Schmitt and Zuniga-Pflucker 2002) or adult bone
marrow (Schmitt, de Pooter et al. 2004; Huang, Garrett et al. 2005), and human
progenitors isolated from umbilical cord blood (La Motte-Mohs, Herer et al. 2005),
pediatric bone marrow (De Smedt, Hoebeke et al. 2004), or postnatal thymus (van Lent,
Nagasawa et al. 2007), have all been shown to be able to differentiate into T cells using
the OP9-DL1 coculture system, although the yield of T cells from adult bone marrow was
reported to be extremely low (Huang, Garrett et al. 2005). Subsequent studies showed
that retrovirus-mediated transfer of a human CD8
+
T cell receptor (TCR) into human
hematopoietic progenitors derived from umbilical cord blood (Zhao, Parkhurst et al.
2007) or postnatal thymus (van Lent, Nagasawa et al. 2007) followed by OP9-DL1
monolayer coculture demonstrated a promising method of in vitro generation of antigen-
specific cytolytic T lymphocytes (CTLs). It remains to be tested if antigen-specific CD4
+
helper T cells can be generated using a similar approach.
Many studies have shown that CD4
+
helper T cells play an indispensable role in
orchestrating CTLs (CD8
+
T cells) to mount efficient immune responses by providing
“cognate help” (Janssen, Lemmens et al. 2003; Shedlock and Shen 2003). This “cognate
help” role of CD4
+
T cells has also been found to be necessary in tumor immunotherapy;
it has been proposed that the lack of an effective means of engaging CD4
+
helper cells is
one of the major reasons for our inability to achieve a robust and long-lived CTL
response against cancer cells (Pardoll and Topalian 1998; Ostrand-Rosenberg 2005). This
may support the argument that adoptive transfer of antigen-specific CD4
+
T cells, along
35
with CD8
+
T cells, could represent an ideal approach for generating productive antigen-
specific immune responses, although care needs to be taken to ensure that CD4
+
T cells
with negative regulatory function, such as CD4
+
CD25
+
Foxp3
+
regulatory T cells (Zou
2006), are depleted from the CD4
+
population before transfer. In this study, we tested the
concept of in vitro generation of antigen-specific CD4
+
T cells by coculturing OP9-DL1
cells with adult bone marrow cells transduced to express a CD4
+
TCR. Considering that
OP9 cells lack the expression of MHC class II molecules, which may limit its ability to
support CD4
+
T cell development (Zuniga-Pflucker 2004), we also investigated the effect
of CD4-TCR/MHC-class-II interactions on the generation of functional CD4
+
cells by
ectopic expression of I-A
b
in OP9-DL1 cells (designated as OP9-DL1-IA
b
). We
demonstrated that CD4
+
TCR retroviral transfer could be used to engineer adult bone
marrow cells to produce antigen-specific CD4
+
T cells in vitro. Moreover, the existence
of CD4-TCR/MHC-class-II interactions in the coculture system could improve the
functional response of these cells to generate cytokines.
2.2 Materials and Methods
2.2.1 Mice
Six- to eight-week old female C57BL/6 (B6) mice were purchased from Charles
River Breeding Laboratories. All animal procedures were approved by the Department of
Animal Resources of the University of Southern California.
36
2.2.2 Construct preparation
The cDNAs of the alpha and beta chains of the murine I-A
b
were PCR-amplified
from appropriate mouse clones (ATCC mammalian gene collection, ATCC numbers
10324451 for alpha chain and 10470166 for beta chain) using specific primers (alpha
chain: sense, 5’-CGCCGAGATCTCTCGAGATGCCGCGCAGCAGAGCTCTGATTC-
3’, antisense, 5’-GGCGGAATTCTCATAAAGGCCCTGGGTGTCTGGAGGTG-3’;
beta chain: sense, 5’-CACAACCATGGCTCTGCAGATCCCCAGCCTCC-3’, antisense,
5’- GCAGGTCGACTCACTGCAGGAGCCCTGCTGGAGGAG-3’; restriction sites for
alpha chain, BglII and EcoRI, and for beta chain, NcoI and SalI, are shown by
underlining). The resulting alpha chain was inserted downstream of the viral LTR
promoter in the MIG retroviral vector (Van Parijs, Refaeli et al. 1999), followed by
insertion of the beta chain downstream of internal ribosome entry site (IRES) to replace
the EGFP gene. We designated the final construct MIAb.
2.2.3 Cell lines
The OP9-MIG and OP9-DL1 cell lines were described previously by Schmitt et
al. (Schmitt and Zuniga-Pflucker 2002). An ecotropic murine leukemia virus
glycoprotein (Eco)-pseudotyped MIAb retrovirus (MIAb/Eco) was generated by co-
transfecting HEK293T cells with the retroviral packaging vector pCL-Eco and a plasmid
expressing a mouse MHC class II protein (MIAb) using a standard calcium phosphate
precipitation technique. OP9-MIG or OP9-DL1 expressing I-A
b
were generated by spin
infection of OP9-MIG or OP9-DL1 with MIAb/Eco retroviruses in the presence of
37
10µg/ml polybrene for 90 min at 2,500 rpm and 25
o
C using a Sorvall Legend 7
centrifuge. The efficiency of infection was determined by staining and flow cytometry
analysis using Phycoerythrin (PE)-conjugated anti-mouse I-A
b
antibody (clone AF6-
120.1, BD Biosciences) (around 50%). Cells expressing I-A
b
were sorted by FACS
sorting, and then subcloned to generate clonal cell lines, OP9-MIG-IA
b
and OP9-DL1-
IA
b
. All of the OP9 derived cell lines were cultured in a 10-cm dish in OP9 medium
(αMEM (Gibco) supplemented with 20% FBS (Sigma), 10 U/ml of penicillin, 100 µg/ml
of streptomycin, and 2 mM glutamine).
2.2.4 Mouse bone marrow stem cells and OP9 cells coculture
Retroviral vector MOT2 was generated by linking cDNAs encoding OT2 TCR α
and β chains with an internal ribosome entry site and cloning this into the MSCV-based
retroviral vector under control of the viral LTR promoter (Yang, Qin et al. 2002). Bone
marrow (BM) cells were harvested from B6 female mice treated with 5-FU for five days
to enrich the stem cells. Collected bone marrow cells were cultured for 4 days in RPMI
1640 medium (Cellgro) containing 10% FBS (Sigma), 10 U/ml of penicillin, 100 µg/ml
of streptomycin and 2 mM glutamine with 20 ng/ml recombinant murine IL-3
(Peprotech), 50 ng/ml recombinant murine IL-6 (Peprotech) and 50 ng/ml recombinant
murine stem cell factor (Peprotech). On day 2 and day 3, the cells were spin infected with
MOT2/Eco retrovirus for 90 min at 2500 rpm and 25
o
C as described above. On day 4, the
transduced BM cells were collected and transferred onto 80%-90% confluent OP9-MIG,
OP9-DL1, OP9-MIG-IA
b
, or OP9-DL1-IA
b
cell monolayers in 10-cm dishes with OP9
38
medium supplemented with 5 ng/ml murine IL-7 (Peprotech) and 5 ng/ml human Flt-3
ligand (Peprotech). BM cells were collected for surface marker analysis and transferred
to new dishes containing OP9-MIG, OP9-DL1, OP9-MIG-IA
b
, or OP9-DL1-IA
b
at the
indicated time points.
2.2.5 Flow cytometry
Surface staining was performed by blocking the cells with anti-mouse
CD16/CD32 (clone 2.4G2, BD Pharmingen) followed by incubation with fluorochrome-
conjugated antibodies. FITC-, PE- or PE-Cy5- conjugated antibodies specific for mouse
Sca-1, CD117 (c-Kit), CD4, CD8, CD25, CD44, CD69, CD62L, Thy1, CD45R/B220,
TCRVα2, and TCRVβ5.1,5.2 were purchased from BD Biosciences. Intracellular staining
of TCRVα2 and TCRVβ5.1,5.2 was performed using the Cytofix/Cytoperm Kit from BD
Pharmingen and following the manufacturer’s protocol. All of the flow cytometry
analysis was done with a FACSort (BD Bioscience) instrument.
2.2.6 T cell stimulation and functional assays
For the anti-CD3/CD28 co-stimulation assay, a 24-well dish was pre-coated with
300 µl/well of anti-mouse CD3 (5 µg/ml in PBS, BD Pharmingen) at 4
o
C overnight. On
the next day, OP9-cocuculted cells were seeded at 1×10
6
cells per well in 1 ml of T cell
culture medium (RPMI 1640 medium (Cellgro) containing 10% FBS (Sigma), 10 U/ml of
penicillin, 100 µg/ml of streptomycin, and 2 mM glutamine) in presence of 1 µg/ml anti-
mouse CD28 (BD Pharmingen) at 37
o
C in a humidified 5% CO
2
incubator. Three days
39
later, the culture supernatants were collected for IFN-γ and IL-2 ELISA assays and the
stimulated cells were collected for flow cytometry analysis. For the peptide-based
stimulation assay, 1×10
6
single positive (van Lent, Nagasawa et al.) CD4
+
T cells were
seeded per well together with 2×10
6
spleen cells harvested from naïve B6 female mice as
antigen-presenting cells (APCs) loaded with 1µg/ml ovalbumin peptide (OVAp
329-337
,
designated as OVAp2) for three days at 37
o
C in a 5% CO
2
incubator and were then
analyzed.
2.2.7 IFN-γ and IL-2 ELISA
ELISA plates (96-well) were pre-coated with 50 µl per well of 1 µg/ml anti-
mouse IFN-γ or anti-mouse IL-2 (BD Pharmingen) in carbonate buffer at 4
o
C overnight.
The plates were then washed six times with deionized water (DI water) followed by
blocking with 100 µl per well of dilution buffer (2% borate buffered saline (BBS) and
0.002% sodium azide) for 30 min at 37
o
C. After six washes, sample supernatants were
two-fold serially diluted in dilution buffer, added to the plates at a final volume of 50 µl
per well, and incubated for 3 h at 37
o
C. The plates were then washed ten times and
incubated for 45 min at room temperature (RT) with 50 µl per well of 1 µg/ml
biotinylated detecting antibody (BD Pharmingen) in dilution buffer. After ten washes,
streptavidin-conjugated horseradish peroxidase (1:200 in dilution buffer) (R & D
systems) were added at 50 µl per well for 30 min at RT. Finally, the plates were washed
ten times, 50 µl per well of tetramethylbenzidine (TMB) substrate solution (KPL) was
added, and the plates were incubated for 5 to 30 min at RT. The reaction was stopped by
40
adding 50 µl per well of 2 M H
2
SO
4
. The absorbance at the wavelength of 450 nm
(OD
450
) was measured using a plate reader (Molecular Devices).
2.2.8 Statistical analysis
The significance of the difference between groups in the experiments measuring
cytokine production was evaluated by analysis of variance
followed by a one-tailed
Student t
test.
2.3 Results
2.3.1 Construction of OP9 cells expressing a MHC class II protein
Our initial interest was to generate antigen-specific CD4
+
T cells in vitro using an
OP9-DL1 culture system. Direct introduction of cDNAs of a TCR into human
hematopoietic progenitor cells (HPCs) by retroviral transduction and further culture of
the cells on OP9-DL1 is a method that has been shown to generate functional antigen-
specific CD8
+
T cells (van Lent, Nagasawa et al. 2007; Zhao, Parkhurst et al. 2007). We
wanted to investigate whether this approach could be used to produce antigen-specific
CD4
+
T cells. In recognition of the fact that OP9 cells lack the expression of MHC class
II molecules, we were also interested in studying whether enforced expression of a MHC
class II molecule on OP9 cells could affect the development and function of antigen-
specific CD4
+
T cells generated in this in vitro culture system.
We chose the well-defined OT2 TCR as a model CD4
+
TCR in this study
(Barnden, Allison et al. 1998). This TCR recognizes residues 329-337 of chicken
41
ovalbumin presented by I-A
b
, a murine MHC class II molecule. We analyzed OP9
stromal cells for their expression of MHC class II proteins by flow cytometry and found
that surface expression of I-A
b
was undetectable (Fig. 2.1, control). Thus, we decided to
evaluate the role of ectopic expression of I-A
b
in OP9-DL1 stromal cells on the
generation of OT2 CD4
+
T cells in vitro. The alpha and beta chains of cDNAs of murine
I-A
b
were linked by an internal ribosome entry site (IRES) sequence and cloned into a
murine stem cell virus-based retroviral vector (Van Parijs, Refaeli et al. 1999) (Fig.
2.1A). Retrovirus encoding two chains of I-A
b
was used to transduce OP9-DL1 cells and
the cells that expressed a high level of I-A
b
were sorted. The sorted cells were subcloned
Figure 2.1: : Construction of OP9 stromal cells to express murine MHC class II protein
(I-A
b
) by retroviral transduction. (A) Schematic representation of a retroviral construct
encoding alpha and beta chains of murine I-A
b
. LTR: long-terminal repeats; IRES:
internal ribosome entry site. Expression is driven by the viral LTR promoter. (B) Flow
cytometry analysis of OP9 stormal cell lines (OP9-MIG-IA
b
and OP9-DL1-IA
b
) for their
expression of I-A
b
by surface staining. Solid line: OP9-MIG-IA
b
or OP9-DL1-IA
b
cells;
Shaded area: parental OP9-MIG or OP9-DL1 cells.
42
and one clone, designated OP9-DL1-IA
b
, was selected as a stromal line for the rest of the
in vitro culture study (Fig. 2.1B, right). A control OP9-MIG-IA
b
cell line lacking the
expression of Delta-like-1 was similarly generated using an OP9-MIG (OP9 transduced
to express GFP alone) cell line (Fig. 2.1B, left).
2.3.2 Generation of TCR-positive T cells using adult bone marrow
progenitors in OP9 coculture systems
It has been shown that primitive fetal liver cells can be used as hematopoeitic
progenitor cells to efficiently generate T lymphocytes when cocultured with OP9-DL1
stromal cells in the presence of IL-7 and Flt3-L (Schmitt and Zuniga-Pflucker 2002).
Although adult bone marrow cells were also reported to be able to give rise to T cells
using similar culture conditions (Schmitt, de Pooter et al. 2004; Balciunaite, Ceredig et
al. 2005; Huang, Garrett et al. 2005; Zakrzewski, Kochman et al. 2006), the yield was
much lower than that of fetal liver cells (Schmitt, de Pooter et al. 2004; Balciunaite,
Ceredig et al. 2005; Huang, Garrett et al. 2005; Zakrzewski, Kochman et al. 2006). We
repeated this culture process using either bulk or sorted (Lin
-
Sca-1
+
c-Kit
+
) bone marrow
cells from wild-type mice (4-6 weeks old) and compared their capacities to generate T
cells with either OP9-DL1 or OP9-DL1-IA
b
cells. B lymphocyte differentiation of
marrow cells was completely blocked when the cells were cultured with either stromal
cell lines (OP9-DL1 or OP9-DL1-IA
b
) (data not shown). Generation of TCR-positive
cells (either double positive (DP) or single positive (SP) stage) was barely detectable
(data not shown), which is consistent with the report by Huang et al. (Huang, Garrett et
43
al. 2005). No marked difference in T cell development was observed using either bulk or
sorted marrow cells. We found that expression of I-A
b
in the OP9-DL1 cells did not
significantly alter the differentiation patterns of marrow cells under our detection
conditions (data not shown). It is not too surprising that the adult bone marrow cells
behaved differently from the fetal progenitor cells and could not efficiently differentiate
to TCR-positive T cells in the current coculture system, as several studies have shown
that different environments were required for adult and fetal lymphopoiesis (Crompton,
Outram et al. 1998; Carvalho, Mota-Santos et al. 2001; Douagi, Vieira et al. 2002;
Huang, Garrett et al. 2005). It seems possible that the current culture conditions have not
been optimized for adult marrow cells and identification of new conditions such as
cytokine concentration (Balciunaite, Ceredig et al. 2005; Huang, Garrett et al. 2005) and
stromal coculture environments could potentially improve the efficiency of the process.
One of the key steps in generating a T cell is the successful rearrangement of the TCR
genes. Thus, we reasoned that the delivery of a pre-arranged TCR gene into adult bone
marrow cells might facilitate T cell development under the OP9-DL1 culture condition.
2.3.3 Generation of TCR-specific CD4 T cells in OP9 coculture systems
We next investigated the idea of generating TCR-specific CD4
+
helper T cells in
OP9 coculture systems by retroviral introduction of a CD4
+
TCR gene to adult bone
marrow cells followed by coculture with OP9 cells expressing Delta-like-1 and/or a
murine MHC class II protein. We used a mouse stem cell virus-based retroviral vector
encoding the alpha and beta chains of an I-A
b
-restricted OT2 CD4
+
TCR gene linked by
44
an IRES sequence to deliver the TCR gene into the adult bone marrow cells (Fig. 2.2A,
designated MOT2) (Yang and Baltimore 2005). The transgene expression was controlled
by the viral LTR promoter. We harvested adult bone marrow cells enriched with
hemapoietic progenitor cells from wild-type mice and exposed them to MOT2 vectors.
Intracellular staining was performed to measure the transduction efficiency. The OT2
TCR uses Vα2 and Vβ5 TCR family chains and we used the antibodies specific for these
chains for flow cytometry analysis. Approximately 45% of the total bone marrow cells
were modified to co-express OT2 TCR alpha and beta chains (Fig. 2.2B).
Figure 2.2: Retroviral transduction of adult bone marrow cells to express OT2 CD4
+
T
cell receptor (TCR). (A) Schematic representation of a retroviral construct encoding
alpha and beta chains of OT2 TCR. LTR: long-terminal repeats; IRES: internal ribosome
entry site. Expression was driven by the viral LTR promoter. (B) Flow cytometry
analysis of viral transduced bone marrow cells for their expression of OT2 TCR by
intracellular staining for TCR alpha chain using anti-Vα2 antibody and for TCR beta
chain using anti-Vβ5 antibody. Non-transduced bone marrow cells were included as
negative controls.
45
The transduced cells were subsequently cocultured on various OP9 cells (OP9-
MIG, OP9-MIG-IA
b
, OP9-DL1, and OP9-DL1-IA
b
). Cell expansion, which is evidence
of hematopoietic differentiation, was observed for cells cultured on OP9-MIG, OP9-DL1,
and OP9-DL1-IA
b
, but not on OP9-MIG-IA
b
cells (Fig. 2.3A). The distinct kinetics and
rate of cell expansion on the various stromal cells was also detected (Fig. 2.3A). OP9-
MIG only supported limited expansion and the expansion did not start until day 17 of
coculture. Significant expansion by both OP9-DL1 and OP9-DL1-IA
b
was observed after
day 9 of coculture. However, MOT2-transduced bone marrow cells cocultured on OP9-
DL1 exhibited a stronger expansion (> 4-fold) than the OP9-DL-IA
b
coculture. An
apparent plateau in cellularity was obtained for the OP9-DL1-IA
b
coculture after an
initial 2-week culture. It seems that OP9-DL1-IA
b
stromal cells have a reduced capacity
to support expansion of adult bone marrow cells, probably due to their elevated potency
in facilitating hematopoietic differentiation (see below).
We monitored the surface markers of the transduced bone marrow cells
developing in the cocultures. The markers (Sca-1
+
c-Kit
+
) were used to track the possible
presence of hematopoietic progenitors. We found that both OP9-DL1 and Op9-DL1-IA
b
could initially increase the percentage of Sca-1
+
c-Kit
+
cells, followed by a gradual
decline (Fig. 2.3B). Most Sca1
+
c-Kit
+
cells disappeared after 3 weeks of coculture,
indicating that the majority of the hematopoietic progenitors had undergone
differentiation. The markers B220
-
Thy1
+
were employed to monitor the progress of T cell
differentiation. As expected, OP9 cells lacking the expression of a notch ligand could not
support hematopoietic T cell differentiation. In contrast, most cells developed on either
46
OP9-DL1 or OP9-DL1-IA
b
cells were committed to the T cell lineage after 2-week
cocultures (Fig. 2.3C).
Figure 2.3: Coculture of OT2 TCR-transduced bone marrow cells with various OP9 cells
(OP9-MIG, OP9-IA
b
, OP9-DL1, and OP9-DL1-IA
b
). Results shown is one representative
set of data from three independent experiments. (A) The expansion rate of transduced
bone marrow cells following coculture on OP9 cells. Total cellularity from OP9-MIG
(■), OP9-IA
b
(●), OP9-DL1 (▲), or OP9-DL1-IAb (▼) coculture was measured at the
indicated time points. (B) Phenotypic analysis of Sca-1 and c-Kit double-positive
transduced bone marrow cells following coculture on OP9 cells. Percentages of Sca-1
+
c-
Kit
+
cells from various OP9 cells (the labeling was the same as (A)) were determined by
flow cytometry analysis during the course of coculture at the indicated time points. (C)
Phenotypic analysis of committed T cell population (Thy1
+
B220
+
) from transduced bone
marrow cells following coculture on OP9 cells. Percentages of Thy1
+
B220
+
cells from
various OP9 cells (the labeling was the same as (A)) were determined by flow cytometry
analysis during the course of coculture at the indicated time points. (D) The dot plots
show the flow cytometry analysis of surface co-expression of CD3 and OT2 TCR alpha
chain from transduced bone marrow cells following coculture on either OP9-DL1 or
OP9-DL1-IA
b
at the indicated time points.
47
We analyzed the TCR expression by co-staining the CD3 and OT2 TCR alpha
chains. For MOT2-transduced bone marrow cells cultured on control OP9 cells lacking
the expression of Delta-like-1 (either OP9-MIG or OP9-IA
b
), we could not detect the
surface expression of the OT2 TCR even after 3 weeks of coculture (data not shown). In
contrast, OT2 TCR expression was readily detectable as early as day 9 of coculture on
transduced cells differentiating on either OP9-DL1 or OP9-DL1-IA
b
stromal cells (Fig.
2.3). Interestingly, it appeared that the expression of I-A
b
could facilitate T cell
development (5.94% of OT2 in OP9-DL1 vs 10.21% of OT2 in OP9-DL1-IA
b
on day 9).
After three weeks of coculture, more than 50% of the total cells were OT2 TCR-positive.
It should be noted that virtually all of the CD3
+
cells expressed the transduced TCR,
suggesting that the rearrangement of endogenous TCR could be a limiting step for in
vitro T cell development and the ectopic provision of pre-arranged TCR could partially
overcome this limitation.
The phenotypic progression from CD25
-
CD44
+
(DN1) to CD25
+
CD44
+
(DN2) to
CD25
+
CD44
-
(DN3), and finally CD25
-
CD44
-
(DN4, also called pre-DP (double positive)
(Petrie and Zuniga-Pflucker 2007)), is a characteristic differentiation pattern for early
immature T-cell precursors that are double-negative (DN) for CD4
+
and CD8
+
expressions (Godfrey and Zlotnik 1993; Ceredig and Rolink 2002; Bhandoola, von
Boehmer et al. 2007; Petrie and Zuniga-Pflucker 2007). Previous study showed that
different DN differentiation kinetics occurred when fetal liver and adult bone marrow
were used as the progenitor cells cocultured on OP9-DL1 cells (Huang, Garrett et al.
2005). We carefully monitored this aspect of differentiation and found that both OP9-
48
DL1 and OP9-DL1-IA
b
could support the progressive differentiation of TCR-transduced
bone marrow progenitors from DN1 to DN4 (Fig. 2.4A). At day 17 of both cocultures,
TCR-transduced cells were predominately DN3 and DN4. At the same time point and the
same conditions of the culture, we (data not shown) and others (Zakrzewski, Kochman et
al. 2006) found that DN2 and DN3 dominated if wild-type, non-transduced bone marrow
cells were cultured with Delta-like-1-expressing OP9 cells, suggesting that the enforced
expression of pre-arranged TCR genes could accelerate the development of DN cells in
such in vitro culture conditions. We also noticed that expression of I-A
b
could facilitate
the DN differentiation, as the major population (52%) of cells cocultured with OP9-DL1-
IA
b
had advanced to the DN4 stage by day 14 of coculture, compared to only 21% of the
cells cocultured with OP9-DL1 (Fig. 2.4A).
Flow cytometry analysis was further performed to track the expression of CD4
+
and CD8
+
on MOT2-transduced cells at various time points during the coculture period
(Fig. 2.4B). Transduced bone marrow cells cocultured on either OP9-DL1 or OP9-DL1-
IA
b
could give rise to CD8
+
CD4
+
immature DP T cells as early as day 17 or day 14 of
coculture, respectively, indicating that expression of a MHC class II protein is
dispensable for differentiation into DP cells under this culture condition. However, the
presence of I-A
b
had the effect of promoting differentiation and resulted in DP cells
(44%) accounting for the majority of cultured MOT2-transduced cells in OP9-DL1-IA
b
after a three-week coculture (Fig. 2.4B). Despite potential help from the early expression
of TCR and the presence of I-A
b
, this differentiation process is still much slower than that
49
Figure 2.4: Development of CD4
+
T cells from TCR-transduced bone marrow cells
cocultured with either OP9-DL1 or OP9-DL1-IA
b
cells. Result shown is one
representative set of data from three independent experiments. (A) Phenotypic analysis of
double-negative development of transduced bone marrow cells following coculture with
OP9 cells at indicated time points by surface co-staining of CD25 and CD44 expressions.
(B) Phenotypic analysis of CD4
+
and CD8
+
expressions on transduced bone marrow cells
cocultured with OP9 cells at indicated time points by surface co-staining of CD4 and
CD8. (C) The total numbers of SP CD4
+
T cells generated from the OP9 coculture. The
two million of bone marrow cells were transduced with OT2 vector and then initiated the
OP9 coculture.
50
of the fetal liver progenitors, in which DP cells can be detected as early as after one week
of coculture (Schmitt and Zuniga-Pflucker 2002). In addition to DP cells, we also
detected a significant population of mature SP CD4
+
T cells (9.48% from OP9-DL1
coculture; 12.01% from OP9-DL1-IA
b
coculture). Approximately 54 and 7 million of SP
CD4
+
T cells were obtained from OP9-DL1 and OP9-DL1-IAb cocultures, respectively
(original cell input of bulk bone marrow cells was 2 million; Fig. 2.4C). These CD4
+
T
cells predominately (> 95%) expressed the OT2 TCR, detected by staining using
antibodies against TCR Vα2 chain and Vβ5 chain (data not shown). No regulatory T cell
populations could be identified from these SP CD4
+
T cells generated from both
cocultures by co-staining of CD4, Vβ5, and Foxp3 (data not shown).
2.3.4 Functional Analysis of TCR-specific CD4 T cells generated by OP9
coculture
In light of fact that mature SP CD4
+
T cells emerged in OP9 cocultures, we went
on to evaluate whether these T cells could undergo TCR signaling in response to
stimulation. Adult bone marrow progenitor cells, transduced by a MOT2 vector and
cultured with either OP9-DL1 or OP9-DL1-IA
b
for about 3-4 weeks, were seeded in an
anti-CD3-coated plate and stimulated with anti-CD28 antibody. We observed that these T
cells proliferated under the condition of this polyclonal anti-CD3/CD28 stimulation (data
not shown), indicating that these cells might have acquired the feature of functional
maturity. Following the stimulation with antibodies, the SP CD4
+
T cells were greatly
expanded (from 9~10% to 43~44%) (Fig. 2.4B and 2.5A). When we gated on these SP
51
CD4
+
T cells, we found that the majority of them expressed the OT2 TCR, as assessed by
TCR Vα2 staining (Fig. 2.5A). The level of OT2 TCR expression was similar for the
CD4
+
T cells cocultured either on OP9-DL1 or OP9-DL1-IA
b
cells. Phenotypic analysis
of these T cells showed that these stimulated cells displayed the typical activated T cell
markers (CD25
+
CD44
+
CD69
+
), as compared to unstimulated control cells (Fig. 2.5B).
IFN-γ and IL-2 ELISAs were performed to measure the capacity of these CD4
+
T cells to
secrete cytokines upon polycloncal stimulation. As shown in Fig. 2.5C, IFN-γ and IL-2
secretions were detected for CD4
+
T cells generated from both cocultures; no production
of either IFN-γ or IL-2 was detected for non-stimulated cells (data not shown).
Interestingly, we found that CD4
+
T cells from the OP9-DL1-IA
b
coculture produced
significantly more IFN-γ (>35 folds) than that of the OP9-DL1 coculture. The same trend
was observed for IL-2 secretion, although to a lesser extent.
To determine if these CD4
+
T cells generated in vitro could mount antigen-
specific immune responses, we cultured these TCR-transduced and SP-sorted cells with
OVAp2 peptide-loaded antigen-presenting cells (APCs) harvested from a mouse spleen.
MOT2-transduced cells were able to recognize OVAp2-pulsed APCs, as evidenced by
the appearance of T cell activation markers (CD25
+
CD44
+
CD69
+
) (Fig. 2.5D). IL-2
secretion was observed for peptide-stimulated cells (Fig. 2.5E); no IL-2 secretion was
detected for either non-stimulated cells or cells cocultured with APCs without peptide-
pulsing (data not shown). The cells were able to respond to a peptide concentration of 1
µg/mL, which was equivalent to the sensitivity of the response of OT2 transgenic T cells
to the OVAp2 peptide (Yang, Qin et al. 2002; Yang and Baltimore 2005). Similar to the
52
observation from polyclonal stimulation, markedly higher production of IL-2 was
detected for T cells developed on OP9-DL1-IA
b
cells, as compared to IL-2 secreted from
cells generated on OP9-DL1 coculture. However, no production of IFN-γ could be
detected for TCR-transduced T cells generated either from the OP9-DL1 or OP9-DL1-
IA
b
coculture.
Figure 2.5: Functional analysis of antigen-specific CD4 T cells generated from TCR-
transduced bone marrow cells cocultured with either OP9-DL1 or OP9-DL1-IA
b
cells.
Cocultured bone marrow cells were stimulated for 22 days with either anti-CD3/CD28
antibodies (A-C) or with antigen-presenting cells pulsed with OVAp2 peptide (D-E). (A)
Flow cytometry analysis of the expansion of SP CD4 T cells upon stimulation for 3 days.
OT2 TCR expression was analyzed by gating on the CD4
+
CD8
-
population (R1-gated,
solid line). Shaded area: staining with an isotype control antibody. (B and D) Flow
cytometry analysis of surface expression of CD25, CD44 and CD69 before stimulation
(shaded area) or after stimulation (solid line) on gated SP CD4 T cells. IFN-γ (C and E)
and IL-2 (C) productions in response to anti-CD3/CD28 stimulation were determined by
ELISA.
53
2.4 Discussion
The purpose of this study is to evaluate a strategy to generate antigen-specific
CD4
+
helper T cells in vitro by culturing adult bone marrow cells transduced to express a
CD4 TCR with OP9 stromal cells expressing a notch ligand, Delta-like-1. In vitro
generation of antigen-specific CD4
+
T cells is of great interest to both fundamental study
of T cell biology and practical immunotherapeutic applications. For instance, there is
accumulating evidence showing that CD4
+
T cells play an indispensable role in antitumor
immunity (Pardoll and Topalian 1998; Nishimura, Iwakabe et al. 1999; Baxevanis,
Voutsas et al. 2000; Marzo, Kinnear et al. 2000; Janssen, Lemmens et al. 2003; Shedlock
and Shen 2003; Ostrand-Rosenberg 2005). The availability of a large quantity of CD4
+
T
cells recognizing the tumor antigen can potentially enhance the current therapy of
adoptive transfer of antitumor CD8
+
T cells to achieve maximal therapeutic efficacy
(Moeller, Haynes et al. 2005; Gattinoni, Powell et al. 2006). We focused on adult bone
marrow cells for this study because from the therapeutic point of view, they represent the
most easily accessible hematopoietic progenitor cells and can be frequently and safely
isolated.
Our method relies on a recently reported OP9-DL1 coculture system, in which the
ectopic expression of a notch ligand in a bone marrow stromal cell line, OP9, was found
to be sufficient to support the development of hematopoietic progenitors into T cells in
vitro (Schmitt and Zuniga-Pflucker 2002; Zuniga-Pflucker 2004; de Pooter and Zuniga-
Pflucker 2007). However, OP9-DL1 cells lack the expression of MHC class II molecules,
which may limit their capacity to support the development of CD4
+
T cells (Zuniga-
54
Pflucker 2004). This prompted us to also evaluate the effect of the expression of MHC
class II proteins in OP9-DL1 cells on generating antigen-specific CD4
+
T cells. It was
reported that hematopoietic progenitors derived from fetal liver had dramatically different
kinetics of differentiation from that of progenitors originating from adult bone marrow
(Crompton, Outram et al. 1998; Carvalho, Mota-Santos et al. 2001; Douagi, Vieira et al.
2002; Huang, Garrett et al. 2005). We verified that the yield of T cells generated from
bulk adult bone marrow cells cocultured on OP9-DL1 cells was extremely low; most of
the development of the cells was halted at the DN2 and DN3 stages (Huang, Garrett et al.
2005). When adult progenitor cells were transduced to express pre-arranged OT2 TCR
alpha and beta chains, a majority of the cells (>70%) were committed to the T cell linage
within two weeks of cocultures. CD3 and TCR expressions were detected as early as day
9 of cocultures. The increased commitment of T cells is likely due to the earlier
expression of the OT2 TCR, leading to the bypass of endogenous TCR rearrangement.
This observation is consistent with the result by van Lent et al. (van Lent, Nagasawa et
al. 2007), in which earlier expression of a human TCR in human hematopoietic
progenitors from postnatal thymus drastically enhanced T cell development.
Several interesting features were observed in T cell development in the TCR
transduction cocultures with the expression of the murine MHC class II protein, I-A
b
. We
first observed different expansion kinetics in the presence of I-A
b
. Transduced bone
marrow cells started marked expansion earlier (day 9) in OP9-DL1-IA
b
coculture, but
quickly reached a plateau at day 14. In contrast, a slower expansion occurred in the OP9-
DL1 coculture at day 10, followed by a rapid expansion after day 17. We therefore
55
observed a reduced expansion capacity of OP9 cells upon an enforced expression of I-A
b
;
it remains to be determined whether the interaction between the OT2 TCR and I-A
b
plays
a role in limiting hematopoietic expansion. It was also found that I-A
b
expression in the
OP9-DL1 cell line could accelerate T cell development. Approximately 10% of bone
marrow cells became CD3
+
OT2
+
T cells by day 9 of the OP9-DL1-IA
b
coculture,
compared to around 6% of these T cells appearing in the OP9-DL1 coculture. A close
examination of the development of DN cells revealed that 52% of OP9-DL1-IA
b
-
cocultured bone marrow cells reached the DN4 stage by day 14, whereas only 21% of the
OP9-DL1-cocultured cells were able to arrive at the same stage. Similar acceleration was
also observed in the development of single-positive cells. A majority of the bone marrow
cells (>45%) turned into CD4
+
CD8
+
SP cells in the OP9-DL1-IA
b
coculture at day 22,
while only 18% of the cells from the OP9-DL1 coculture displayed the SP phenotype.
Further experiments are needed in order to elucidate the exact reason for the accelerated
development, although we suspect that TCR signaling induced by I-A
b
engagement may
partially contribute to such an observation.
In vitro culture of OT2 TCR-transduced bone marrow cells resulted in a large
majority (~90%) of OT2 TCR-expressing CD4
+
T cells. Such a dominant representation
of TCR-specific T cells was also reported previously when human CD8
+
TCRs were
introduced to hematopoietic progenitors (van Lent, Nagasawa et al. 2007). In fact, similar
over-representation of TCR-cloned T cells was observed in both TCR-transgenic mice
(von Boehmer 1990) and TCR-retrogenic mice (mice reconstituted by hematopoietic
stem cells transduced by a retroviral vector carrying a TCR gene) (Yang, Qin et al. 2002;
56
Arnold, Burton et al. 2004; Yang and Baltimore 2005). In contrast, transduction of
mature T cells with TCR-retroviral vectors can only generate a modest population of
antigen-specific T cells (Kessels, Wolkers et al. 2001; Morgan, Dudley et al. 2006;
Heemskerk, Hagedoorn et al. 2007). The mispairing between the alpha and beta chains of
the transduced TCR with the beta and alpha chains of endogenous TCRs was proposed to
be the major reason for the low efficiency of redirecting the specificity of mature T cells
by the TCR-transduction method (Cohen, Zhao et al. 2006; Kuball, Dossett et al. 2007).
Thus, our result and others’ results (van Lent, Nagasawa et al. 2007; Zhao, Parkhurst et
al. 2007) clearly suggest that modification of hematopoietic progenitors with a TCR-
retroviral vector represents an efficient way to generate antigen-specific T cells both in
vitro (van Lent, Nagasawa et al. 2007; Zhao, Parkhurst et al. 2007) and in vivo (Yang,
Qin et al. 2002; Arnold, Burton et al. 2004; Yang and Baltimore 2005).
We tested the functional properties of SP CD4
+
T cells generated from the TCR
transduction cocultures. Upon polyclonal anti-CD3/CD28 stimulation, these cells
proliferated, displayed typical surface markers of activated T cells, and secreted the
cytokines IFN-γ and IL-2, indicating that these T cells reached functional maturity and
were able to initiate TCR signaling in response to stimulation. As compared to CD4
+
T
cells developed from the OP9-DL1 coculture, a much higher production of cytokines
(IFN-γ and IL-2) were detected for T cells generated from the OP9-DL1-IA
b
coculture,
suggesting that the provision of interaction between the OT2 TCR and I-A
b
in the
coculture might enhance the functional development of CD4
+
T cells. To examine
whether our OP9 coculture could generate regulatory T cells, which might contribute to
57
the observed discrepancy of cytokine production of cells cultured on OP9-DL1 versus
those cultured on OP9-DL1-IA
b
, we performed intracellular staining of Foxp3 and could
not detect any population of T cells that were CD4
+
Vβ5
+
Foxp3
+
. Experiments are also
underway to identify the molecular difference between these two groups of T cells and to
elucidate the detailed molecular mechanism for the enhanced function. In response to
peptide stimulation, we observed antigen-specific responses by these SP CD4
+
T cells, as
manifested by both activation marker staining and IL-2 secretion. Similar to the results of
polyclonal stimulation, markedly higher production of IL-2 was observed for OT2 CD4
+
T cells cocultured with OP9-DL1-IA
b
. Interestingly, under the current condition of
antigen-specific stimulation, we were unable to detect the production of IFN-γ. We
speculate that the lack of certain costimulatory signals (Kroczek, Mages et al. 2004;
Ward and Kaufman 2007) during the stimulation may result in the failure of IFN-γ
secretion and are designing experiments to test this hypothesis.
In conclusion, we demonstrated an example of in vitro generation of antigen-
specific CD4
+
T cells by retroviral transduction of adult bone marrow cells to express a
CD4
+
TCR. Ectopic expression of a MHC class II protein could accelerate the
development of TCR-transduced bone marrow cells, resulting in functionally improved
antigen-specific CD4
+
T cells. Although our data shows that this represents a promising
approach to obtain CD4
+
T cells with desired specificity in vitro, there is much left to
learn about this stromal system we engineered. Will the provision of MHC class II
molecules on OP9-DL1 have a similar effect on other hematopoietic progenitors, such as
fetal liver cells? We did not observe a significantly altered development pattern for non-
58
transduced bone marrow cells cocultured with either OP9-DL1 or OP9-DL1-IA
b
. Could
this be simply due to the inefficient nature of coculture using bone marrow cells,
disallowing the opportunity to capture the difference? We are addressing this question by
coculturing wild-type progenitors from fetal livers. We are also in the process of fully
examining the functional potential of these CD4
+
T cells by adoptive transfer of these
cells to mice followed by the measurement of the immune response upon antigen-specific
immunization.
2.5 Acknowledgements
We thank April Tai and Lili Yang for critical reading of the manuscript. This
work was supported by a National Institute of Health grant AI068978.
59
Chapter 3: HIV-1 Gag-specific immunity induced by a lentivector-
based vaccine directed to dendritic cells
Portions of this Chapter are adapted from:
Bingbing Dai, Lili Yang, Haiguang Yang, Biliang Hu, David Baltimore and
Pin Wang, Proc. Nat. Acad. Sci. USA (2009) 106: 20382–20387
Lentivectors (LVs) have attracted considerable interest for their potential as a
vaccine delivery vehicle. In this study, we evaluate in mice a dendritic cell (DC)-directed
LV system encoding the Gag protein of human immunodeficiency virus (LV-Gag) as a
potential vaccine for inducing an anti-HIV immune response. The DC-directed specificity
is achieved through pseudotyping the vector with an engineered Sindbis virus
glycoprotein capable of selectively binding to the DC-SIGN protein. A single
immunization by this vector induces a durable HIV Gag-specific immune response. We
investigated the antigen-specific immunity and T-cell memory generated by a
prime/boost vaccine regimen delivered by either successive LV-Gag injections or a DNA
prime/LV-Gag boost protocol. We found that both prime/boost regimens significantly
enhance cellular and humoral immune responses. Importantly, a heterologous DNA
prime/LV-Gag boost regimen results in superior Gag-specific T-cell responses as
compared with a DNA prime/adenovector boost immunization. It induces not only a
higher magnitude response, as measured by Gag-specific tetramer analysis and
60
intracellular IFN-γ staining, but also a better quality of response evidenced by a wider
mix of cytokines produced by the Gag-specific CD8
+
and CD4
+
T cells. A boosting
immunization with LV-Gag also generates T cells reactive to a broader range of Gag-
derived epitopes. These results demonstrate that this DCdirected LV immunization is a
potent modality for eliciting anti-HIV immune responses.
3.1 Introduction
Recombinant adenovirus-based vectors (rAd), used either alone or as a booster
immunization after priming with a DNA plasmid, are among the most potent viral vectors
for inducing human immunodeficiency virus (HIV)-specific T-cell responses in animals
and humans (Barouch and Nabel 2005; Lasaro and Ertl 2009). However, a phase 2b trial
that used recombinant adenovirus serotype 5 (rAd5) vectors as the HIV vaccine carrier
failed to show efficacy (Barouch 2008; McElrath, Rosa et al. 2008). This trial result is
consistent with a preclinical study in which a rAd5-based vaccine expressing a simian
immunodeficiency virus (SIV) Gag antigen failed to lower setpoint viral loads after SIV
challenge of rhesus monkeys (Casimiro, Wang et al. 2005). A recent study by Barouch
and coworkers has shown that a heterologous prime/boost vaccine regimen using a newly
identified rAd26 vector could elicit a strong and high quality immune response in non-
human primates (NHP), resulting in markedly reduced viral loads and decreased AIDS-
related mortality (Liu, O’Brien et al. 2009). This study highlights the importance of
exploring viral vector-based vaccine modalities for development of an effective HIV
vaccine.
61
Efficient antigen delivery to antigen-presenting cells (APCs) and their subsequent
presentation to stimulate virus-specific T cells is vital for the success of a T-cell-based
vaccine. Dendritic cells (DCs) are the most powerful APCs to initiate and maintain
immune responses of T cells (Banchereau and Steinman 1998) and therefore become one
of the major target cells for the HIV vaccine development (Dorrell 2005). Immunization
by adoptive transfer of autologous DCs loaded in vitro with inactivated HIV particles
induced anti-virus immunity in animals (Yoshida, Hayashi et al. 1990) and humans (Lu,
Arraes et al. 2004). However, this is a labor-intensive, personalized medicine approach,
which limits its prospect as a vaccine design to deal with the worldwide AIDS pandemic.
A direct method is to target the delivery of HIV immunogens to DCs in vivo (Tacken,
Vries et al. 2007). Steinman and coworkers reported a strategy to conjugate HIV Gag p24
and p41 onto an antibody to DEC-205, a relatively DC-restricted surface protein, as a
means to load antigens into DCs in vivo for generating an immune response
(Trumpfheller, Finke et al. 2006; Trumpfheller, Caskey et al. 2008). Although with
coadministration of appropriate adjuvants, a strong Gag-specific CD4
+
T-cell response
was elicited (Trumpfheller, Finke et al. 2006), it remains a challenge for this antibody
fusion vaccine to evoke CD8
+
cytotoxic T cells, which are essential for controlling HIV
replication.
We have developed a DC-targeted, lentivector (LV)-based system for delivery of
genetic vaccines in vivo (Yang, Yang et al. 2008). LV is known to be an efficient vehicle
for genetic modification of DCs in vitro (Dullaers and Thielemans 2006), and direct
injection with LV enveloped with glycoproteins with broad tropism is able to induce
62
CD8
+
T-cell responses (Esslinger, Chapatte et al. 2003; Kim, Majumder et al. 2005). To
fully harness the immuno-stimulatory potency of DCs and mitigate off-target effects, we
synthesized a LV enveloped with a Sindbis virus-derived glycoprotein engineered to be
specific to the DC-specific surface protein DC-SIGN [also known as CD209]
(Geijtenbeek, Torensma et al. 2000); DC-SIGN has also been explored by others as the
target receptor of DCs for protein antigen delivery (Tacken, Vries et al. 2007). In our
prior studies, we used ovalbumin (OVA) as a model antigen and found that a single-
round immunization with this vector could result in substantial antigen-specific T-cell
and antibody response (Yang, Yang et al. 2008). In the present study, we show that the
significant HIV Gag-specific immune response can be elicited by this DC-directed LV
used alone or with other modalities.
3.2 Materials and Methods
3.2.1 Mice and vaccination schedule
Six- to eight-week old female BALB/c mice were purchased from Charles River
Breeding Laboratories. All animal procedures were approved by the Department of
Animal Resources of the University of Southern California. The DNA animals were
injected with 50µg of each plasmid DNA intrsmuscularly at 3 weeks intervals for three
injections in total. The ADV and LV mice were injected with replication-defective
adeonoviral vector (10
10
viral particles (v.p.)) or lentiviral vector (5×10
6
transfection
units (TU)) encoding the antigen respectively. The DNA/ADV and DNA/LV animals
63
were given the three DNA injections as described above, followed by the ADV or LV
boost three weeks after the last DNA injection. The LV/LV animals were primed with the
lentiviral vector and boosted with the same vector after four weeks. The DNA, LV and
LV/LV animals were bled and analyzed two weeks after the last injection, the ADV mice
were sacrificed three weeks after the injection, while the immunogenic analyses were
performed 10 days after the final injection for DNA/ADV, DNA/LV animals.
3.2.2 Plasmid construction and lentivector production
SVGmu was prepared as previously described. FUW-gag was constructed from
FUGW by replacing the GFP with the cDNA of HIV-1 gag. Lentivectors were prepared
by transient transfection of 293T cells using a standard calcium phosphate precipitation
protocol. 293T cells cultured in 15-cm tissue culture dishes (Corning or BD Biosciences)
were transfected with the appropriate lentiviral transfer vector plasmid (37.5 mg), along
with 18.75 mg of the envelope plasmid (SVGmu) and the packaging plasmids
(pMDLg/pRRE and pRSV-Rev). The viral supernatants were harvested 48 and 72 h post-
transfection and filtered through a 0.45-mm filter (Corning). To prepare concentrated
viral vectors for in vivo study, the viral supernatants were ultracentrifugated at 50,000g
for 90 min. The pellets were then resuspended in an appropriate volume of cold PBS.
3.2.3 Peptides and peptide pools
The H2-K
d
-restricted immunodominant CTL epitope contained in the p24 portion
of the Gag protein consists of amino acids 197 to 205 (AMQMLKETI). This single
64
peptide was dissolved in dimethyl sulfoxide at 8 mg/ml. Gag peptides are 15 amino acids
long, overlapping by 10-11 amino acids, and spanning the entire HIV-1 consensus
subtype B Gag sequence. Peptides were dissolved in DMSO at 10 mg/ml, and kept at -80
˚C.
3.2.4 MHC class I tetramer staining and phenotypic analysis
The phycoerythrin (PE)-conjugated major histocompatibility complex (MHC)
class I H2-K
d
-AMQMLKETI tetramer complex was obtained from Beckman Coulter. At
indicated time points after vector injection, tetramer staining was performed on mouse
spleen cells. Surface staining was performed by blocking the cells with anti-mouse
CD16/CD32 (clone 2.4G2, BD Pharmingen) followed by incubation with fluorochrome-
conjugated antibodies. FITC-, PE-, PE-Cy5- or APC- conjugated antibodies specific for
mouse CD8, CD44, CD62L were purchased from BD Biosciences. All of the flow
cytometry analysis was done with a FACSort (BD Bioscience) instrument.
3.2.5 Intracellular cytokine staining (ICCS) and multicolor ICS
Mice spleen cells (1×10
6
/sample) were incubated with 4 µg/ml of the HIV Gag
peptide (AMQMLKETI) and 2 µg/ml antibody to CD28 (BD Pharmingen) for 1 h at
37°C in 96-well round-bottom microtiter plate in RPMI medium supplemented with 10%
FBS (Sigma), 10 U/ml of penicillin, 100 µg/ml of streptomycin, and 2 mM glutamine.
Brefeldin (BFA, Sigma-Aldrich) was added at a final concentration of 10 µg/ml, and cells
were incubated for another 4 h. Cells were blocked with anti-mouse CD16/CD32
65
followed by stained with anti-mouse CD8a and anti-mouse CD4 antibodies,
permeabilized in 100 µl of Cytofix/Cytoperm solution at 4°C for 20 min, washed with
Perm/Wash solution, and stained with PE-conjugated anti-mouse gamma interferon (IFN-
γ) at 4°C for 30 min, followed by flow cytometric analysis. Eight-color ICS assays used
pooled HIV-1 Gag peptides. Cells were incubated with the viability dye ViViD and
stained by the following monoclonal antibodies: anti-CD4-PerCP, anti-CD8-APC-Cy7,
anti-CD3-Alexa488, anti-IL-2-PE, anti-IFN-γ-APC, anti-TNF-α-PE-Cy7. All of the
antibodies were purchased from BD Bioscience.
3.2.6 ELISAs
Enzymelinked immunosorbent assay (ELISA) plates were coated with 100 µl of
Galanthus nivalis lectin (10 µg/ml) overnight at 4°C. The lectin solution was removed
from the wells and blocked with 200 µl of PBS containing 10% fetal bovine serum
(PBSS) for 2 h at room temperature. The plates were washed twice with PBS containing
0.2% Tween 20 (PBS-T), and then 100 µl of supernatant from cells transfected with
FUW-gag was added to each well, and wells were incubated for an hour at room
temperature. The plates were washed with PBS-T five times, and then the sera from
immunized mice from different groups were added with threefold dilution (in PBSS) for
1 h. The plates were washed with PBS-T five times, and then 100 µl of 1:10000-diluted
secondary antibody-conjugated horseradish peroxidase was added, and mixtures were
incubated for 1 h and washed with PBS-T five times. Finally, 100 µl per well of
tetramethylbenzidine (TMB) substrate solution (KPL) was added, and the plates were
66
incubated for 45 min at 37°C. The reaction was stopped by adding 100 µl per well of 2 M
H
2
SO
4
. The absorbance at the wavelength of 450 nm (OD
450
) was measured using a plate
reader (Molecular Devices).
3.2.7 Gamma interferon (IFN-γ) ELISpot assay
Elispot assays were performed for IFN-γ with kits from Millipore according to the
manufacturer’s instructions. Briefly, 96-well MultiScreen-IP plates (Millipore,
MSIPS4510) were coated overnight at 4 ˚C with 100 µl of anti-mouse IFN-γ antibody (10
µg/ml in PBS). The antibody was decanted and the plate was blocked with RPMI
medium containing 10% FBS at 37 ˚C for 2 h. Spleen cells from mice were plated at
1×10
5
cells/well in 150 µl complete medium with peptides and incubated for 18 h at 37
˚C and 5% CO
2
. HIV-1 Gag single peptide (4 µg/ml) or pools of peptides (at a final
concentration of 3 µg/ml for each peptide) were used for this assay. The plates were then
washed and 0.5 µg/ml of biotinylated anti-IFN-γ antibody (BD Pharmingen) was added
to the plates, which was incubated at room temperature for 2 h. Plates were then washed
and incubated with 1000-fold-diluted streptavidin-alkaline phosphate conjugated
antibody (Chemicon) for 45 minutes at room temperature. After a final wash, IFN-γ
producing cells were identified by spots development by addition of BCIP/NBTplus
substrate (Millipore).
67
3.3 Results
3.3.1 Immune Responses Generated by Various Routes of Vaccine
Administration
We constructed a Gag-encoding lentiviral backbone plasmid by insertion of Gag
cDNA into FUW (Lois, Hong et al. 2002) downstream of a human ubiquitin C promoter,
a vector designated FUWGag (Fig. 3.1A). LV encoding the Gag immunogen and
pseudotyped with the DC-directed envelope SVGmu was generated in 293T cells by
transient transfection with various plasmids and is designated LV-Gag. We first tested a
range of vector doses [1.25×10
6
~10×10
6
transduction units (TU)] for immunization of
naive mice through footpad injection and found that a dose of 5×10
6
TU generated the
highest percentage of Gag-specific CD8
+
T cells 2 weeks after vaccination. We then
assessed the immunogenic response to this vector dose via different administration
routes. Naive mice were immunized with a single injection by the s.c. (s.c.), footpad
(f.p.), intramuscular (i.m.), i.p. (i.p.), or intradermal (i.d., at the base of tail) route. Gag-
specific T-cell responses were monitored by tetramer analysis and intracellular cytokine
staining (ICCS). The f.p. and i.d. injections resulted in the strongest Gag-tetramer
+
CD8
+
T-cell responses (~3%, Fig. 3.1B, left) 2 weeks post immunization, which is in consistent
with these being the best routes to target skin DCs. The i.p. injection gave the lowest
responses. When splenocytes harvested from vaccinated animals were restimulated in
vitro with the Gag dominant peptide (AMQMLKETI), a similar trend for the pattern of
IFN-γ producing CD8
+
T cells was observed (Fig. 3.1B, right).
68
Figure 3.1: Comparison of immune responses generated from different injection routes
after a single immunization. (A) Schematic representation of a lentiviral backbone
construct encoding the full sequence of a HIV-1 subtype B Gag antigen. R, U5, and ΔU3
are components of the long terminal repeat (LTR) and ΔU3 contains the self-inactivating
deletion; SD: splicing donor; SA: splicing acceptor; ψ and ΔGag: the encapsulation
sequence; RRE: the Rev-responsive element; Ubi: human ubiquitin-C promoter; WPRE:
woodchuck hepatitis virus posttranductional regulatory element. (B) Five groups of
BALB/c mice were immunized with 5×10
6
TU (Transduction Units) of LV-Gag by a s.c.,
footpad (f.p.), intramuscular (i.m.), i.p., or intradermal (i.d.) injection route. Two weeks
post immunization, spleen cells were harvested and analyzed for the frequency of Gag-
specific CD8
+
T cells by H2-K
d
-AMQMLKETI-PE tetramer and CD44 staining. Spleen
cells were also restimulated in vitro with the HIV-1 Gag peptide (AMQMLKETI).
Intracellular cytokine staining (ICCS) was performed to assess the IFN-γ response. (C)
Sera from different groups of mice were harvested 2 weeks postimmunization. IgG and
IgM antibody responses against HIV-1 Gag were detected by ELISA. Each group
consisted of three mice.
69
HIV Gag-specific serum IgG and IgM could be detected 2 weeks
postimmunization with the LV-Gag. The highest IgG titers were obtained from the f.p.,
i.d., and s.c. routes (Fig. 3.1C, left). Interestingly, the IgM titer showed a reverse trend, in
which f.p. and i.d. injections yielded lower IgM production (Fig. 3.1C, right). This
suggests that immunization through these two injection routes yields a significant CD4
+
T-cell response, resulting in efficient isotype switching to convert IgM into IgG. Due to
its potential to induce superior response, the f.p. injection route was chosen for the
subsequent prime/boost and other functional studies.
3.3.2 Enhanced Gag-Specific Immunity by Prime/Boost Regimens
To further characterize the efficacy of the LV-Gag immunization, four cohorts of
mice were injected with PBS, empty LV (lacking the Gag transgene), bone marrow-
derived DCs (BMDCs) loaded with the HIV-1 Gag dominant peptide and matured with
lipopolysaccharide (LPS), or LV-Gag. Two weeks postinjection, we assessed IFN-γ
secreting CD8
+
T cells in freshly harvested splenocytes restimulated in vitro with the Gag
peptide for all of the comparison groups. We observed that the LV-Gag immunized mice
displayed a significant fraction of CD8
+
T cells secreting IFN-γ, with a statistically
significant difference (P<0.01) when compared to the three comparison groups (Fig.
3.2A). The fact that the empty vector was not different from the PBS control suggests
that the Gag-specific CD8
+
T cells elicited by LV-Gag results from the delivery of the
vector-encoded transgene, rather than being elicited by Gag protein that might be carried
70
within the vector particles. In addition, no significant level of epitope-specific responses
was elicited by adoptive transfer of in vitro loaded DCs, which indicates that DC-directed
delivery of Gag antigen by the LV in vivo is a much more potent vaccination method.
Figure 3.2: DC-directed LV can effectively boost HIV-1 Gag-specific immune response.
(A) BALB/c mice were immunized with PBS (●), BMDCs (1×10
6
) loaded by the HIV-1
Gag peptide (AMQMLKETI) (■), empty LV lacking the Gag transgene (5×10
6
TU) (▲),
or LV-Gag (5×10
6
TU) (▼). The immune responses of spleen cells upon restimulation
with the Gag dominant peptide were estimated by IFN-γ ICCS 2 weeks postinjection (*,
P<0.01; **, P<0.001) (B–D) Four vaccine groups received PBS, single immunization of
LV-Gag (LV-Gag once), LV-Gag prime/LV-Gag boost (LV-Gag/LV-Gag), or DNA
prime/LV-Gag boost (DNA/LV-Gag). Splenocytes from vaccinated animals were
analyzed for Gag-specific response by H2-K
d
-AMQMLKETI PE tetramer staining (B),
IFN-γ ICCS (C), and mouse serum ELISAs for IgG and IgM (D). The data shown are
mean values of triplicates ± SD.
71
We next explored the utility of the LV-Gag vector in prime/boost settings. Three
groups of mice received either one dose of LV-Gag vector, a dose of LV-Gag vector
prime followed by a homologous LV-Gag vector boost, or a DNA prime followed by a
LV-Gag vector boost. Two weeks after the final injection, tetramer-positive and IFN-γ
producing CD8
+
T cells were quantified by flow cytometry (Fig. 3.2B). Both assays
showed an enhanced anti-Gag CD8
+
T cells response in the prime/boost animals
compared to the single dose LV-Gag immunization group. Splenocytes from the different
groups of animals were also cocultured with Gag peptide and then examined for IFN-γ
production by an ELISPOT assay (Fig. 3.2C). Obvious enhancement of IFN-γ secretion
was seen in the prime/boost groups, with 4- to 5-fold greater responses than for the single
dose LV-Gag mice. We further measured the titers of HIV Gag-specific IgG and IgM
antibodies in the sera from these animals and found that sera from LV-Gag/LV-Gag mice
and DNA/LV-Gag mice showed higher responses to Gag protein than the single dose
LV-Gag mice (Fig. 3.2D). Collectively, our data demonstrate that the DC-directed LV is
an effective booster of responses initiated by either DNA or LV itself, enhancing CD8
+
T-cell as well as antibody responses.
3.3.3 Comparison of T-Cell Responses Elicited by Lentivector and
Adenovector
We conducted experiments to compare DC-directed LV with the extensively
studied rAd5 for their ability to induce the Gag-specific immune responses. Several
72
groups of naive mice were immunized with a DNA prime/LV-Gag boost, LV-Gag
prime/LV-Gag boost, or DNAprime/rAd5-Gag boost. Following the last immunization,
comparable frequencies of IFN-γ producing and Gag-specific CD8
+
T cells were detected
in splenocytes of the prime/boost vaccine groups (Fig. 3.3A). Presumably due to the high
rAd5 vaccine dose (10
10
VP) used in this study, we found that a single rAd5-Gag
immunization was about as good as the prime/boost regimens.
Figure 3.3: Comparison of magnitude, kinetics and memory responses of Gag-specific
CD8
+
T cells after immunization with LV-Gag and rAd5-Gag. Six groups of BALB/c
mice received the following vaccination regimens: PBS, LV-Gag (LV, 5×10
6
TU), rAd5-
Gag (rAd5, 10
10
VP), LV-Gag prime/LV-Gag boost (LV/LV), DNA prime/LV-Gag boost
(DNA/LV), and DNA prime/rAd5-Gag boost (DNA/rAd5). Vaccine-induced HIV Gag-
specific immune responses were analyzed by: (A) percentage of IFN-γ or Gag-tetramer-
positive CD8
+
T cells (*, P < 0.05; **, P < 0.005); (B) kinetics of the total frequency of
IFN-γ producing CD8
+
T cells of LV-Gag and rAd5-Gag groups on indicated time points
after immunization; and (C) division of central memory (T
CM
, CD44
high
CD62L
+
) and
effector memory (T
EM
, CD44
high
CD62L
-
) CD8
+
T cells of LV-Gag (LV) and rAd5-Gag
(rAd5) groups by surface staining.
73
Kinetic analysis of the early responses to single immunization showed that the
magnitude of Gag-specific CD8
+
T-cell immunity with LV-Gag immunization declined
after week 2 to approximately 1% IFN-γ producing CD8
+
T cells by 4 weeks post-
vaccination (Fig. 3.3B). In contrast, the primary response to the rAd5-Gag vaccine
reached a higher level at week 2, and active T cells were continuously expanded through
week 4 (Fig. 3.3B).
The memory phenotype of the Gag-specific CD8
+
T cells elicited by different
regimens was studied by scoring the memory differentiation markers CD44 and CD62L.
After gating on Gag-tetramer
+
CD8
+
T cells among splenocytes from LV-Gag vaccinated
mice, approximately 30% of them exhibited the central memory phenotype of
CD44
hi
CD62L
+
, which was higher than the approximately 12% obtained from mice
immunized with the rAd5-Gag vector (Fig. 3.3C). The LV-Gag immunized cells also
displayed a discrete very high CD62L population lacking in the rAd5-Gag immunized
mice. This result suggests that the DC-targeted LV is more potent than rAd5 for
induction of high quality memory T cells.
3.3.4 Multifunctional CD4
+
and CD8
+
T-Cell Responses Elicited by
Lentivector
We examined the capacity of individual HIV-specific T cells to produce multiple
cytokines, a parameter which was shown to correlate with a cell’s ability to protect
against infection in certain models (Darrah, Patel et al. 2007). We selected the LV-
Gag/LV-Gag, DNA/LV-Gag, and DNA/rAd5-Gag immunization regimens for the study
74
because they were able to generate sufficiently high levels of responses to allow a reliable
multifunctionality analysis. Splenocytes harvested from vaccinated animals were
restimulated with a pool of 123 overlapping peptides covering the entire Gag protein.
Intracellular cytokine levels were measured by multiparameter flow cytometry to assess
the ability of single cells to produce various combinations of IFN-γ, interleukin (IL)-2
and tumor necrosis factor (TNF)-α. As shown in Fig. 3.4A, although the DNA/rAd5-Gag
elicited CD4
+
T cells that were single-positive for IFN-γ, IL-2, or TNF-α, and double-
positive for IFN-γ and TNF-α, there was no detectable level of these cells that were IFN-
γ
+
IL-2
+
, IL-2
+
TNF-α
+
, or IFN-γ
+
IL-2
+
TNF-α
+
. In contrast, both the LV-Gag/LV-Gag and
DNA/LV-Gag regimens generated substantial percentages of CD4
+
T cells that were
IFN-γ
+
/TNF-α
+
, IFN-γ
+
/IL-2
+
, and IL-2
+
/TNF-α
+
. Especially, the DNA/LV-Gag group
induced a high frequency of CD4
+
T cells secreting three cytokines simultaneously (6.4%
of the responding cells). The distribution of Gag-specific CD8
+
T cells by various
regimens showed the same pattern (Fig. 3.4A). Compared with the DNA/rAd5-Gag
immunization, a substantially greater proportion of the LV-Gag/LV-Gag- or
DNA/LVGag- elicited CD8
+
T cells were able to secrete multiple cytokines, with
approximately 3% of responding cells from the DNA/LV-Gag vaccine producing three
cytokines. Interestingly, we found that CD4
+
T cells were more multipotent than CD8
+
T
cells. Examining the IL-2 secreting CD4
+
T cells, we see that although the overall
frequency of such cells was highest following DNA/rAd5-Gag immunization, only a
slight portion of them secreted more than one cytokine (i.e., 2.8% of them are IL-2
+
TNF-
α
+
) (Fig. 3.4B). On the contrary, at least half of the IL-2 secreting CD4
+
T cells from both
75
DNA/LV-Gag and LV-Gag/LV-Gag vaccination were multicytokine producers, and a
notable portion of them were able to generate three cytokines. Thus, both the CD4
+
and
CD8
+
T cells induced by immunization involving DC-directed LV were more
polyfunctional than those generated by the DNA/rAd5-Gag vaccine.
Figure 3.4: Generation of multifunctional CD4
+
and CD8
+
responses by prime/boost
immunization regimens. Splenocytes of DNA/rAd5-Gag (DNA/rAd5), LV-Gag/LV-Gag
(LV/LV) and DNA/LV-Gag (DNA/LV) groups of BALB/c mice were stimulated with
the pooled HIV-1 Gag peptides (2.5 µg/mL for each peptide) for 6 h, and analyzed by an
eight-color ICCS assay to assess: (A) the fraction of total responding CD4
+
or CD8
+
T
cells expressing each of the seven possible combinations of IFN-γ, IL-2, and TNF-α; and
(B) the frequency and proportion of responding CD4
+
T cells expressing all three
cytokines (IFN-γ
+
IL-2
+
TNF-α
+
: γ+2+α+), two cytokines (IFN-γ
-
IL-2
+
TNF-α
+
: γ-2+α+ ;
or IFN-γ
+
IL-2
+
TNF-α
-
: γ+2+α-), or one cytokine (IFN-γ
-
IL-2
+
TNF-α
-
: γ-2+α-).
76
3.3.5 Breadth of T-Cell Responses Induced by Various Immunization
Regimens
To assess the breadth of the induced T-cell responses, we generated a peptide matrix as
shown in Fig. 3.5A (Gaviolia, Cellinia et al. 2008). A library of peptides covering the
entire HIV-1 Gag protein was divided into 23 pools named P1-P23, with each peptide
present in two independent pools. The splenocytes of mice immunized with LV-Gag/LV-
Gag, DNA/LV-Gag, and DNA/rAd5-Gag were stimulated by one of the peptide pools,
and then assayed by IFN-γ ELISPOT. In contrast to the T cells from DNA/rAd5-Gag
immunized mice, those from LV-Gag/LV-Gag mice responded to many peptides. Taking
an ELISPOT cut-off at 80 SFC (spot forming cells)/0.1 million cells, mice immunized
with LV-Gag/LV-Gag responded to eight peptide pools (P4, P5, P6, P9, P10, P15, P17,
and P18) (Fig. 3.5 B and C), while the DNA/LV-Gag and DNA/rAd5-Gag mice only
vigorously responded to three pools (P4, P5, and P17) (Fig. 3.5 B and C). We were able
to verify the response of LV-Gag/LV-Gag T cells to 11 individual peptides derived from
these responding eight peptide pools. However, when the DNA/rAd5-Gag did respond to
a peptide pool, its response was higher than that of the mice receiving LV-Gag. There
were 15 different peptide pools identified as nonreactive (refers to the ELISPOT reading
<20 SFC/0.1 million cells) to cells from the DNA/rAd5-Gag immunized mice. However,
none of the pools were found to be nonreactive for either the LV-Gag/LV-Gag or
DNA/LV-Gag induced T cells (Fig. 3.5B). We also conducted an ICCS analysis of the
LV-Gag/LV-Gag splenocytes stimulated by two representative peptide pools (P6 & P10),
77
Figure 3.5: Breadth of HIV-1 Gag-specific responses to LV-Gag-based vaccination. (A)
A library of 123 15-mer peptides spanning the entire HIV-1 subtype B Gag sequence was
divided into 23 pools (P1–P23) as indicated by the peptide matrix table. (B and C) Spleen
cells of DNA/rAd5-Gag, LV-Gag/LV-Gag, and DNA/LV-Gag groups of BALB/c mice
were harvested, stimulated with one of peptide pools for 18–24 h, and assayed by IFN-γ
ELISPOT. Each group consisted of three mice. Number of highly reactive peptide pools
versus number of nonreactive peptide pools for each group of mice was summarized as
shown in (B). The threshold for defining a nonreactive peptide pool was based on the
ELISPOT readout comparable with the control PBS group. A peptide pool was defined as
highly reactive when its stimulated response was 5 times higher than that of the control
PBS reading and could be obviously detected by an ICCS assay.
and found that the ratio of Gag-specific CD8
+
vs. CD4
+
T-cell responses for both pools
was approximately 3:1 (data not shown). The above results indicate that the Gag-specific
T cells generated by LV-Gag-involved vaccination regimens (LV-Gag/LV-Gag and
78
DNA/LV-Gag) can recognize a broader range of epitopes as compared to the T-cell
response induced by the DNA/rAd5-Gag strategy. The DNA/rAd5-Gag-immunized mice
gave a high total response but one much more focused on immune-dominant
determinants.
3.4 Discussion
We have previously shown that one can build a replication deficient LV that
targets a gene of interest directly to DCs in an animal to induce antigen-specific immune
responses (Yang, Yang et al. 2008). In this study, we examine a special case, the
production of T cells and antibodies against the Gag protein of HIV. This is of particular
interest because attempts to make a vaccine against HIV have failed thus far and a new
and more effective vector system is needed (Barouch 2008; Walker and Burton 2008).
When this vector was used to deliver Gag immunogen (LV-Gag), a significant quantity of
Gag-specific CD8
+
T cells could be detected upon a single injection. We performed a
direct comparison of the LV-Gag with rAd5-Gag with the respect to magnitude, kinetics
and the memory nature of the induced cellular immune response. A single round
immunization of rAd5-Gag induced a stronger immunity than LV-Gag at the chosen
doses. The time-course measurement showed that LV-Gag resembled a usual immune
response of expansion and contraction, but rAd5-Gag provoked a persistent response.
This persistent immunization of rAd-Gag vector might stem from the continuously active
transcription of adenovirus vector at the site of injection (Tatsis and Ertl 2004) and could
79
generate a challenge for generating high quality memory T cells (Lasaro and Ertl 2009).
This unusually prolonged response of rAd5-Gag can compromise its utilities for repeated
immunization [such a protocol was used by Merck’s STEP trial (McElrath, Rosa et al.
2008)] because the sustained APCs and neutralizing antibodies may inhibit responses
from further homologous vaccination. As compared with the rAd5-Gag vector, LV-Gag
induced a greater percentage of Gag-specific memory T cells that were of the central
memory phenotype (T
CM
: CD44
high
CD62L
+
).
Heterologous prime/boost strategies have been well studied for AIDS vaccines,
especially with a rAd5-based vector (Wu, Kong et al. 2005). Unlike the adenoviral
vector, the DC-directed LV is less likely to be restricted by the preexisting immunity,
thus we tested its application for both homologous and heterologous vaccination
regimens. We demonstrated that the DNA/LV-Gag as well as LV-Gag/LV-Gag displayed
a remarkable enhancement of vaccine efficacy for generation of HIV-specific T-cell and
antibody responses. Although generation of a robust CD8
+
T-cell response is one
requirement for a HIV vaccine, the magnitude itself is not necessarily predictive of a
superior control of HIV infection in many individuals (Kaufmann, Bailey et al. 2004).
Therefore, we further investigated the functional potency and breadth of LV-Gag-induced
T-cell responses. The CD8
+
T cells from the DNA/rAd5-Gag regimen were primarily
IFN-γ
+
, TNF-α
+
, and IFN-γ
+
TNF-α
+
cells, with few of them secreting IL-2. The ability of
CD8
+
T cells to generate IL-2 could be significant because it should allow them to
survive and expand (Gattinoni, Klebanoff et al. 2005). Moreover, the central memory
CD8
+
T cells which home to lymphoid organs are thought to produce IL-2, while the
80
effector counterparts are restricted to peripheral tissues and primarily secrete IFN-γ (Sun,
Schmitz et al. 2006). Promisingly, our data indicate that there is a significant portion of
HIV-specific CD8
+
T cells generated by either DNA/LV-Gag or LV-Gag/LV-Gag that
are IL-2 producers. Notably, approximately 3% of the cytokine-producing DNA/LV-
Gag-induced CD8
+
T cells produced three cytokines.
CD4
+
T cells, especially the polyfunctional ones, are of great importance to
vaccine responses (Yamamoto, Iwamoto et al. 2009). Consistent with the results of Sun et
al. (Sun, Schmitz et al. 2006), we observed that IFN-γ and TNF-α production dominated
the CD4
+
T-cell population induced by DNA/rAd5-Gag immunization. The functional
profiles of Gag-specific CD4
+
T cells elicited by DNA/LV-Gag and LV-Gag/LV-Gag
were different from that of DNA/rAd5-Gag. This may not be too surprising, considering
that LV-Gag and rAd5-Gag target different cell populations through distinct cellular
receptors and are likely to mediate different forms of antigen presentations. IL-2
+
TNF-α
+
cells represented the highest portion of CD4
+
T cells in the DNA/LV-Gag and LV-
Gag/LV-Gag groups, with IFN-γ
+
IL-2
+
, IFN-γ
+
TNF-α
+
, and IFN-γ
+
IL-2
+
TNF-α
+
Gag
specific CD4
+
T cells at detectable levels. Several studies on the SIV-macaque model
revealed that prolonged survival of infected monkeys was associated with the
preservation of SIVspecific CD4
+
T cells producing IFN-γ, IL-2, and TNF-α (Kaufmann,
Bailey et al. 2004; Letvin, Mascola et al. 2006; Liu, O’Brien et al. 2009). The frequency
of CD4
+
T cells producing three cytokines simultaneously was also positively related to
protection against Leishmania major infection (Darrah, Patel et al. 2007). Furthermore,
CD4
+
T cells were reported to be indispensable for secondary CD8
+
T-cell expansion,
81
and the depletion of them during the priming phase led to deficient development of
functional CD8
+
T-cell memory (Janssen, Lemmens et al. 2003; Gattinoni, Klebanoff et
al. 2005). The role of CD4
+
T cells is particularly important in a prime/boost vaccination,
because CD4
+
T cells help establishment of CD8
+
T-cell functionality and expansion in
the boost phase of immunization (Shedlock and Shen 2003). Balanced CD8
+
and CD4
+
T-cell responses are thought to be highly desirable for vaccine effectiveness, and were
suggested by Liu et al. to explain the efficient priming by rAd26 vectors (Liu, O’Brien et
al. 2009). This cytokine profile study reveals that DNA/LV-Gag is very effective regimen
to produce both multifunctional CD8
+
and CD4
+
HIV-specific T cells in mice.
We further assessed the breadth of antigen recognition displayed by vector-
induced T cells. Our experiments showed that there were three peptide pools that elicited
the most vigorous responses for all three groups of mice given a prime/boost regimen.
Although magnitude of DNA/rAd5-Gag response to the three dominant peptide pools
was the highest, there was a greater diversification of immunogen recognition by the
DNA/LV-Gag and LV-Gag/LV-Gag regimens. We speculate that through the DC
targeting, the vaccination involved with LV-Gag might load and present antigens more
efficiently in the DCs, allowing the generation of broader responses (Wu, Kong et al.
2005). Our polyfunctional study also supports the notion that this wider epitope response
might be the result of a better CD4
+
T-cell response.
In summary, we report an effort to evaluate an anti-HIV vaccination involving a
LV directed to DCs. We found that both the DNA/LV-Gag and LV-Gag/LV-Gag
vaccination regimens elicited multifunctional CD4
+
and CD8
+
Gag-specific T cells, and
82
the DNA/LV-Gag method generated the highest frequencies of CD4
+
and CD8
+
cells
secreting three cytokines simultaneously. Homologous or heterologous immunization
using LV-Gag-induced T cells recognizing a wide range of Gag epitopes. This study in
mice demonstrates that this DC-targeted LV is a promising vector system and should
warrant further investigations in NHP to continue the evaluation of its potential for future
human HIV/AIDS vaccine development.
3.5 Acknowledgements
We thank Dr. Gary Nabel for providing reagents and Dr. Larry Corey for an
initial discussion of the breadth of antigen responses. This research was generously
supported by grants from the National Institutes of Health, the Skirball Foundation, and a
Grand Challenges in Global Health grant from the Bill and Melinda Gates Foundation.
83
Chapter 4: PD-1/PD-L1 Blockade Can Enhance HIV-1 Gag-
specific T Cell Immunity Elicited by Dendritic Cell-Directed
Lentiviral Vaccines Exhaustion of CD8
+
T cells and upregulation of programmed death 1 (PD-1), a
negative regulator of T cell activation, is one of characteristic features of individuals
chronically infected with human immunodeficiency virus type 1 (HIV-1). In a previous
study, we showed in mice that a dendritic cell (DC)-directed lentiviral vector system
(DCLV) encoding the HIV-1 Gag protein (DCLV-Gag) was an efficient vaccine modality
to induce durable Gag-specific T cell immune responses. In this study, we demonstrate
that blocking of the PD-1/PD-L1 inhibitory signal via an anti-PD-L1 antibody (αPD-L1)
generated an enhanced HIV-1 Gag-specific CD8
+
immune response following a single
round of DCLV immunization. DCLV also synergized with αPD-L1 as a booster regimen
to the homologous prime immunization, resulting in rapid expansion of Gag-specific
CD8
+
T cells. The prime/boost regimen combined with PD-1 blockade generated Gag-
specific CD8
+
T cells comprising several valuable features: improved ability to produce
multiple cytokines, responding to a broader range of Gag-derived epitopes, and long-
lasting memory. The increased cellular immune response generated by DCLV
immunization combined with αPD-L1 was correlated with an improved viral control
following a vaccinia virus challenge. Taken together, our studies offer evidence to
support the use of PD-1/PD-L1 blockade as an adjuvant modality to enhance antigen-
specific immune responses elicited by T cell-based immunizations such as DCLV.
84
4.1 Introduction
The failure of immune system to control chronic infections and the difficulty in
developing effective vaccines against such infections have been thought to be associated
with defects in cytotoxic T lymphocyte (CTL) activation, owing to continuous antigenic
stimulation (Trautmann, Janbazian et al. 2006). The exhaustion of virus-specific CD8
+
T
cells, which was first reported in lymphocytic choriomeningitis virus (LCMV) infection,
has been observed in other chronic infections as well, including human
immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), hepatitis B virus
(HBV), and hepatitis C virus (HCV) models (Trautmann, Janbazian et al. 2006; Petrovas,
Price et al. 2007; Zhang, Zhang et al. 2007; Larrubia, Benito-Martínez et al. 2011). One
signature of dysfunctional CD8
+
T cells is their upregulation of certain inhibitory
molecules. Programmed death-1 (PD-1), a transmembrane immunoreceptor of CD28
family, has been identified as one of these key negative regulators of T cell functions
(Khoury and Sayegh 2004). PD-1 is expressed by a range of activated immune cells, including CD8
+
and
CD4
+
T cells, B cells, and natural killer (NK) cells. It binds to two known ligands, PD-L1
and PD-L2, with PD-L1 more widely expressed on T cells, B cells, dendritic cells (DCs),
macrophages, and certain tumor cells (Chen 2004). A number of studies have identified
that PD-1 up-regulation is correlated with functional impairment of virus-specific CD8
+
T cells during persistent infections (Day, Kaufmann et al. 2006; D’Souza, Fontenot et al.
2007; Petrovas, Price et al. 2007). Therefore, blocking PD-1-mediated pathway to restore
the function of exhausted antigen-specific CD8
+
T cell provides a therapeutic means to
85
alleviate these infections (Barber, Wherry et al. 2006; Ha, Mueller et al. 2008; Velu,
Titanji et al. 2009). Evidence from LCMV-infected mouse model showed that therapeutic
vaccination with PD-L1 blockade enhanced epitope-specific CD8
+
T cell response and
promoted viral clearance (Ha, Mueller et al. 2008). In a SIV-infected macaque study,
Velu et al. observed expansion of SIV-specific polyfunctional CD8
+
T cells, as well as B
cell proliferation and an increase in antibody titer following the treatment with PD-1
blockade (Velu, Titanji et al. 2009). The blockade was demonstrated to be effective
during both the early and the late infections. In a study attempting to evaluate the
potential role of PD-1/PD-L1 pathway in HIV infection, PD-1 expression level was
positively associated with HIV-specific CD8
+
T cell impairment and plasma viraemia
(Day, Kaufmann et al. 2006). Thus, targeting the PD-1/PD-L immunosuppressive
pathway can be a potent strategy for treating chronic infections and development of
effective vaccines (Macatangay and Rinaldo 2009).
PD-1 induces an inhibitory signal through the phosphylation of its
immunoreceptor tyrosine-based switch motif (ITSM) within its cytoplasmatic tail (Ha,
West et al. 2008). Subsequent recruitment of Src homology 2 domain-containing
phosphatases (SHIPs) leads to direct dephosphorylation of other signaling intermediats,
and therefore inhibits certain downstream cellular signaling events. The up-regulation of
PD-1 expression and its engagement to its ligands reduce the ability of T cells to undergo
activation, proliferation, and cytokine production (Shin, Yoshimura et al. 2005).
Moreover, PD-1 molecule is shown to be involved in regulating T cell tolerance and
autoreactive B cells (Fagarasan and Honjo 2000; Yang, Riella et al. 2011). Evidence
86
show that PD-1 mRNA is highly expressed in CD4
+
CD25
+
regulatory T cells and anergic
T cells, and PD-1-deficient mice exhibit augmentation of autoantibodies to heart tissue
and develop dilated cardiomyopathy (Najafian and Khoury 2003; Sharpe, Wherry et al.
2007). Thus, the PD-1/PD-L pathway plays a critical role in regulating a balanced T cell
response.
Dendritic cells (DCs) are professional antigen presenting cells that are responsible
for mounting and modulating the adaptive immune response (Trumpfheller, Finke et al.
2006). Numerous approaches have been developed to generate various forms of potent
DC-based vaccines, such as targeting specific DC populations (Trumpfheller, Finke et al.
2006), involvement of molecular ajuvants (Arruda, Chikhlikar et al. 2004; Bonifaz,
Bonnyay et al. 2004; MacLeod, McKee et al. 2011), administration of molecules to
inhibit signaling involved in attenuating DC activation (Peggs, Quezada et al. 2008). In
our previous studies, we reported a lentiviral vector (LV) enveloped with a Sindbis virus-
derived glycoprotein engineered to be specific for DCs (Yang, Yang et al. 2008), this
vector system was designated as DCLV. The DCLV-based vaccine was able to elicit
potent HIV Gag-specific immune responses (Dai, Yang et al. 2009). In this study, we
evaluated a combinational vaccine strategy that involves a DCLV-based vaccine along
with blocking the PD-1/PD-L1 pathway. For PD-1 blockade to take place, we
administered an antibody specific to mouse PD-L1 (αPD-L1) to suppress the interaction
between PD-L1 and its cognate receptor PD-1. When used at the primary phase of
immunization delivered by DCLV encoding HIV-1 Gag (DCLV-Gag), αPD-L1
significantly enhanced the generation of Gag-specific and cytokine-producing CD8
+
T
87
cells. We further demonstrated that a second dose of DCLV-Gag combined with αPD-L1
could robustly boost a homologous priming response. The responding Gag-specific CD8
+
T cells displayed an increased ability to produce multiple cytokines, a broader T cell
profile capable of recognizing multiple Gag epitopes, and enhanced capacity to control
virus infection following a vaccinia virus challenge.
4.2 Materials and Methods
4.2.1 Mice and vaccination procedure
Six- to eight-week old female BALB/c mice were purchased from Charles River
Laboratories (Wilmington, MA). All animal procedures were performed in accordance
with the guidelines set by the National Institute of Health and the University of Southern
California on the Care and Use of Animals. For the single dose DCLV vaccination
protocol, 5×10
6
TU (Transduction Units) of DCLV-Gag was given through footpad
injection along with administration of rat anti-mouse PD-L1 antibody (clone 10F.9G2,
BioXcell, West Lebanon, NH) or rat IgG2b isotype antibody control. For the DCLV-Gag
prime/DCLV-Gag boost with PD-L1 blockade vaccinations, mice were primed with the
DCLV-Gag (5×10
6
TU) via footpad injection, and four weeks thereafter, they were
boosted with the same vector and dose via subcutaneous injection (at the base of tail) in
combination with the treatment of anti-mouse PD-L1 or isotype control antibodies. For
the antibody treatment, 200 µg of anti-mouse PD-L1 or isotype control antibodies were
88
administered intraperitoneally every 3 days with totaling 3 times, starting on the day of
vector immunizaiton.
4.2.2 Lentiviral vector production
The plasmid encoding the DC-targeted envelope SVGmu was constructed as
previously described (Yang, Yang et al. 2008). FUW-Gag was constructed by insertion of
the cDNA of a HIV-1 subtype B Gag into the lentiviral backbone plasmid FUW
downstream of the human ubiquitin C promoter (Dai, Yang et al. 2009). To efficiently
produce this LV (termed DCLV-Gag), human embryonic 293T cells were seeded at 18
million per 15-cm tissue culture dish (BD Biosciences, San Jose, CA) and grown for 18-
20 hours. 293T cells were then co-transfected with the lentiviral backbone plasmid FUW-
Gag, the SVGmu-encoding envelope plasmid, and the packaging plasmids
(pMDLg/pRRE and pRSV-Rev) using a standard calcium phosphate precipitation
protocol. The viral supernatant was harvested twice at both 48 and 72 hour post-
transfection, combined and filtered through a 0.45 µm filter (Corning, NY). The
concentrated viral pellets were obtained after ultracentrifugation of the viral supernatants
at 50,000×g for 90 minutes, and were then resuspended in an appropriate volume of cold
HBSS for in vivo study.
4.2.3 Gag peptide and peptide pools
The immunodominant H2-K
d
-restricted CD8
+
T cell epitope (AMQMLKETI, aa
197-205) is located in the p24 portion of the Gag protein. This peptide was synthesized
89
(GenScript, Piscataway, NJ) and dissolved in dimethyl sulfoxide (DMSO) at 8 mg/mL.
The Gag peptide libraries contain 123 overlapping 15-mer peptides, and span the entire
HIV-1 subtype B Gag sequence. Individual peptides in the libraries were dissolved in
DMSO at 10 mg/mL, and stored at -80 ˚C. The HIV-1 Gag libraries were divided into 23
pools of 11 to 12 peptides as illustrated (Figure 5A).
4.2.4 Tetramer staining
The phycoerythrin (PE)-conjugated major histocompatibility complex (MHC)
class I tetramer H2-K
d
-AMQMLKETI was obtained from Beckman Coulter (Fullerton,
CA). Spleen cells and lymph node cells were harvested from vaccinated and control
mice. In some experiments, blood was drawn from the retro-orbital venous plexus of
mice periodically. Following lysis buffer treatment to remove red blood cells, the surface
staining was performed by blocking the Fcγ receptors of cells with an anti-mouse
CD16/CD32 antibody (clone 2.4G2, BD Biosciences), followed by incubating the cells
with tetramer in conjunction with FITC-anti-CD44 and PE-Cy5-anti-CD8 (BD
Biosciences) antibodies. The cells were washed and resuspended in PBS. Samples were
analyzed using either the FACSort or the FACSCalibur instrument (BD Biosciences).
4.2.5 Intracellular cytokine staining
Splenocytes (1×10
6
/sample) were cultured for 5 hours at 37°C in a 96-well round-
bottom plate in RPMI medium supplemented with 10% FBS (Sigma, St. Louis, MO), 10
U/mL of penicillin, 100 µg/mL of streptomycin, and 2 mM glutamine. The HIV-1 Gag
90
peptide (AMQMLKETI) (4 µg/mL) was added to stimulate T cells for 6 hours, and
GolgiPlug (0.67 µL/mL) was supplemented in the culture to accumulate intracellular
cytokines. After washing, restimulated cells were incubated with anti-mouse CD16/CD32
antibody, followed by surface stained with anti-mouse CD8 and anti-mouse CD4
antibodies. Cells were then permeabilized in 100 µL of Cytofix/Cytoperm solution (BD
Bioscience) at 4°C for 20 minutes, washed with Perm/Wash buffer (BD Bioscience), and
stained with PE-conjugated anti-mouse IFN-γ at 4°C for 30 minutes. The flow cytometry
analysis was carried out using the FACSort instrument from BD Biosciences.
4.2.6 Multiparameter intracellular cytokine staining
Spleen cells (1×10
6
/sample) were stimulated with the HIV-1 Gag peptide libraries
(2.5 µg/mL for each peptide) in the presence of costimulatory anti-CD28 antibody (2
µg/mL, BD Biosciences) and anti-CD49d (2 µg/mL, BD Biosciences) for 6 hours at 37°C
in a 96-well round-bottom plate. Brefeldin A (BFA, Sigma) was added (10 µg/mL) for
the last 4 hours to inhibit cytokine exporting. The multiparameter intracellular cytokine
staining procedure was similar to the procedure of above intracellular cytokine staining,
except that the cells were stained with the following surface monoclonal antibodies: anti-
CD4-PerCP, anti-CD8-APC-Cy7, anti-CD3-Alexa488, and intracellular monoclonal
antibodies: anti-IL-2-PE, anti-IFN-γ-APC, anti-TNF-α-PE-Cy7. The data was acquired
on a BD LSR II flow cytometry (BD Biosciences). All of the staining antibodies were
purchased from BD Bioscience.
91
4.2.7 IFN-γ enzyme-linked immunospot assay
The enzyme-linked immunospot assay (ELISPOT) was conducted with the
Millipore IFN-γ ELISPOT kit (Billerica, MA) according to the manufacturer’s
instruction. Briefly, 96-well MultiScreen-IP plates were coated with 100 µL/well of anti-
mouse IFN-γ antibody (10 µg/mL in PBS) and stored overnight at 4 ˚C. The plate was
decanted and blocked with RPMI medium containing 10% FBS at 37 ˚C for 2 hours.
Splenocytes from mice were plated at 1×10
5
cells/well in 150 µL complete medium with
addition of e HIV-1 Gag single peptide (2 µg/mL) or pools of peptides (at a final
concentration of 3 µg/mL for each peptide) for this assay. After 18 hours incubation at 37
˚C, plates were washed, and coated with 0.5 µg/mL of biotinylated anti-IFN-γ detection
antibody (BD Biosciences) for 2 hours at room temperature. Plates were further washed
and incubated with the 1000-fold-diluted streptavidin-alkaline phosphate conjugate for 45
minutes at room temperature. After a final extensive wash, spots were identified by
addition of BCIP/NBTplus substrate, and the number of IFN-γ producing cells was
quantified by an Zeiss ELISPOT reader.
4.2.8 Vaccinia virus and challenge of mice
Vaccinia virus encoding Gag (vv-Gag, NIH AIDS Research & Reference Reagent
Program, Rockville, MD, contributed by Dr. D Kuritzkes) was propagated in CV-1 cells,
and the virus titer was evaluated by the standard plaque forming assay on CV-1 cells.
Groups of BALB/c mice received DCLV-Gag prime followed by DCLV-Gag boost with
or without PD-1 blockade vaccinations. Two weeks after the boost immunization, mice
92
were challenged with 10
7
PFU of vv-Gag intraperitoneally (i.p.). The animals were
inspected daily to monitor the body weight for 7 days. On day 7 after challenge, mice
were sacrificed, and the ovaries were harvested, disrupted by homogenization. Vaccinia
virus titers in ovaries were evaluated by plating serial log dilutions of extracts on a
density of 5×10
5
CV-1 indicator cells for 48 hours. After 2 days, the media was removed,
CV-1 cell monolayer was stained with 0.1% crystal violet (Sigma, St. Louis, MO), and
vaccinia virus plaques were quantified.
4.3 Results
4.3.1 PD-1 blockade in combination with DCLV immunization increases
frequency of Gag-specific CD8
+
T cells
To examine the potential role of PD-1/PD-L1 pathway on eliciting antigen-specific T cell
immunity, we first investigated the effect of in vivo blockade of the PD-L1 molecule on
the generation of T cell immune responses from a DCLV-based vaccine for HIV-1 Gag.
This provides a means to evaluate its impact on generating a primary immune response
and eventually a memory (Velu, Titanji et al. 2009). BALB/c mice were immunized with
5×10
6
Transduction Units (TU) of DCLV-Gag, in the presence of anti-PD-L1 blocking
antibody (αPD-L1), or an isotype antibody as a control. The antibodies were administered
for three times (50 µg each time) at three-day interval, starting from the day of vector
93
immunization. Two weeks after vaccination, the animals were sacrificed, and cells from
spleens and lymph nodes were collected for analysis of Gag-specific T cell responses. A
Figure 4.1 In vivo PD-L1 blockade in combination with DCLV immunization increased
frequency of HIV-1 Gag-specific CD8 T cells. BALB/c mice were immunized via
footpad injection with 5×10
6
TU of DCLV-Gag, along with intraperitoneal (i.p.) injection
of anti-mouse PD-L1 or isotype antibodies three times at every three days, starting from
the date of immunization. Two weeks after vector vaccination, the number of Gag-
specific CD8
+
T cells in splenocytes was measured by H2-K
d
-AMQMLKETI-PE
tetramer and CD44 staining. (A). The frequency of IFN-γ-producing cells was assessed
by ELISPOT (B). Representative FACS plots showing the Gag tetramer
+
CD8
+
T-cell
population in spleen (SP) and lymph nodes (LN) were depicted in (C). Each group
consisted of three mice. The p values are calculated using Student’s t test.
94
staining assay was conducted utilizing a major histocompatibility complex (MHC) class I
tetramer specific for a Gag epitope (AMQMLKETI). In consistent with our previous
study (Dai, Yang et al. 2009), DCLV immunization alone induced potent expansion of
Gag-specific CD8
+
T cells in spleens (~3%) (Fig 4.1A and C). Notably, in vivo PD-1
blockade by αPD-L1 resulted in a marked increase in the Gag-specific CD8
+
T cell
frequency (~7%) (Fig 4.1A and C). ELISPOT assay showed that more IFN-γ-secreting
cells were present in the group with PD-1 blockade in respond to in vitro stimulation with
the Gag epitope peptide (Fig 4.1B). We then investigate tetramer-positive CD8
+
T cells in
lymph nodes (LN), which is the reservoir of memory T cells. Although Gag-specific
CD8
+
T cells were minimally detectable in LN of mice immunized with DCLV alone, a
substantial number of Gag-specific T cells (~1.6%) were detected in LN of mice
immunized with DCLV along with αPD-L1 (Fig 4.1C). These data suggested that PD-1
blockade can promote the generation of antigen-specific T cells induced by DCLV-based
vaccination.
4.3.2 PD-L1 blockade enhances DCLV-based booster immunization for
generating Gag-specific T cell immunity
In our previous study, we have shown that DCLV could significantly increase
CD8
+
T cell responses when used as a booster following the primary DCLV vaccination
(Dai, Yang et al. 2009). In this study, we devised a prime/boost vaccination regime to
determine the efficacy of DCLV combined with the PD-L1 blockade to boost a primary
antigen-specific memory immune response. BALB/c mice were primed with a footpad
95
injection of DCLV-Gag, followed by a booster injection consisting of a second dosage of
DCLV-Gag with either a αPD-L1 or an isotype antibody treatment (Fig 4.2A). A group
of control mice were vaccinated with PBS at all injection points and used as controls. To
assess the immune response during this combinational vaccination process, blood
samples from all groups were obtained at indicated time points, and Gag tetramer staining
were performed on peripheral blood mononuclear cells (PBMC) (Fig 4.2A). In agreement
with our previous study (Dai, Yang et al. 2009), the initial immunization reached a peak
response at week two, and the boost injection could greatly enhance the primed immunity
(Fig 4.2B). Although a similar level of Gag-specific CD8
+
T cell responses was observed
in both groups after the prime vaccination, a boost DCLV-Gag immunization with PD-1
blockade exhibited a 7-fold increase in the frequency of Gag-specific CD8
+
T cells,
whereas a booster with DCLV-Gag alone only increase the number of specific CD8
+
T
cells by two folds (Fig 4.2B). Ten days after boost injection, cells from spleens and
lymph nodes were assayed by Gag tetramer staining and IFN-γ ELISPOT. By examining
the cellular response in spleen cells, we found that mice immunized with PD-L1 blockade
showed a significant advantage in numbers of antigen-specific (~20%), and IFN-γ-
producing (~8000 SFC/million cells) CD8
+
T cells over that without αPD-L1 (Fig 4.2C
and D). PD-L1 blockade resulted in 1.5% of Gag tetramer
+
T population and around 1000
IFN-γ secreting spots per million cells among total CD8
+
T cells extracted from lymph
nodes, both of which were markedly higher than the CD8
+
T cell responses in lymph
nodes of vaccinated mice without αPD-L1 treatment (Fig 4.2C and D).
96
Figure 4.2: DCLV immunization combined with PD-1 blockade efficiently boosted
primary Gag-specific T cell immunity. (A) Schematic representation of immunization
and analysis protocol. (B-D) BALB/c mice were primed with 5×10
6
TU of DCLV-Gag,
and boosted with DCLV-Gag in conjugation with αPD-L1 ( ) or isotype antibody (▲)
treatment for three times; the control group were primed and boosted with PBS (♦). (B)
Frequencies of Gag tetramer
+
CD8
+
T cells were determined in PBMC samples from
different groups at the indicated time points. Ten days post-boost immunization, spleen
and lymph node cells were harvested, and T cells were evaluated for Gag-specific CD8
+
T cell expansion by tetramer staining (C) and IFN-γ secretion by ELISPOT (D). The data
shown are mean values of triplicates ± s.d. The p values are calculated using Student’s t
test.
97
4.3.3 DCLV prime/boost regimen combined with PD-L1 blockade elicits
multifunctional CD8
+
T cell responses
Increasing evidence points to the correlation of polyfunctional HIV-specific CD8
+
T cells frequencies with delay of disease progression (Sun, Schmitz et al. 2006; Liu,
Ewald et al. 2008). Blocking the PD1/PD-L1 pathway was suggested to be beneficial to
restoration of CD8
+
T cell function. Therefore we next attempted to characterize the
functional repertoire of antigen-specific T cells generated by different vaccination
protocols, in term of their ability to produce cytokines. Spleen cells were harvested day
10 post-boost immunization from mice vaccinated with DCLV prime/DCLV boost with
the treatment of either an αPD-L1 or an isotype antibody, followed by restimulation with
a library of overlapping HIV-1 Gag peptides. A polychromatic flow cytometry assay was
performed on the restimulated spleen cells to simultaneously assess the production of
three cytokines (IFN-γ, IL-2 and TNF-α). The pattern (%) of cytokine-secreting cells in
both groups was similar, with IFN-γ secretion dominated, followed by TNF-α, and much
less of IL-2. PD-1 blockade in conjunction with DCLV prime/boost regimen increased
the total frequency of IFN-γ
+
(from ~3% to ~9%) and TNF-α
+
(from ~1% to ~4%) cells
within the CD8
+
T cell population, while had a minimal effect on the frequency of cells
producing IL-2 (less than 1% in both groups) (Fig 4.3B, Day 10). Generally, comparing
with the prime/boost alone, PD-1 blockade produced larger subsets of Gag-specific CD8
+
T cells that secreted more than one cytokine simultaneously. The number of CD8
+
T cells
producing three cytokines from αPD-L1-treated mice was almost four times of that from
98
the isotype control group (Fig 4.3C, Day 10). Typically, we observed an individual mice
treated by PD-1 blockade generated around 3% of Gag-specific CD8
+
T cells capable of
producing both IFN-γ and TNF-α cytokines, which was in contrast to 0.6% of the
counterpart in the group without PD-1 blockade (Fig 4.3A and C, Day 10). These results
demonstrated a significant polyfunctional advantage of cellular immune response resulted
from prime/boost vaccination with PD1 blockade over that without blockade.
Figure 4.3: Multifunctionality of CD8
+
T cell immune responses induced by DCLV
prime/DCLV boost in combination with PD-1 blockade. Splenocytes harvested from
DCLV/DCLV+αPD-L1, DCLV/DCLV+isotype, and the PBS control groups of BALB/c
mice were in vitro restimulated with the HIV-1 Gag peptide pools for 6 hours. (A)
Representative flow cytometry plots show distribution of Gag-specific cytokine-secreting
CD8
+
T cells as a percentage of total CD8
+
T cells 10 days after the boost injection. (B)
The percentages of cytokine-secreting CD8
+
T cells at day 10 and day 68 post-boost
injection were shown. (C) Cytokine co-expression subsets at day 10 and day 68 post-
boost immunization were expressed as a percentage of total CD8
+
T cells. Each group
consisted of three mice.
99
4.3.4 DCLV-based prime/boost vaccination combined with PD-L1 blockade
induces long-term immunity
To assess the persistence of Gag-specific immune response, we conduct a separate
experiment consisting of three groups of mice that receive the same vaccine modalities as
Fig. 4.2, but were analyzed for 2 months after the boost injection (Fig 4.4A). The Gag-
specific CD8
+
T cell responses in blood samples from each group were longitudinally
monitored. The numbers of Gag-specific CD8
+
T cells in PBMC samples of both groups
decreased during the first three weeks and reached a stable value through day 68, with an
ultimate number of ~8% of tetramer-positive CD8
+
T cells from αPD-L1-treated mice
and ~2% from non-treated mice (Fig 4.4B). To exam whether PD-L1 blockade resulted in
prolonged immune responses, at day 68 post-boost immunization, tetramer staining was
performed on splenocytes from each group, and the multifunctional immune response
was analyzed in a similar manner as Fig. 4.2. In comparison with the cellular immunity
10 days post-boost immunization, the frequency of Gag tetramer-positive CD8
+
T cells in
the αPD-L1-treated mice at day 68 declined, but maintained a considerable number of
8.4% (Fig 4.4B). In contrast, only 2.2% of Gag-specific CD8
+
T cells were observed two
months following boost in splenocytes of non-treated mice (Fig 4.4C). Upon stimulation
with the HIV-1 Gag peptide pools, the total responding CD8
+
T cells in the PD-L1
blockade group were greater in cytokine-secreting frequency than those from the group
without blockade (Fig 4.3B, Day 68). Moreover, the Gag-specific CD8
+
T cells
maintained their polyfunctionality with an average of 1% IFN-γ
+
/TNF-α
+
, and 0.5% of
100
IFN-γ
+
/IL-2
+
cells (Fig 4.3C, Day 68). The above findings confirmed a better
preservation of HIV 1-specific CD8
+
T cell frequency as well as polyfunctionality by a
vaccine regimen with PD-L1 blockade.
Figure 4.4: DCLV prime/DCLV boost in combination with PD-1 blockade generated
prolonged immunity. (A) Schematic representation of experimental procedures. (B) The
percentage of Gag tetrmer-positive CD8
+
T cells in PBMC was determined at the
indicated time points. Day 0 denotes the date that mice were boosted with DCLV-Gag
plus αPD-L1 or isotype antibody treatment. (C) Representative FACS plots for measuring
the population of tetramer-positive cells in spleen at day 10 and day 68 post-boost
immunization.
101
4.3.5 DCLV-based prime/boost vaccination combined with PD-L1 blockade
induces broad HIV-1 Gag-specific T cell responses
Due to the genetic diversity of HIV-1 in nature, inducing broad T cell responses is
a desirable feature of AIDS vaccine (Kong, Huang et al. 2003; Wu, Kong et al. 2005).
We next assessed the role of PD-1 blockade in inducing the breadth of T cell responses. T
cell epitopes for the Gag immunogen could be mapped by virtue of a cross-linked peptide
matrix as we described previously. A library of peptides, consisting of 123 peptides
covering the entire HIV-1 B Gag sequence, was divided into 23 pools named P1-P23.
Each 15 amino acid-long single peptide was presented in two independent peptide pools
(Fig 4.5A). The splenocytes from animals immunized with PBS, DCLV/DCLV+αPD-L1,
or DCLV/DCLV+isotype antibody 10 days post-boost injection were isolated, exposed to
each of the 23 peptide pools, and were then assessed by the IFN-γ ELISPOT assay. As
the readout of T cells from PBS mice were less than 16 SFC per 0.1 million cells, we
defined a critical value of 80 SFC/0.1 million (five times of background readout) as a
baseline to a positive response. It was found that mice immunized with DCLV plus PD-1
blockade positively responded to ten peptide pools (P4, P5, P6, P7, P8, P15, P16, P17,
P22, P23), while the response of the parallel group without PD-1 blockade was directed
to only four pools (P4, P5, P16, P17) (Fig 4.5A and B). Moreover, it’s noticeable that the
overall T cell response elicited by vaccination with PD-1 blockade is greater than that
from the prime/boost only. The aforementioned results indicated that PD-1 blockade with
102
DCLV prime/boost strategy induced an increased function of virus-specific CD8
+
T cells
responding to multiple epitopes.
Figure 4.5: DCLV prime/DCLV boost in combination with PD-1 blockade broadened the
profile of vaccine-specific T cell responses. (A) 123 of 15-mer peptides spanning the
entire HIV-1 clade B Gag sequence were divided into 23 pools (P1-P23) as indicated by
the peptide matrix. (A & B) Spleen cells harvested from DCLV/DCLV+αPD-L1,
DCLV/DCLV+isotype, or the control group of BALB/c mice were pooled, stimulated in
vitro with one of peptide pools for 18-24 hours, and assayed by IFN-γ ELISPOT. A
peptide pool is regarded as a positive group when its ELISPOT readout is 5 times higher
than that of the control group. Positive reactive peptide pools were summarized in (A).
Each group consisted of three mice.
103
4.3.6 DCLV-based vaccination combined with PD-1 blockade provides
better protection against vaccinia virus challenge
To assess whether the magnitude of Gag-specific immune responses predicts
resistance to infection, we sought to evaluate the protection efficacy of distinct vaccine
strategies against recombinant vaccinia virus challenge. A recombinant vaccinia virus
carrying the full length of HIV-1 Gag gene (vv-Gag) was used as a challenge model.
Three groups of mice, including unvaccinated mice, mice vaccinated with the DCLV-
based prime/boost protocol alone, or DCLV-based prime/boost with PD-L1 blockade,
were challenged intraperitoneally with vv-Gag (10
7
PFU) two weeks after last
vaccination. Although we didn’t notice the loss of body weight in all three groups, at day
7 following challenge, mice treated with either vaccination regimen developed a
significant lower level of vaccine virus titer in ovaries as compared with the unvaccinated
group (Fig 4.6, P<0.01 in both). PD-1 blockade afforded a 100-fold reduction of vaccinia
virus titer comparing to that without PD-1 blockade. In two of the six mice treated with
PD-1 blockade, viral load declined to a hardly detectable level. The better protection in
PD-1 blockade group is likely due to a stronger cytotoxic T lymphocyte (CTL) immune
response, as vaccinia viruses do not incorporate Gag proteins, but presents Gag-derived
peptides on the surface of infected cells(Arruda, Chikhlikar et al. 2004; Song, Liu et al.
2007). These data confirmed the stronger antiviral immunogenicity afforded by PD-1
blockade in combination with a DCLV-based prime/boost regimen.
104
Figure 4.6: Immunization delivered by DCLV prime/DCLV boost in combination with
PD-1 blockade could protect animals from recombinant vaccinia virus challenge.
BALB/c mice were immunized with PBS, DCLV-Gag prime/DCLV-Gag boost alone, or
DCLV-Gag prime/DCLV-Gag boost along with PD1 blockade. Two weeks following the
boost immunization, all the groups were challenged with 10
7
PFU of vv-Gag. Vaccinia
virus titers in paired ovaries harvested 7 days after challenge were determined on CV-1
cells. Data are expressed as mean +/- SD from groups of 4-6 mice. The p values are
calculated using Student’s t test.
4.4 Discussion
Cellular immune response is of pivotal importance because the effector T cells are
the guards through which the immune system eliminates virus-infected cells and controls
virus infection. An ideal AIDS vaccine is expected to elicit sufficient CTL to kill the
105
HIV-infected cells. Many strategies to enhance immunizations have been tested towards
achieving this AIDS vaccine goal. For example, elevated vaccination efficiency was seen
when immunizations were combined with either cytokines/chemokines (IL-2, IL-7, IL-15,
MIP-1α, MIP-3β) capable of regulating survival, expansion or trafficking of immune
cells (Sumida, McKay et al. 2004; Song, Liu et al. 2007; Colombetti, Lévy et al. 2009),
or reagents that can activate positive T cell receptor (TCR) costimulatory molecules
(CD80, CD40). Recent studies also suggested that blocking the signaling of certain
negative costimulatory molecules in T cells could restore cellular immunity (Peggs,
Quezada et al. 2008). In this study, we demonstrated that in vivo blockade of the negative
costimulatory PD1/PD-L1 pathway in combination with DCLV-based immunization
either at the primary phase or at the boost phase could markedly enhance antigen-specific
CD8
+
T cell responses. The prime/boost immunization combined with PD-L1 blockade
improved the quality and function of Gag-specific T cells, prolonged CD8
+
T cell
responses, and facilitated vaccine-induced protection against virus challenge.
There is growing number of studies utilizing LVs as vaccine carriers due to their
sustained endogenous antigen delivery (He, Zhang et al. 2005; Beignon, Mollier et al.
2009; Hu, Tai et al. 2011). In our previous studies, we reported a DCLV-based vaccine
for delivery of HIV-1 Gag protein(Dai, Yang et al. 2009). This vector is enveloped with
an engineered Sindbis virus glycoprotein, which ensures the specific binding to DCs and
the delivery of immunogens persisting for the life time of DCs (Yang, Yang et al. 2008).
Although DCLV-mediated long-term expression of immunogens in DCs can offer
sustained vaccine-specific immunity, it may, on the other hand, causes exhaustion of T
106
cells as observed in chronic HIV infection. HIV-specific CD8
+
T cells in disease
progressors showed up-regulation of PD-1 (Zhang, Zhang et al. 2007). Therefore, we
hypothesized that blocking the PD1/PD-L1 interaction could be synergized with DCLV-
based vaccine. Prior work by Barber et al. showed that antibody blocking of PD-L1 is
more efficient than blocking PD-1 in enhancing T cell responses (Barber, Wherry et al.
2006). Petrovas et al. also demonstrated that in vitro treatment of CD8
+
T cells with αPD-
L1 leads to more efficient cytokine production than the treatment with anti-PD-1
antibody (Petrovas, Price et al. 2007). Thus, we decided to use αPD-L1 as the antibody
blockade to investigate its effect on DCLV-based vaccines.
We first studied a single dose of DCLV immunization to highlight the role of
PD1/PD-L1 in priming a T cell response. There was a previous study attempting to block
the PD-1-mediated pathway with a therapeutic vaccine to augment the antiviral immune
responses in mice chronically infected with LCMV (Ha, Mueller et al. 2008). This
prompted us to also investigate PD-L1 blockade with DCLV as a booster to enhance the
memory responses. In both circumstances, treatment of αPD-L1 resulted in enhanced
expansion of Gag-specific CD8
+
T cells, as well as increased production of IFN-γ by
these cells, which is crucial for inducing the Th1 immunity (Song, Liu et al. 2007; Velu,
Titanji et al. 2009). For an HIV infection, lymphoid tissues are the main sites where
viruses replicate. Therefore, maintenance of virus-specific CD8
+
T cells in lymphoid
organs is of great importance (Petrovas, Casazza et al. 2006). In studies involving either a
single DCLV injection or a prime/boost strategy, Gag-specific CTLs in lymph nodes
were hardly detected without αPD-L1 treatment, whereas blocking the PD1/PD-L1
107
pathway generated a substantially larger number of Gag-specific CD8
+
T cells. These
findings suggest that such a blockade could favor vaccine-specific immune responses to
control HIV infection.
Cytokines play a prominent role in retaining CD8
+
T cells’ effector function and
memory, and providing essential help to antibody production and killer cells.
Polyfunctionality of T cells correlates with the reduction of viraemia and clinical non-
progression (Sun, Schmitz et al. 2006). When the CD8
+
T cells extracted from
prime/boost regimens were stimulated with the HIV-1 Gag peptide pool, the most
dramatic difference in cytokine production by αPD-L1 treatment was the enhanced
number of IFN-γ
+
/TNF-α
+
double producers, which is in agreement with the findings for
LCMV-specific CD8
+
T cells in a therapeutic vaccine study (Ha, Mueller et al. 2008),
and HIV-specific CTL analysis performed on HIV-infected patients. A measurable
increased count of IFN-γ
+
/IL2
+
co-producers was observed in the αPD-L1-treated group,
which is characteristic of a population displaying a cytolytic function with proliferative
ability (Ha, West et al. 2008). A slightly increased proportion of IFN-γ
+
/TNF-α
+
/IL2
+
triple producers were observed with αPD-L1 treatment. A prophylactic vaccine against
infectious disease requires induction as well as maintenance of CTLs (He, Zhang et al.
2005). Thus, we performed kinetic studies of vaccine-specific T cells circulating in the
blood. We monitored the response for two months post-boost immunization and noticed
decreased but still extensive number of epitope-specific memory CD8
+
T cells in the
αPD-L1 group. Moreover, these vaccine-specific CD8
+
T cells retained their
polyfunctionality two months after vaccination, with more IFN-γ
+
/IL2
+
producers and
108
less IFN-γ
+
/TNF-α
+
producers, which is presumably due to the immune system
undergoing contraction and developing memory.
One desirable feature of an AIDS vaccine is to generate CTL that react with
multiple targets on virus or virus-infected cells (Kong, Huang et al. 2003), therefore we
employed peptide pools containing an array of peptides for in-depth understanding of the
functional repertoire of CD8
+
T cells (Arruda, Sim et al. 2006). Our results showed that
the most three potent responsive pools in both groups contain the well-characterized
dominant peptide sequence. Both the diversification and magnitude of immunogens
recognized by DCLV/DCLV with PD-L1 blockade is greater than that by DCLV/DCLV
alone. The vaccine delivered by DCLV/DCLV along with αPD-L1 resulted in a better
control of viral load in mice challenged with a Gag-encoding vaccinia virus. This
superior vaccine-induced protection is presumably due to larger numbers of Gag-specific
CTL fighting against virus-producing cells (Sumida, McKay et al. 2004; Song, Liu et al.
2007). PD1/PD-L1 blockade restores exhausted CD8
+
T cells probably through
regulation of survival and differentiation ability of these cells (Petrovas, Casazza et al.
2006), which is supported by the evidence that the Gag-specific CD8
+
cells up-regulated
the proliferative CD127 markers following the boost vaccination with PD-L1 blockade
(data not shown).
109
4.5 Acknowledgements
We thank Dr. Gary Nabel for providing reagents and Dr. Larry Corey for an
initial discussion of the breadth of antigen responses. We thank Paul Bryson for critical
reading of the manuscript This research was generously supported by grants from the
National Institute of Health, the Bill and Melinda Gates Foundation, and the California
HIV/AIDS Research Program.
110
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Abstract (if available)
Abstract
T cell immunotherapy fell into two categories: passive (adoptive) transfer of in vitro expanded cells, and active expansion of antigen-specific T cells by in vivo immunization. I present three studies to promote T cell immunotherapy and T cell vaccine. In my first study, I described a method to generate autologous antigen-specific CD4⁺ helper T cells in vitro from easily accessible bone marrow cells. T lymphocytes are produced in thymus as the progeny of fetal liver (FL)- and bone marrow (BM)- derived precursors. A murine stromal cell line (OP9-DL1) expressing a notch ligand, Delta-like-1, has been shown to partially mimic the function of thymus and to drive the differentiation of both murine and human hematopoietic progenitors into T cells in vitro. Next, I attempt to develop a specific T-cell vaccine by in vivo gene delivery. Human immunodeficiency virus-1 (HIV-1) is one of the most catastrophic pandemics confronted by mankind with 33 million infections, and there is an urgent need for an effective vaccine. I choose lentiviral vector as the vaccine carrier as it is among the most efficient gene delivery machinery, and can infect both dividing and nondividing cells. In my second study, I evaluate in mice a dendritic cell (DC)-directed LV system encoding the Gag protein of human immunodeficiency virus (LV-Gag) as a potential vaccine for inducing an anti-HIV immune response. The DC-directed specificity is achieved through pseudotyping the vector with an engineered Sindbis virus glycoprotein capable of selectively binding to the DC-SIGN protein. To further optimize this T-cell vaccine system to achieve an increased immune response, in my third study, I attempt to break down the suppressive signaling pathways involved in T cell function. It was found that exhaustion of CD8⁺ T cells and upregulation of programmed death 1 (PD-1), a negative regulator of T cell activation, is a characteristic feature of individuals chronically infected with HIV-1. In this project, I demonstrate that blocking of the PD-1/PD-L1 inhibitory signal via an anti-PD-L1 antibody (αPD-L1) generated an enhanced HIV-1 Gag-specific CD8⁺ immune response following a both a single round of DC-targeting LV immunization and a homologous prime/boost regimen.
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Asset Metadata
Creator
Dai, Bingbing (author)
Core Title
Engineering viral vectors for T-cell immunotherapy and HIV-1 vaccine
School
Andrew and Erna Viterbi School of Engineering
Degree
Doctor of Philosophy
Degree Program
Materials Science
Publication Date
03/29/2012
Defense Date
11/04/2011
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
gene delivery,HIV/AIDS vaccine,lentiviral vector engineering,OAI-PMH Harvest,PD1/PD1L pathway,stem cell development
Language
English
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Electronically uploaded by the author
(provenance)
Advisor
Wang, Pin (
committee chair
), Arnold, Donald B. (
committee member
), Goo, Edward K. (
committee member
)
Creator Email
bingbingdai@gmail.com
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https://doi.org/10.25549/usctheses-c3-406
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UC11288428
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etd-DaiBingbin-560.pdf
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406
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Dissertation
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Dai, Bingbing
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texts
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The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
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Tags
gene delivery
HIV/AIDS vaccine
lentiviral vector engineering
PD1/PD1L pathway
stem cell development