University of Southern California Dissertations and Theses

Targeting Langerhans cells: a human papillomavirus type 16 immune evasion mechanism

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Targeting Langerhans cells: a human papillomavirus type 16 immune evasion mechanism

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TARGETING LANGERHANS CELLS:
A HUMAN PAPILLOMAVIRUS TYPE 16 IMMUNE EVASION MECHANISM

by

Laura Marie Fahey

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

May 2009

Copyright 2009 Laura Marie Fahey

ii
Dedication

Mum and Dad: I did it! I couldn’t have made it this far without all your love,
encouragement, and support. You guys made it happen for me. I am forever grateful!

iii
Acknowledgments

To Adam Raff, my best friend and favorite lab partner: I couldn’t have done it
without you by my side. Since you came into my life I have become a better person and a
better scientist. Thank you for your strength, support, and love. I love you with all my
heart.
To my family and friends on the East and West coasts: earning my Ph.D. hasn’t
always been easy, as you all know, so thank you for your continued support. It means
everything to me.
To the members of the Kast laboratory: you have witnessed all my struggles and
accomplishments. Thank you for your advice and support over the last five years. I feel
so lucky to have become a member of the Kast lab family.
To my mentor, W. Martin Kast: thank you for pushing me, believing in me, and
always being there for me. You have given me confidence in myself as a scientist and I
can never thank you enough.

iv
Table of Contents

Dedication ii
Acknowledgments iii
List of Tables vi
List of Figures vii
Abbreviations ix
Abstract xiii
Chapter 1. Introduction 1
Human Papillomavirus Taxonomy and Genome 1
HPV Life Cycle 3
High-risk HPV, Pathogenesis, and Cervical Cancer 4
HPV VLP 7
Antigen Presenting Cells and Adaptive Immune Responses 8
Natural HPV Immunity 12
Mechanisms of Immune Escape by HPV 14
Project Goals and Significance 24

Chapter 2. HPV can Escape Immune Recognition Through 26
LC Phosphoinositide 3-Kinase Activation
Introduction 26
Results 30
Discussion 43
Chapter 3. A Major Role for the Minor Capsid Protein 47
of HPV16 in Immune Escape
Introduction 47
Results 49
Discussion 58
Chapter 4. Annexin A2 is a Candidate Receptor for HPV16 on LC 61
Introduction 61
Results 63
Discussion 70

v

Chapter 5. Reversal of HPV-Specific T cell Immune Suppression 74
Through Toll-like Receptor 8 Agonist Treatment of LC Exposed
to HPV16
Introduction 74
Results 76
Discussion 88
Chapter 6. Discussion and Future Directions 95
Chapter 7. Materials and Methods 105
Antibodies and Agonists 105
Donor Material 106
DC and LC Generation 106
VLP Production 107
VLP ELISA 109
VLP Western Blot Analysis 109
VLP Electron Microscopy 110
Activation Assay 110
Flow Cytometry 111
Cytokine and Chemokine Analysis 112
Migration Assay 112
In Vitro Immunization Assay 113
ELISPOT Assay 114
Western Blot Analysis 114
HPV16L1L2 VLP Binding Assay 115
L2
108-120
Peptide Pulldown Assay 116
Silver Staining Assay 118
HPV16 VLP Uptake Assay 118
Statistical Analysis 119
Bibliography 120

vi
List of Tables

Table 1. Classification of Human Papillomaviruses 2
Table 2. Human papillomavirus proteins 3
Table 3. Synthetic imidazoquinolines and the respective receptor(s) 75

vii
List of Figures

Figure 1. HPV viral particles 2
Figure 2. Schematic of VLP 8
Figure 3. Signal transduction pathways initiated in APC after 29
encountering pathogens
Figure 4. Human monocyte-derived LC express langerin, 30
CD1a, and E-cadherin
Figure 5. After HPV16L1L2 VLP encounter, LC down-regulate 33
MAPK pathways
Figure 6. After HPV16L1L2 VLP encounter, DC activate NF-κB 36
while LC do not
Figure 7. After exposure to an intact HPV16L1L2 VLP, LC activate 39
PI3K but down-regulate p-Akt
Figure 8. Activation of PI3K by HPV16L1L2 VLP-exposed LC 41
inhibits surface marker expression
Figure 9. Inhibition of PI3K allows HPV16L1L2 VLP-stimulated LC 43
to induce an HPV-specific CD8
+
T cell response
Figure 10. Potential signal transduction pathways initiated in LC 45
after exposure to HPV16L1L2 VLP
Figure 11. Differential expression of surface markers on LC after 50
exposure to HPV16 VLP
Figure 12. Differential secretion of cytokines and chemokines by 52
LC exposed to HPV16 VLP
Figure 13. HPV16 VLP induce LC migration 54
Figure 14. LC exposed to HPV16 VLP induce differential activation 56
of HPV16-specific CD8
+
T cells
Figure 15. HPV16L1L2 VLP induce an immune suppressive signal 57
transduction cascade in LC

viii
Figure 16. The HPV16 L2
108-126
peptide inhibits binding of 64
HPV16L1L2VLP to LC
Figure 17. PolyHistidine immunoblot analysis of eluates 65
isolated from a pulldown assay
Figure 18. Silver stain analysis of eluates isolated from a 66
pulldown assay
Figure 19. Annexin A2 immunoblot analysis of eluates isolated 67
from a pulldown assay
Figure 20. SLPI inhibits uptake of HPV16L1L2 VLP by LC but 69
not uptake of HPV16L1 VLP
Figure 21. Human monocyte-derived LC express TLR7 and TLR8 76
Figure 22. Differential expression of surface markers on DC and 79
LC stimulated with imidazoquinolines
Figure 23. 3M-002 and resiquimod highly induce the secretion of 83
Th1 associated cytokines and chemokines by LC previously
incubated with or without HPV16L1L2 VLP
Figure 24. 3M-002 and resiquimod induce the up-regulation of CCR7 86
and migration of LC exposed to HPV16L1L2 VLP towards CCL21
Figure 25. 3M-002 and resiquimod induce an HPV16 epitope-specific 88
CD8
+
T cell immune response through the activation of LC exposed
to HPV16L1L2 cVLP

ix
Abbreviations

aa, amino acid
ANOVA, analysis of variance
ATF, activating transcription factor
APC, antigen presenting cells
Btk, bruton tyrosine kinase
CCL, c-c chemokine ligand
CCR, c-c chemokine receptor
CFDA-SE, carboxyfluorescein diacetate, succinimidyl ester
CIN, cervical intraepithelial neoplasia
CREB, cAMP responsive element binding protein
CRB, CREB binding protein
CTL, cytotoxic T lymphocytes
cVLP, chimeric virus-like particle
DC, dendritic cells
DC-SIGN, dendritic cell-specific ICAM-grabbing non-integrin
EBV, epstein-barr virus
ELISA, enzyme-linked immunosorbent assay
ELISPOT, enzyme-linked immunosorbent spot
ERK, extracellular signal-regulated protein kinase
FDA, Food and Drug Administration
GM-CSF, granulocyte macrophage-colony stimulating factor

x
GSK, glycogen synthase kinase
HCMV, human cytomegaloviurs
HDAC, histone deacetylases
HIS, histidine
HIV, human immunodeficiency virus
HLA, human leukocyte antigen
HPV, human papillomavirus
IARC, International Agency for Research on Cancer
ICAM, inter-cellular adhesion molecule
IFN, interferon
IKK, IκB kinase
IL, interleukin
IP-10, interferon-gamma induced protein-10
IRF, interferon regulatory factor
ISGF, IFN stimulatory gene factor
JAK, janus kinase
JNK, c-Jun N-terminal kinase
LC, Langerhans cells
LMP, low molecular mass protein
LN, lymph node
LPS, lipopolysaccharide
MAPK, mitogen-activated protein kinase

xi
MCP, monocyte chemoattractant protein
MEKK3, MAPK/extracellular signal-regulated kinase kinase kinase 3
MHC, major histocompatibility complex
MIP, macrophage inflammatory protein
MKK, MAPK kinase
mRNA, messenger RNA
NK, natural killer
NF-κB, nuclear factor-κB
p38K, p38 stress-activated proteins kinases
pap, Papanicolaou
PBS, phosphate buffered saline
P/CAF, p300-CBP-associated factor
PDK1, phosphoinositide-dependent kinase 1
PI3K, phosphoinositide 3-kinase
PKC, protein kinase C
PP2A, protein phosphatase 2A
PTEN, phosphatase and tensin homolog
pRb, retinoblastoma protein
RANTES, regulated upon activation, normal T-cell expressed, and secreted
SD, standard deviation
SEM, standard error of the mean
Ser, serine

xii
SGK, serum- and glucocorticoid-regulated kinase
SLPI, secretory leukocyte protease inhibitor
SSC, squamous skin cancer
ssRNA, single stranded RNA
STAT, signal transduction and transcription
TAK-1, transforming growth factor–β-activated kinase 1
TCR, T-cell antigen receptors
TAP, transporter associated with antigen presentation
TGF-β
1
, transforming growth factor-β
1

Th, T helper
T
H
3, CD4
+
T helper 3
Thr, threonine
TLR, Toll-like receptor
TNF, tumor necrosis factor
T
R
1, T regulatory 1
TRAIL, tumor necrosis-related apoptosis-inducing ligand
Tregs, regulatory T cells
Tyk2, tyrosine kinase 2
Tyr, tyrosine,
VIN, vulvar intraepithelial neoplasia
VLP, virus-like particle

xiii
Abstract

High-risk human papillomaviruses (HPV) infect the epithelial layer of cervical
mucosa and are causally associated with the generation of cervical cancer. Although most
women infected with HPV clear their lesions, the long latency period from infection to
resolution indicates that HPV has evolved immune escape mechanisms. Langerhans cells
(LC) are the resident antigen-presenting cells at the site of infection and therefore are
responsible for initiating an immune response against HPV. However, LC exposed to
HPV16L1L2 virus-like particles do not induce an HPV-specific T cell response,
suggesting that LC are targeted by HPV to evade immune detection. Herein we describe a
novel immune escape mechanism of HPV16 that targets LC function and identify
therapeutic compounds to overcome the HPV16 induced suppression of LC. We
demonstrate that LC incubated with HPV16L1L2 virus-like particles up-regulate
phosphoinositide 3-kinase activation while down-regulating mitogen-activated protein
kinase and nuclear factor-κB pathways. When phosphoinositide 3-kinase activation is
inhibited and LC are subsequently exposed to HPV16L1L2 virus-like particles, LC
initiate a potent HPV-specific response, revealing that phosphoinositide 3-kinase
activation in LC is an escape mechanism utilized by HPV16. Importantly, we also show
that the minor capsid protein L2 is responsible for the induction of this immune escape of
HPV16 through the manipulation of LC. Additionally, using pulldown assays we
demonstrate that the N-terminus of L2 associates with annexin A2. Inhibiting the
interaction between HPV16 L2 and annexin A2 disrupts the internalization of
HPV16L1L2 virus-like particles by LC, indicating that annexin A2 is the L2 receptor for

xiv
HPV16, which likely initiates the immune escape mechanism of HPV16 through LC.
Furthermore, we identify two molecules, 3M-002 (TLR8 agonist) and resiquimod
(TLR8/7 agonist) that can overcome the phenotypic and functional suppression of LC
previously exposed to HPV16L1L2 virus-like particles. Collectively, our studies
delineate a novel HPV16 immune escape mechanism and direct the future development
of therapeutics for treatment of HPV infections and HPV-induced cervical lesions.

1
Chapter 1. Introduction

Human Papillomavirus Taxonomy and Genome
Human papillomaviruses (HPV) are members of the papillomaviradae family.
HPV are 55nm icosahedral-shaped, non-enveloped, non-lytic, species-specific, double-
stranded DNA viruses that infect the epithelium of skin and mucosa (Fig. 1). More than
100 HPV genotypes have been identified based on the homology of genomic DNA
(Stoler, 2000) and of these over 40 genotypes can infect anogenital mucosa (de Villiers,
2001). HPV that infect anogenital mucosa can be divided into low-risk HPV types (such
as HPV6 and 11), which are found in benign condylomas, and high-risk types (such as
HPV16, 18, 31, and 45) which are found in and are causally associated with generation of
cervical cancer [(Table 1), (Munoz et al., 2003; Walboomers et al., 1999; zur Hausen,
1991)].
HPV have a simple closed circular genome that is about 8000 base pairs in length
and consists of three regions: the early, late, and long control region. The early gene
products (E1, E2, E4, E5, E6, and E7) control viral DNA replication, transcription,
genome persistence, and cellular transformation (zur Hausen, 1996). The late gene
products, L1 and L2 are the major and minor capsid proteins, respectively (Kirnbauer et
al., 1992; Kirnbauer et al., 1993; Zhou et al., 1994). They control proper capsid protein
assembly, DNA packaging, and virus infectivity [(Table 2), (Finnen et al., 2003;
Holmgren et al., 2005; Mallon et al., 1987; Zhou et al., 1991)]. The long control region
contains cis-response elements and therefore is bound by cellular and viral transcription
factors that augment the viral life cycle. There is evidence that mutational changes or

2
recombination are very rare events in HPV genomes. It has been estimated that
mutational events occur at similar frequencies in HPV genomes as they do in the human
genome, about 2.5 x 10
–8
mutations per nucleotide (de Villiers et al., 2004).

Figure 1. HPV viral particles. A. Illustration of a HPV virion composed of the major
capsid protein L1 and minor capsid protein L2 surrounding the double stranded circular
7.9 kb genome. B. Image of a HPV viral particle composed of the L1 and L2 proteins
assembled into pentamers (Buck et al., 2008). C. Image of a HPV viral particle composed
of L1 and L2 proteins, depicting L2 density in red (Buck et al., 2008).

Groups Genotypes Site of
Infection
Clinical
Manifestations
Cutaneous
(Low-Risk)
HPV 1, 2, 3, 4, 10, 28, &
41,
Skin Warts
Cutaneous
(High-Risk)
HPV 5, 8, 9, 12, 14, 15, 17,
19, 20, 21, 22, 23, 24, 25,
36, 37, 38, 47, & 49
Skin Flat lesions, warts, &
squamous skin cancer
Mucosal
(Low-Risk)
HPV 6, 11, 40, 42, 43, 44,
54, 61, 70, 72, 81, & 84
Anogenital &
Oral Mucosa
Warts
Mucosal
(High-Risk)
HPV 16, 18, 31, 33, 35,
39, 45, 51, 52, 56, 58, 59,
66, 68, 73, & 82
Anogenital &
Oral Mucosa
Flat Lesions,
intraepithelial neoplasia,
& cancer

Table 1. Classification of Human Papillomaviruses.

3

Protein
Cellular
localization
Function
E1 Nuclear Helicase, ATPase, ATP-binding protein
E2 Nuclear DNA binding proteins, Transcription factor
E4 Cytoplasmic
Interacts with cytoskeletal proteins,
Allows for viral assembly
E5 Cytoplasmic Prevents cell differentiation
E6 Nuclear Prevents cell differentiation
E7 Nuclear Prevents cell-growth arrest & differentiation,
L1 Nuclear Structural & Capsid assembly
L2 Nuclear Capsid assembly, DNA packaging, & Infection

Table 2. Human papillomavirus proteins.

HPV Life Cycle
The life cycle of HPV is unique in that it is completely dependent on the cellular
differentiation of the host cell, keratinocytes. The replication cycle of HPV, from the time
of infection to the release of virions, is about 3 weeks, which corresponds to the time
required for basal cells to differentiate into keratinocytes and then desquamate (Stanley et
al., 2007). HPV initially infects basal cells, which are a single layer of undifferentiated
proliferating cells, via a microabrasion in the epithelium. Upon infection of the basal cells
the viral genome is amplified from 1-10 to about 50-100 genomes per cell. (Stanley et al.,
2007). The subsequent stage of viral growth is episomal maintenance, in which the
infected cell and virus replicate concurrently. At this point the early viral genes are
expressed at very low levels. E1 and E2 form a complex that bind to the origin of
replication and recruit cellular polymerases and accessory proteins to mediated DNA

4
replication (Doorbar, 2006). Next, the infected cell exits the cell cycle and differentiates.
This is the stage when viral gene expression and viral DNA amplification are highly up-
regulated. After genome amplification the viral copy number per a cell is at least 1000,
indicating that there are approximately one thousand virions produced per cell. Finally,
once the infected cell reaches the distal layer of the epithelium the late viral genes, L1
and L2, are expressed, virion assembly occurs, and virion release occurs upon
desquamation (Doorbar, 2006; Stanley et al., 2007).

High-risk HPV, Pathogenesis, and Cervical Cancer
Mucosotropic high-risk HPV are sexually transmitted viruses that can lead to the
development of proliferative, epithelial lesions including the development of cervical,
vaginal, anal, and penile cancers (Parkin, 2006). The majority of women will acquire a
genital HPV infection at some point in their lifetime (Syrjänen et al., 1990), and although
most women will clear the infection, the average time for clearance is about a year
(Woodman C. B. et al., 2001). Conversely about 15% of women that have high-risk HPV
infections cannot initiate an effective immune response against HPV (Stanley et al.,
2007) and persistence of a high-risk HPV infection is a major risk factor in the
development of cervical cancer (Schlecht et al., 2001). A study conducted by the
International Agency for Research on Cancer (IARC) found that HPV16 and HPV18
DNA are found in more than 70 percent of HPV positive biopsies obtained from cervical
cancer patients (Castellsague et al., 2006). HPV16 is the most common type of mucosal
high-risk HPV, with an overall prevalence of 58.9 percent. (Munoz et al., 2003).

5
High-risk HPV can induce the generation of cancer because of the specific
activity of their oncoproteins, E6 and E7. During a normal HPV infection the viral
genome is present as an episome, however in the majority of HPV-induced cancers a
section of the viral genome integrates into the host chromosome, which leads to an
increase in genome instability of these infected cells (Duensing and Munger, 2002). This
integration is a terminal event in the life cycle of HPV. Most commonly, the virus
integrates into the host’s genome at the E2 gene, which leads to the alteration in the
expression of E2. The full length E2 protein, when highly expressed, suppresses the
expression of E6 and E7 proteins. Thus, when the E2 gene is disrupted the protein is non-
functional, leading to an increase and overexpression of E6 and E7 proteins (Leggatt and
Frazer, 2007; Stubenrauch and Laimins, 1999). E6 and E7 proteins account for the
transforming capabilities of the high-risk HPV genotypes by binding to and inactivating
the tumor-suppressor gene products p53 and retinoblastoma protein (pRb), respectively
(Dyson et al., 1989; Werness et al., 1990). Additionally, E7 associates with and directs
class I histone deacetylases (HDAC) to promote cell growth (Brehm et al., 1999;
Longworth and Laimins, 2004) and can induce centrosomal abnormalities (Duensing et
al., 2001). Furthermore, E6 has a C-terminal PDZ ligand domain, allowing E6 to bind to
and stimulate proteasomal degradation of proteins that have PDZ motifs such as hDlg and
hSribble, both of which are thought to be involved in the regulation of cell growth and
attachment (Zeitler et al., 2004). However, even though E6 and E7 can disrupt normal
cell function and induce genomic instability, the aberrant expression of high-risk
oncogenes is not sufficient for carcinogenesis. Nonetheless, over expression of E6 and E7

6
can lead to a precancerous state that allows for secondary genetic changes following
infection. Cancer usually occurs in individuals that have persistent infections and who
continue to express oncogenes for years to decades.
Cervical cancer is the second most common cancer among women worldwide,
killing approximately a quarter of a million women each year (Lowndes, 2006; Parkin,
2006). It is principally a cancer of women in developing countries because there is a lack
of resources and routine Papanicolaou (Pap) smear examinations. Approximately eighty
percent of cervical cancer cases occur in developing countries (Castellsague et al., 2006).
Merck has developed the first Food and Drug Administration (FDA) approved
preventative HPV vaccine, Gardasil. The vaccine is based upon virus-like particles (VLP)
composed of the major capsid proteins from HPV 6, 11, 16, and 18. It has been
demonstrated in phase III clinical trials to be nearly 100% effective as a prophylactic
vaccine for HPV-induced lesions (The FUTURE II Study Group, 2007; The FUTURE II
Study Group, 2007). However, even with the use of Gardasil it has been predicted that it
will take decades to be able to detect a quantifiable effect on cervical cancer rates in the
population (Dasbach et al., 2006; Ryding et al., 2008). Most importantly it should be
noted that Gardasil is not a therapeutic treatment for the millions of women that are
currently infected with HPV and no therapeutic options currently exist to promote HPV
clearance in persistent infections. Therefore, there remains a clear need to develop an
intervention to facilitate immune mediated clearance of the virus and reduce the
percentage of women whose lesions progress to cervical cancer.

7
HPV VLP
The life cycle of HPV is dependent on the differentiation of keratinocytes in the
squamous epithelium and because of this it is difficult to produce large quantities of HPV
virions in vitro. Therefore, as an alternative to HPV virions, HPV VLP have been
developed for structural and immunological analysis of HPV. HPV VLP look and act
similar to native HPV, however they do not contain any genomic material, rendering
them non-transforming. There are three types of VLP that are currently being used in the
field, L1 VLP, L1L2 VLP, and chimeric VLP (cVLP) (Fig. 2). When the major capsid
protein L1 is expressed, it can self-assemble into a L1 VLP with a 72-pentamer
icosahedral structure (Kirnbauer et al., 1992). If both L1 and the minor capsid protein L2
are simultaneously expressed, the proteins assemble into L1L2 VLP that contain up to 72
L2 proteins per VLP (Buck et al., 2008; Kirnbauer et al., 1993; Zhou et al., 1991).
Importantly, due to the fact that HPV virions are composed of both L1 and L2 proteins,
HPV L1L2 VLP are morphologically equivalent to HPV virions while VLP comprised of
L1 alone are not. cVLP are composed of both capsid proteins with the addition of the E7
protein fused to either L1 or L2. cVLP are used in vitro to deliver E7 to antigen
presenting cells to induce an anti-HPV CD8
+
T cell response (Greenstone et al., 1998).

8

Figure 2. Schematic of VLP. A. VLP composed of the L1 protein. B. VLP composed
of L1 and L2 proteins. C. Chimeric VLP composed of the L1 protein and an L2-E7
fusion protein. D. Electron micrograph of the 55 nm icosahedral L1L2 VLP. Bar = 100
nm.

Antigen Presenting Cells and Adaptive Immune Responses
Dendritic cells (DC) consist of a family of potent antigen presenting cells (APC)
that are found in nearly all tissues (Banchereau and Steinman, 1998). DC can be divided
into subsets defined by their phenotype, function, and location. The first major division of
DC is between plasmacytoid and conventional DC (Heath et al., 2004). Plasmacytoid DC
are critical in the production of interferon (IFN)-α, and -β, likely contributing to the
control of viral replication and the activation of other DC subsets. Conventional DC
subsets can be further subdivided into two groups: blood-derived DC and tissue-derived
DC. The blood-derived DC reside in the spleen and lymph nodes while tissue-derived DC
are located in the peripheral non-lymphoid organs. Tissue-derived DC can be further
subdivided into unique subsets: dermal DC that reside in the dermis and deeper tissues
and Langerhans cells (LC) that reside in the epidermis of skin and mucosa. LC are

9
arranged in a three-dimensional network between keratinocytes in the epithelium. Of
note, transforming growth factor-β
1
(TGF-β
1
), produced by epidermal keratinocytes, is
essential for the development of LC in vivo from CD14
+
cells (Borkowski et al., 1997). In
vitro, cells with either DC or LC phenotypic and functional characteristics can be
generated from human blood derived monocytes that have been cultured in the presence
of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4
or with GM-CSF, IL-4, and TGF-β
1
, respectively (Geissmann et al., 1998; Romani et al.,
1996). LC differ from DC in that they express high levels of E-cadherin, a homotypic
adhesion molecule that anchors LC to neighboring keratinocytes, CD1a, which expresses
microbial lipids and is a member of the group 1 CD1 proteins, and langerin, a type II C-
type lectin receptor that binds mannose and related sugars. LC also contain rod or racket-
shaped intra-cytoplasmic Birbeck granules, which are poorly understood however they
might function in antigen presentation (Merad et al., 2008). In addition, epidermal LC do
not express the dendritic cell-specific inter-cellular adhesion molecule (ICAM)-grabbing
non-integrin (DC-SIGN) (Klechevsky et al., 2008). Therefore, the location and
phenotypic expression profiles are specific for each DC subset, indicating that the subsets
may respond differently to various antigens.
The primary functions of APC in the periphery are recognition, internalization
processing, and presentation of foreign antigens to naïve T lymphocytes in regional
lymph nodes (LN), thereby inducing an adaptive immune response (Banchereau and
Steinman, 1998; Larsen et al., 1990). When APC encounter foreign antigens they
undergo a process called ‘maturation’ and it is associated with phenotypic and functional

10
changes of APC, including up-regulation of co-stimulatory molecules CD80 and CD86,
MHC class I and II molecules, chemokine receptors (such as CCR7), secretion of
cytokines and chemokines, and migration to regional LN (Albert et al., 1998; Banchereau
and Steinman, 1998; De Smedt et al., 1996; Larsen et al., 1990). This migration is
directed by the secondary lymphoid tissue chemokine (SLC/CCL21) and macrophage
inflammatory protein (MIP)-3β (CCL19) via binding to CCR7 (Hirao et al., 2000; Saeki
et al., 1999). Migration of mature APC to draining LN is critical for the presentation of
antigens to unprimed T cells, consequently leading to the induction of an antigen specific
cell-mediated immune response (Banchereau and Steinman, 1998; Larsen et al., 1990).
Thus, APC residing in the peripheral non-lymphoid organs act as a bridge between the
innate and adaptive immune response.
Mature APC and T cells interact through the major histocompatibility complex
(MHC) located on the surface of APC and T-cell antigen receptors (TCR) on T cells.
There are two types of MHC molecules that peptides can be presented on, MHC class I
and MHC class II. Of note, in humans MHC is referred to as human leukocyte antigen
(HLA). MHC class I molecules are ubiquitously expressed on nucleated cells and usually
present intracellular antigens while MHC class II molecules are found primarily on APC
and present extracellular antigens. However exogenous antigens can also be presented on
MHC class I molecules by cross-presentation (Albert et al., 1998; Heath et al., 2004).
CD8
+
T cells recognize MHC class I molecules while CD4
+
T cells recognize MHC class
II molecules. When naïve CD8
+
T cells interact with mature APC and recognize a peptide
presented on MHC class I molecules they become cytotoxic T lymphocytes (CTL), which

11
can directly kill a target cell expressing the corresponding antigen and secrete pro-
inflammatory cytokines (Banchereau and Steinman, 1998; Heath et al., 2004). When
naïve CD4
+
T cells recognize mature APC their activation and function depends on the
microenvironment surrounding the interaction. CD4
+
T cells become T helper (Th)1 cells
when they interact with and recognize mature APC in the presence of IL-12. Th1 cells
primarily secrete IL-2 and IFN-γ, which aid in the activation of macrophages, natural
killer (NK) cells and CD8
+
T cells, thereby driving cell-mediated immunity. Notably, in
the absence of CD4
+
Th1 cells, CTL memory is decreased thereby reducing the potential
for a secondary response. On the contrary, when CD4
+
T cells recognize mature APC in
the presence of IL-4 they differentiation into Th2 cell, which can secrete IL-5, IL-4, IL-
10 and IL-13. These Th2 derived cytokines function to activate eosinophils, drive B cells
to make the appropriate antibodies, and inhibits Th1 mediated immune responses
(Banchereau and Steinman, 1998).
In order to generate an effective T cell response, there must exist other signals and
interactions besides that of MHC molecules and TCR molecules, such as the interaction
between co-stimulatory molecules, CD80 and CD86, on APC with CD28 on the surface
of T cells and signals from cytokines and chemokines. It has been demonstrated that APC
displaying peptides on MHC molecules in the absence of co-stimulatory molecules and
cytokines and chemokines have the ability to both anergize T cells (Tan et al., 1993) and
generate regulatory T cells (Tregs) (Jonuleit et al., 2000).

12
Natural HPV Immunity
The resolution of an HPV infection is immune mediated as demonstrated by
persistent HPV infections and enhanced progression of HPV-induced cervical lesions in
immuno-deficient patients (Laga et al., 1992; Petry et al., 1994). Protective immunity
against an HPV infection is controlled by the interplay between the non-specific innate
and antigen-specific adaptive arms of the immune system. The innate immune effectors,
monocytes, macrophages, polymorphic leukocytes, and NK cells detect pathogens and
act as the first line of defense by producing immunoregulatory molecules (IFN-α, -β, -γ,
TGF-β, tumor necrosis factor (TNF)-α, and IL-1, IL-16, IL-10, IL-12, and IL-15) which
can directly control virally infected cells and shape the activation of the adaptive immune
response (Stern, 2005). The importance of innate immunity in the clearance of HPV-
induced lesions is highlighted by the presence of macrophages in regressing lesions
(Coleman et al., 1994) and by the production of TNF-α in HPV-induced cervical lesions
(Routes et al., 2005). TNF-α not only induces inflammation but it also suppresses viral
replication. Additionally, TNF-α initiates the activation and migration of LC
(Cumberbatch et al., 2000). Considering that LC are the immune sentinels at the site of an
HPV infection, the activation of LC is vital to induce an adaptive immune response
against HPV.
It is well established that CD8
+
T cells are critical in mediating the immune
clearance of HPV infections. Many studies have investigated the presence and
importance of CD8
+
T cells in HPV-induced lesions, providing evidence of naturally
occurring HPV-specific CD8
+
T cell immunity. Specifically, HPV16 and HPV18 specific

13
CTL responses have been identified in patients with HPV16 or HPV18 induced cervical
lesions but not in healthy donors (Evans et al., 1997; Todd et al., 2004). Memory CTL
responses were also identified in patients with HPV16-induced cervical lesions but were
absent in healthy donor controls (Ressing et al., 1996). Additionally, a recent study
demonstrated that HPV16 E6 epitope-specific memory CD8
+
T cells are present and
continue to circulate for months, if not years, after an HPV16 infection has cleared and is
no longer detectable (Wang et al., 2008). Taken together, these studies strongly suggest
that CTL responses, specific for HPV epitopes, are crucial in restricting the progression
of and inducing the clearance of HPV-induced lesions.
For an effective CTL response to occur it is important to have a robust CD4
+
Th1
response. CD4
+
Th1 cells play an important role in antiviral immunity, specifically in
mediating the expansion of antigen-specific CTL. There is evidence that CD4
+
Th1 cells
play a role in the clearance and control of HPV infections. Coleman et al., found that the
immune cell infiltrate in regressing HPV-induced genital warts is largely CD4
+
T cells
(Coleman et al., 1994). In addition to the clearance of warts, HLA-DR, -DQ, and –DP
restricted CD4
+
tumor infiltrating lymphocytes specific for HPV16 and HPV18 have been
identified in patients with cervical cancer (Höhn et al., 1999; Piersma et al., 2008). It was
also shown in multiple studies that strong memory CD4
+
Th1 responses against HPV16
E2, E6 and HPV18 E6 antigens can be frequently detected in healthy donors. These
memory CD4
+
Th1 responses are likely the result of previous effective anti-HPV immune
response that have been cleared (de Jong et al., 2002; Gallagher and Man, 2007; Welters

14
et al., 2003). Collectively, these studies illustrate that CD4
+
T cells, like CD8
+
T cells,
play a critical role in the natural immunity against HPV.

Mechanisms of Immune Escape by HPV
The majority of high-risk HPV infections are cleared by an HPV-specific cell-
mediated immune response, however a small percentage of high-risk HPV infections
persist and this can lead to the generation of cervical cancer. The slow clearance rate and
lack of an effective immune response indicates that HPV is escaping immune detection.
Experimental evidence suggests that this immune evasion is due to the lack of
recognition of viral particles and viral infected cells. It has also been demonstrated that
viral proteins primarily drive immune escape mechanisms by disrupting immune cell
functions, consequently disabling host immunity.
Low profile of HPV life cycle
The replicative life cycle of HPV has co-evolved with the differentiation of
epithelial keratinocytes making it difficult for the immune system to detect a viral
infection. This is due to several factors. First, the host immune system has minimal
exposure to HPV because HPV DNA replication and expression of early viral proteins
mainly occurs in the nucleus of host cells and late proteins are only produced in the distal
layers of the epithelium where virions are assembled and then shed (Frazer, 2004).
Additionally, throughout an HPV infection there is little to no pro-inflammatory signals
and antigens are not released into the milieu because HPV induces cell proliferation
rather than viral induced cytolysis or necrosis. Therefore the micro-environment during

15
an HPV infection lacks the necessary signals for the induction of an innate and/or
adaptive immune response (Kupper and Fuhlbrigge, 2004). Furthermore, there is no
blood-borne phase of the HPV life cycle so the immune system outside of the epithelium
has little opportunity to detect the virus.
Modulation of antigen processing and presentation
In order for T cells to recognize infected cells there must exist an interaction
between their TCR and peptide bound MHC molecules on the surface of infected cells.
Consequently, many viruses have targeted the disruption of antigen processing and MHC
function as a means of immune evasion, an example of such viruses are Adenovirus,
Human Cytomegalovirus (HCMV), Epstein-Barr Virus (EBV), and Human
Immunodeficiency Virus (HIV) (Farrell and Davis-Poynter, 1998; Früh et al., 1999;
Tortorella et al., 2000).
It is also well established that HPV disrupts the processing and presentation of
viral antigens, leading to the suppression of a cell-mediated immune response. Multiple
studies have found that HPV-induced lesions exhibit a loss or down-regulation in MHC
class I molecules and this loss is associated with clinical progression of HPV-induced
lesions (Bontkes et al., 1998; Cromme et al., 1994; Keating et al., 1995). The down-
regulation of MHC class I is correlated with the reduced expression of the transporter
associated with antigen presentation (TAP) 1 and 2 and proteasome subunits low
molecular mass protein (LMP) 2 and 7 (Cromme et al., 1994; Evans et al., 2001; Keating
et al., 1995). As their names suggest, TAP and LMP are involved in the processing and
transport of peptide/MHC class I complex to cell surface. As a result of altered

16
expression in TAP and LMP proteins the immunogenic peptides from both HPV E6 and
E7 proteins are not efficiently processed or presented by HPV positive tumor cells or
cervical cell lines (Bauer and Lipford, 2000; Evans et al., 2001). The mechanisms
responsible for disrupting antigen processing and presentation have been in part
attributed to HPV proteins E5 and E7.
Papillomavirus E5 is a hydrophobic protein localized in the endoplasmic
reticulum and the Golgi apparatus of the host cell (Burkhardt et al., 1989; Pennie et al.,
1993). The function of E5 has yet to be clearly defined but it has been demonstrated to
interfere with the acidification of the Golgi apparatus and endosomes (Schapiro et al.,
2000; Straight et al., 1995). Because the stability of peptide bound MHC class I complex
is pH dependent (Reich et al., 1997), E5 mediated alkalinization of the Golgi apparatus
and endosomes likely leads to the disruption in transport of peptide bound MHC class I
complexes to the cell surface (Ashrafi et al., 2005; Marchetti et al., 2002). Additionally, it
has recently been demonstrated that HPV16 E5 also forms a complex with calnexin and
the heavy chain of MHC class I, which also mediates the retention of MHC class I
complex in the endoplasmic reticulum of cells (Gruener et al., 2007). Moreover, HPV16
E5 has been shown to disrupt MHC class II antigen presentation. Zhang et al.
demonstrated that E5 can prevent the breakdown of the invariant chain, a chaperone
protein required to load peptides onto MHC class II molecules, by inducing the
alkalinization of endosomal compartments. This is because the degradation of invariant
chain is dependent on an acidic microenvironment. E5-mediated alkalinization in turn

17
blocks the formation of peptide loaded MHC class II complexes, which correlates to the
down-regulation of surface expression of MHC class II (Zhang et al., 2003).
In addition to E5, E7 has also been demonstrated to suppress MHC class I
molecules from being expressed. The HPV16 E7 protein has been shown to repress the
promoter for the MHC class I heavy chain, while HPV18 E7 can both suppress the
promoter for the MHC class I heavy chain and repress the bidirectional promoter that
controls the expression of TAP1 and LMP2 (Georgopoulos et al., 2000). Thus, HPV E7
regulates the transcription and therefore protein expression of essential molecules
involved in antigen presentation, resulting in reduction of viral antigen presented on the
surface of infected cell. Collectively, these studies are evidence that both E5 and E7 play
a role in hindering proper antigen processing and presentation by MHC class I and II
molecules on HPV-infected cells, thereby aiding in HPV immune evasion.
Disruption of IFN responses
IFNs are part of the innate immune response and are one of the first lines of
defense against viral infections. Type I IFN, IFN-α and IFN-β, interfere with viral
replication, enhance MHC class I expression, attract and induce the activation of immune
cells such as macrophages, DC, and NK cells, and have anti-proliferative activities. They
act by binding specific receptors on target-cells, which induce signal transduction
cascades that lead to the synthesis of proteins, which aid in mediating IFN functions.
Because type I IFN are critical in mounting an immune response against viral infections,
HPV and many other viruses has evolved ways to disrupt the IFN induced signaling
pathways.

18
Clinically, E7 expression in HPV-induced lesions is associated with IFN-α
resistance (Barnard et al., 2000). The mechanism by which HPV16 E7 inhibits IFN-α
signaling is by binding to interferon regulatory factor (IRF)-9, thereby inhibiting the
translocation of IRF-9 to the nucleus and binding to the IFN stimulatory gene factor
(ISGF)-3 transcription complex, leading to an inhibition in INF-α signaling (Barnard and
McMillian, 1999; Barnard et al., 2000). Another mechanism by which E7 interrupts IFN
signaling is through inhibiting IRF-1 activity. IRF-1 is a transcription factor for the IFN-β
promoter and is a critical mediator in IFN signaling. E7 disrupts the function of IRF-1 by
directly binding to it and by recruiting histone deacetylases to the IFN-β promoter,
consequently inhibiting transcription (Park et al., 2000). Additionally, Nees et al. used
cDNA microarrays to examine global alterations in gene expression in differentiating
cervical keratinocytes after transfection with HPV16 E6 and E7. They found that HPV16
E6 and E7 work in concert to down-regulated IFN-response genes (Nees et al., 2001).
HPV16 E6 has been demonstrated to inhibit IFN responses by binding to and inactivating
IRF-3, a potent transcriptional activator that binds to the IFN-β promoter (Ronco et al.,
1998). HPV18 E6 has also been shown to impair INF signaling cascades by physically
associating with tyrosine kinase 2 (Tyk2). This interaction prevents Tyk2 from binding to
the IFN-α receptor, which in turn impairs Jak-STAT activation and IFN-α mediated
signaling (Li et al., 1999). Thus E6 and E7 inhibit the induction of INF inducible genes
that enable IFN host resistance to HPV infection. This disruption and therefore lack of an
IFN response during high-risk HPV infections may also be partly responsible for the

19
observed decrease in NK cell activity in persistent HPV infections (Garzettii et al., 1995;
Malejczyk et al., 1993).
Alteration in the expression of cytokines and chemokines
Cytokines and chemokines are small immunomodulating molecules that are
critical in the development and function of both the innate and adaptive immune
response. They are often secreted during a viral infection to activate and direct the
migration of immune cells. Altered expression of cytokines and chemokines during a
viral infection would compromise the ability of the host to respond to the pathogen.
There is extensive evidence that one method by which HPV escapes immune detection is
via the alteration in expression of cytokines and chemokines in infected tissues.
The pro-inflammatory cytokine, IL-18 plays an important role in antiviral and
antitumor immunity. It does so by a variety of functions, including stimulation of T cell
proliferation, IFN-γ secretion, and induction of the Th1 immune response (Dinarello,
1999). In relation to HPV infections, IL-18 expression and activity has been shown to be
inhibited by both HPV16 and HPV18 (Cho et al., 2001; Lee et al., 2001). Cho et al.
demonstrated that the down-regulation in IL-18 is likely due to the direct interaction
between HPV E6 and IL-18 (Cho et al., 2001). Additionally, it has been identified that
both HPV16 E6 and E7 inhibit IL-18 function by competitively binding to the IL-18 α-
chain of the receptor, inhibiting the interaction and activation of the receptor by IL-18
(Lee et al., 2001). Considering that IL-18 can modulate IFN-γ secretion, which can drive

20
NK cell activities, it is possible that HPV16 and HPV18 may impede NK cell activity
indirectly through the down-regulation of IL-18 activity, however this has yet to be
demonstrated.
Nuclear factor (NF)-κB is a transcriptional activator that controls the expression
of a number of immune response genes including chemokines such as IL-8, Regulated
upon Activation, Normal T-cell Expressed, and Secreted (RANTES), and Interferon-
gamma induced protein (IP)-10 (Huang and McCance, 2002). Each of these chemokines
is a potent activator and chemoattractant for immune cells such as T cells, neutrophils,
NK cells, DC, and monocytes. There is experimental evidence that these above stated
chemokines are repressed by HPV16 E6 and E7. Specifically, HPV16 E6 down-regulates
IL-8 promoter activity by competing with and interfering with NF-κB binding to the
transcriptional co-activator, CREB binding protein (CRB)/ p300, which is required for
optimal activation of the IL-8 promoter. E7 has also been shown to repress IL-8 promoter
activity and works in concert with E6 through interfering with NF-κB binding to the
promoter through binding to another co-activator, p300-CBP-associated factor (P/CAF)
(Huang and McCance, 2002). Additionally, HPV16 E6 and E7 have also been shown to
suppress monocyte chemoattractant protein (MCP)-1 expression in primary human
cervical epithelial cells (Kleine-Lowinski et al., 2003). However, the mechanism of how
this suppression occurs has yet to be delineated. Like IL-8, RANTES, and IP-10, MCP-1
is another chemokine that is critical during viral infections because it attracts monocytes,
memory T cells, and NK cells. Therefore, the repression of certain chemokines during
HPV infection may in part explain why there is a decrease in infiltration and activity of

21
immune cells, specifically T cells and NK cells in cervical cancer (de Jong et al., 2004;
Malejczyk et al., 1993).
Macrophage inflammatory protein (MIP)-3α is produced in response to viral
infection by various cells, such as epidermal keratinocytes and macrophages. It is a
potent chemoattractant for LC precursors that express CCR6, the MIP-3α receptor (Dieu-
Nosjean et al., 2000). Notably, human keratinocytes expressing HPV16 E6 and E7
produce decreased amounts of MIP-3α in comparison to cells that do not express E6 or
E7. The observed HPV mediated reduction in MIP-3α secretion resulted in decreased
migration of LC and their precursor cells (Caberg et al., 2009; Guess and McCance,
2005). Suppression of MIP-3α secretion by E6 and E7 negatively effects the recruitment
of LC precursor cells to the site of infection and therefore likely contributes to the
reduced numbers of LC in persistent HPV-induced lesions, aiding in HPV persistence
(Jimenez-Flores et al., 2005).
Inhibition of LC function
As mentioned before, DC and LC play a critical role in the initiation and
modulation of an antiviral immune response because they have the ability to stimulate
resting, naïve T cells and initiate CTL immune responses in vivo (Banchereau et al.,
2000). Because of where LC reside they are the only APC that naturally come into
contact with HPV during an infection. Consequently, LC are responsible for initiating an
immune response against an HPV infection. On the contrary, DC are exposed to HPV
VLP only during vaccination strategies and it has been found in various studies that
HPV16 VLP can bind to and activate DC (de Witte et al., 2008; Lenz et al., 2001; Rudolf

22
et al., 2001), providing evidence that the structural surface components of HPV can
induce the maturation of APC.
While human DC can generate HPV-specific immune responses to HPV16L1L2
VLP (Rudolf et al., 2001), we have previously demonstrated that human LC exposed to
HPV16L1L2 VLP present HPV-derived peptides on their MHC molecules but do not up-
regulate surface markers important for T cell co-stimulation and migration, including
CD86, CD80 and CCR7, secrete pro-inflammatory cytokines, or generate an HPV16
antigen specific CD8
+
T cell response (Fausch et al., 2003; Fausch et al., 2002). Thus, it
can be concluded that even though DC and LC are both potent APC, they have different
phenotypic and functional responses to HPV, suggesting that HPV is evading immune
detection by inhibiting the phenotypic and functional maturation of LC. The molecular
mechanism mediating this immune escape has yet to be elucidated. However, it has been
shown that both DC and LC internalize HPV16L1L2 VLP but do so through different up-
take mechanisms (Fausch et al., 2003) suggesting that HPV16 binds to different cell
surface receptors on DC and LC, which could lead to differential means of internalization
and initiation of differing signal transduction pathways that could either mediate the
immune response or immune escape.
CD4
+
regulatory T cells
CD4
+
regulatory T cells (Tregs) play an important role in immune homeostasis
(Mills, 2004). They have also been shown to be critical in curtailing effective antitumor
responses (Casares et al., 2003; Shimizu et al., 1999). It has been well established that
there are both natural and inducible Tregs. Inducible Tregs, in contrast to natural Tregs,

23
can be antigen-specific and are generated in the periphery from naïve CD4
+
and CD8
+
T
cells after encounter with APC that have sub-optimal activation status, presenting
peptides in the absence of co-stimulatory molecules (Mills, 2004). The inducible CD4
+

Tregs can be divided into CD4
+
T regulatory 1 cells (T
R
1), which secrete high levels of
IL-10 and small amounts of IL-2, IL-4 and TGF-β and CD4
+
T helper 3 cells (T
H
3) that
secrete high levels of TGF-β and small amounts of IL-10 and IL-4 (Mills, 2004).
Recently, multiple studies have identified CD4
+
Tregs in tumor draining LN of cervical
cancer patients, cervical intraepithelial neoplasia (CIN) I, CIN II, and CIN III, and
cervical cancer biopsies (Adurthi et al., 2008; Fattorossi A et al., 2004; Molling et al.,
2007; van der Burg et al., 2007; Visser et al., 2007). Interestingly, van der Burg et al.,
isolated HPV-specific CD4
+
Tregs from lymph node biopsies of cervical cancer patients
that had the ability to suppress responder T cells (van der Burg et al., 2007). In addition,
CD4
+
Treg frequencies were found to be significantly increased in women who had
persistent HPV16 infection. This increase was found in patients who had detectable
HPV16 E7-specific T-helper cell responses, suggesting that HPV16-specific Tregs are
generated in concert with HPV16-specific T effector cells (Molling et al., 2007).
Moreover, human cervical cancers predominately express Th2 associated cytokines, such
as IL-10 and TGF-β (Bais et al., 2005; Sheu et al., 2001), which could partially be due to
the activity of CD4
+
Treg. Collectively, these studies suggest that CD4
+
Tregs are playing
a role in suppressing the immune response mounted against high-risk HPV.

24
Project Goals and Significance
As mentioned previously, the majority of women infected with HPV resolve their
viral infection, however there is a long latency period between infection and clearance.
This indicates that HPV has evolved mechanisms to evade immune system detection and
mechanisms to suppress immune responses against the virus. These immune evasion
tactics allow for the persistence of HPV infections. It is important to note that viral
persistence is a major risk factor in the development of cervical cancer. The incidence of
cervical cancer is a major health concern of today, with more than 490,000 women per
year are diagnosed with cervical cancer globally (Parkin et al., 2005). Despite recent
advances in prophylactic vaccination, no therapeutic is available that initiates viral
clearance to treat the approximately 24 million American women currently infected with
HPV (Dunne et al., 2007). Therefore, the development of therapeutics to treat persistent
HPV infections is imperative to eradicate the world-wide health burden of cervical
cancer.
In order to develop an effective immunotherapeutic for HPV-induced lesions, the
natural immune response generated against HPV and immune escape mechanisms used
by HPV to evade clearance must be fully understood. An effective therapy should not
only bolster the natural cell-mediated immune response but must also overcome or
reverse immune escape mechanisms utilized by the virus. LC are an attractive target for
immunotherapy of high-risk HPV-induced lesions because they are the APC responsible
for initiating an immune response against HPV, they mediate both the innate and adaptive
immunity, and they are targeted by HPV to evade immune detection. However, the

25
mechanism of how HPV16 manipulates LC has yet to be determined. We hypothesize
that HPV16 binds to a receptor on LC that induces an immune suppressive signal
transduction cascade, which prevents the phenotypic and functional maturation of LC,
leading to the immune evasion of HPV16. We aim to; 1) delineate the signal transduction
cascade initiated in LC after exposure to HPV16 VLP which mediates the immune
escape, 2) determine which viral capsid protein, L1 or L2, induces the suppressive signal
transduction cascade, 3) identify the receptor that HPV16 VLP bind to on LC which
initiates the immune escape, and 4) investigate potential small-molecule therapeutic
compounds to reverse the phenotype and function of LC exposed to HPV16 VLP in order
to induce a cell-mediated immune response against HPV16.
These projects are of great significance and have clinical implications as they
directly address a major medical need, namely the eradication of existing HPV infections
that otherwise could lead to the development of cervical cancer. This work is novel
because it attempts to elucidate the mechanism of HPV16 immune escape through LC,
which has not been previously studied, and it also examines small molecules that target
LC as a potential immunotherapeutic strategy, which has never been explored. In
addition, this research is highly relevant because it utilizes human cells and a human
virus to study a human disease, therefore making the study applicable in a clinical setting
if positive results are obtained.

26
Chapter 2. HPV can Escape Immune Recognition Through LC
Phosphoinositide 3-Kinase Activation
1

Introduction
The importance of the immune system in the control of HPV infection and lesion
development is shown by the fact that increased cellular immune responses correlate with a
good clinical prognosis (Eiben et al., 2002). Various direct and indirect mechanisms
utilized by HPV to evade host immunity have been previously described [chapter 1,
(Kanodia et al., 2007)]. However, no mechanism has been identified in which HPV directly
alters immune system activation via its interaction with LC.
After pathogen encounter, APC may initiate multiple different signal transduction
pathways culminating in expression of a variety of different immune response genes (Fig.
3). The signal transduction pathways initiated after antigen encounter in APC have a
profound effect on the type of response the APC displays towards that antigen. APC may
remain non-responsive to the antigen encountered, can stimulate a Th1 response, a Th2
response, or generate an inhibitory immune response. Specifically in APC it has been
demonstrated that pathogens can activate various members of the mitogen-activated protein
kinase (MAPK) family, the NF-κB cascade, and/or the phosphoinositide 3-kinase (PI3K)
cascade to induce a response (Ardeshna et al., 2000; Banchereau and Steinman, 1998; Guha
and Mackman, 2001). The MAPK family is comprised of three groups: the extracellular

1
This work has been published in The Journal of Immunology (Vol. 174, pp.7172-7178,
2005) and I have received permission to use this manuscript as part of my dissertation.
Copyright 2005. The American Association of Immunologists, Inc.

27
signal-regulated protein kinases (ERK) (Boulton et al., 1991), the c-Jun N-terminal kinsases
(JNK) (Kyriakiset al. et al., 1994), and the p38 stress-activated protein kinases (p38K)
(Karin, 1998). The subgroups of the MAPK family differ in substrate specificity and are
activated by upstream MAPK kinases (MKK), which are triggered by various external
stimuli. The ERK pathway mainly responds to mitogens and growth factors while the JNK
and p38K pathways are activated by stress and inflammatory cytokines (Boulton et al.,
1991; Karin, 1998). NF-κB is a dimer (such as p50/p65) composed of a DNA-binding
subunit and a transcription-activating subunit that is usually held inactive in the cytoplasm
by the IκB family or by inactive precursors (such as p100 or p105). Upon cellular activation
by pro-inflammatory cytokines and growth factors, the IκB kinase (IKK) complex is
activated and it subsequently phosphorylates IκB and/or p100, leading to the degradation of
IκB and/or processing of p100. The NF-κB dimer then translocates to the nucleus and
activates gene expression (Israel, 2003). Notably, activated NF-κB regulates the expression
of many genes encoding immune and inflammatory response proteins (Ghosh et al., 1998;
Pettit et al., 1997). Class I
A
PI3K are a subfamily of lipid kinases consisting of regulatory
and catalytic subunits, which are activated by phosphorylation, that control multiple
signaling cascades which regulate differentiation, cell-proliferation, survival, motility, and
cell secretion (Deane and Fruman, 2004; Katso et al., 2001). PI3K activity is mediated
through downstream effectors such as protein kinase C (PKC) and Akt. PI3K has the
capacity to control a diverse set of signaling molecules and has been shown to both
positively and negatively regulate MAPK and NF-κB cascades (Fukao and Koyasu, 2003;
Guha and Mackman, 2002; Kane et al., 1999; Lee et al., 2006; Madrid et al., 2001).

28
Signaling cascades are complex and intricate cross-talk between different signaling
pathways is becoming increasingly evident through the use of specific inhibitors.
Importantly, the signal transduction profile initiated by a particular antigen is specific and
results in a response unique to that antigen. Therefore, various pathogens have evolved
mechanisms to induce or inhibit particular signaling cascades to aid in their escape from
host immunity (Alcami and Koszinowski, 2000; Xu et al., 2001).
It was previously found that human LC may be immunosuppressive after their
encounter with HPV16L1L2 VLP, whereas human DC induced a potent CD8
+
T cell
response (Fausch et al., 2003; Fausch et al., 2002). Those studies did not examine the
signaling cascades initiated by the respective APC after HPV16L1L2 VLP encounter. In the
current study, we determined the intracellular signaling cascades initiated in human DC and
LC after HPV16L1L2 VLP encounter. Since the receptor for HPV16 has not been identified,
we examined three signaling pathways frequently involved in the regulation of immune
responses, the MAPK pathways, the NF-κB pathway, and the PI3K pathway that are all
potential candidates downstream of a putative viral receptor. After HPV16L1L2 VLP
stimulation, the MAPK, NF-κB, and PI3K signaling cascades are initiated in DC
culminating in cellular activation. However when LC are exposed to HPV16L1L2 VLP,
PI3K is activated while MAPK and NF-κB pathways are down-regulated. Furthermore, the
up-regulation of PI3K in LC led to the inactivation of Akt. After inhibition of the PI3K
cascade, LC up-regulated surface activation markers and induced a potent immune response
against HPV16L1L2 VLP-derived antigens. Taken together these data indicate that LC fail
to induce an anti-HPV16 immune response because of the activation of PI3K. These

29
findings define a novel immune escape mechanism utilized by HPV16 through the
manipulation of LC.

Figure 3. Signal transduction pathways initiated in APC after encountering
pathogens. After HPV16L1L2 VLP encounter, DC and LC may initiate multiple signal
transduction pathways resulting in transcription from immune response genes.

30
Results
Phenotypic characterization of human monocyte-derived LC
Because we use human monocyte-derived LC in our studies and not epidermal
derived LC we first wanted to verify the purity of our human monocyte-derived LC
population. To do so, we assessed the presence of surface markers commonly used to
identify human epidermal LC: langerin, CD1a, and E-cadherin. Our results show that LC
generated from human monocytes are a pure population and express LC associated
surface markers, therefore they are phenotypically equivalent to LC found in the
epidermis (Fig. 4).

Figure 4. Human monocyte-derived LC express langerin, CD1a, and E-cadherin.
Monocyte-derived LC were stained with either anti-langerin, anti-CD1a, anti-E-cadherin
(black histograms) or isotype matched negative controls (grey histograms). The cells
were analyzed by flow cytometry. One representative experiment of three is shown.

After HPV16L1L2 VLP encounter, LC down-regulate MAPK pathways
The activation of MAPK pathways has been implicated in the response APC
display towards antigenic stimuli (Ardeshna et al., 2000; Arrighi et al., 2001), therefore
we examined the activation of MAPK pathways in DC and LC after exposure to
HPV16L1L2 VLP. Within minutes after antigen-encounter many signal transduction

31
molecules become phosphorylated and activated, therefore we first determined the level
of phosphorylated signaling molecules in DC or LC cellular extracts by western blot
analysis at 15 min and 45 min post-stimulation. After a 15 min incubation with
HPV16L1L2 VLP, LC showed a profound decrease in phosphorylated ERK1/2 (p-
ERK1/2), phosphorylated MKK4 (p-MKK4), and phosphorylated activating transcription
factor (ATF)2 (p-ATF2) (Fig 5A). MKK4 is a MAPK kinase upstream of JNK and ATF2
is a transcription factor downstream of both JNK and p38 kinase. LPS, a known inducer
of MAPK signaling (Ardeshna et al., 2000), up-regulated p-ATF2 in LC (Fig. 5A and B)
indicating that the LC used can respond to other stimuli. After a 45 min incubation, DC
exposed to HPV16L1L2 VLP up-regulated p-ATF2 similar to LPS stimulation, whereas
LC did not (Fig. 5B). The enhanced activation of signal transduction molecules in
untreated LC as compared to DC can be attributed to the presence of TGF-B
1
mediated
signaling in LC (Fig. 5A). TGF-B
1
, which is required for LC differentiation (Geissmann
et al., 1998; Larrengina et al., 2001), induces activation of the pathways examined (Kim
et al., 2002). However significant washes with PBS ensured that no free TGF-B
1
is
present during the incubations. We next determined the level of cAMP responsive
element binding protein (CREB)-1 binding activity in nuclear extracts from treated DC
and LC using a modified ELISA procedure (Fig. 5C). CREB-1 is a transcription factor
activated downstream of MAPK signaling (Gee et al., 2007; Gee et al., 2006). We
observed a significantly increased level of CREB-1 binding activity in extracts from DC
treated with HPV16L1L2 VLP, similar to the positive control LPS, but not in
HPV16L1L2 VLP-exposed LC (Fig. 5C). When taken together, these data indicate that

32
HPV16L1L2 VLP activate MAPK signaling cascades in DC, whereas MAPK pathways
are suppressed in LC after exposure to HPV16L1L2 VLP.
The up-regulation of surface activation markers is a hallmark of APC activation
and the activation of MAPK signaling has been implicated in their expression (Arrighi et
al., 2001). Therefore, we examined the level of marker expression by DC and LC after
treatment with SB203580, a potent p38K inhibitor (Tong et al., 1997). DC treated with
SB203580 did not up-regulate p-ATF2 after stimulation with LPS (data not shown),
indicating a block in MAPK signaling. DC exposed to HPV16L1L2 VLP up-regulated
MHC class I, CD80, and CD86 compared to untreated DC as assessed by flow cytometry
(Fig. 5D). With the addition of the MAPK inhibitor, DC treated with HPV16L1L2 VLP
did not significantly induce expression of any of the surface activation markers above
untreated levels further indicating the importance of MAPK signaling in the activation of
APC (Fig. 5D). As expected, both LC incubated with the MAPK inhibitor and LC left
untreated did not show a significantly increased level of marker up-regulation after
exposure to HPV16L1L2 VLP (Fig. 5D).
After HPV16L1L2 VLP encounter, DC activate NF-κB, LC do not
Another signaling molecule frequently found activated in APC after pathogen
encounter is NF-κB (Ghosh et al., 1998; Pettit et al., 1997). DC exposed to HPV16L1L2
VLP for 15 min decreased the level of IκB-α, whereas LC exposed to HPV16L1L2 VLP
did not (Fig. 6A). In nuclear extracts from DC, and not LC, treated for 45 min with
HPV16L1L2 VLP we found an increase in NF-κB p50 (Fig. 6B) and NF-κB p65 (Fig.
6C) binding activity. This indicates that the NF-κB cascade was activated in DC and not

33

Figure 5. After HPV16L1L2 VLP encounter, LC down-regulate MAPK pathways.
Western blot analysis of elements of the MAPK pathways in cellular lysates from DC and
LC either untreated or treated with LPS or HPV16L1L2 VLP for A, 15 min or B, 45 min.
The numbers below the blots represents the average density of the band relative to the
band in untreated APC. C, CREB-1 binding activity in nuclear extracts from DC and LC
either untreated or treated with LPS or HPV VLP for 45 min. The experiment was
repeated three times with similar results (*P < .005 by students t-test assuming
homoscedastic, one-tailed distribution). D, Fold change in expression of MHC class I,
CD80, or CD86 as determined by flow cytometry of DC and LC after exposure to
HPV16L1L2 VLP relative to untreated DC and LC, respectively. DC or LC were either
untreated (-) or treated (+SB) with the MAPK inhibitor SB203580 prior to exposure of
HPV16L1L2 VLP. These data are expressed as the mean of three experiments ± SEM.

34
Figure 5. Continued

35
in LC in response to HPV16L1L2 VLP. To explore if NF-κB is partially responsible for
the up-regulation of surface activation markers, we treated DC and LC with BAY11-
7082, an inhibitor of IκB-α processing, thereby inhibiting the activation and translocation
of NF-κB (Pierce et al., 1997). DC treated with BAY11-7082 and LPS showed no
decrease in IκB-alpha degradation (data not shown), which indicates a block in NF-κB
signaling. We treated DC and LC with or without BAY11-7082 and then with or without
HPV16L1L2 VLP. We compared the level of cell surface MHC class I, CD80, and CD86
by flow cytometry after treatment with HPV16L1L2 VLP to the level expressed without
addition of HPV16L1L2 VLP. The results indicate that incubation of DC with the NF-κB
inhibitor limits the expression of the surface activation markers after exposure to
HPV16L1L2 VLP (Fig. 6D), indicating a role for NF-κB in the HPV16L1L2 VLP-
mediated activation of APC. Treatment of LC with BAY11-7082 resulted in no
significant change in marker expression (Fig. 6D).
Activation of PI3K by HPV16L1L2 VLP-stimulated LC inhibits the immune response
Previously it was shown that PI3K activation in response to various stimuli
negatively regulates the production of IL-12 by DC and inhibition of PI3K results in
enhanced immunity (Fukao et al., 2002). After exposure to HPV16L1L2 VLP for 15 min,
LC activated PI3K, whereas DC displayed no detectable increase in p-PI3K (Fig. 7A).
Although Akt is downstream of PI3K, Akt activation decreased in LC shown by a
decrease in the amount of p-Akt (Fig. 7A). In DC treated for 15 min with HPV16L1L2
VLP, we observed a slight increase in p-Akt, indicating an increase in Akt activation.
After a 45 min exposure to HPV16L1L2 VLP, we observed no increase in PI3K

36

Figure 6. After HPV16L1L2 VLP encounter, DC activate NF-κB while LC do not.
A, IκB-alpha expression in cellular lysates from DC or LC either untreated or treated
with LPS or HPV16L1L2 VLP for 15 min. The numbers below the blot represents the
average density of the band relative to the band in untreated APC. B, NF-κB p50 or C,
NF-κB p65 binding activity in nuclear extracts from DC or LC either untreated or treated
with LPS or HPV16L1L2 VLP for 45 min. These experiments were repeated three times
with similar results (*P < .02 by students t-test assuming homoscedastic, one-tailed
distribution). D, Fold change in expression of MHC class I, CD80, or CD86 as
determined by flow cytometry of DC and LC after exposure to HPV16L1L2 VLP relative
to untreated DC and LC, respectively. DC and LC were either untreated (-) or treated
(+BAY) with the NF-κB inhibitor BAY11-7082 prior to exposure with HPV16L1L2
VLP. These data are expressed as the mean of three experiments ± SEM.

37
Figure 6. Continued

38

activation in LC (Fig. 7B). However DC exposed to HPV16L1L2 VLP increased p-PI3K
and increased p-Akt similar to the positive control LPS (Fig. 6B). After a 24 h exposure
to HPV16L1L2 VLP, LC displayed an increased amount of p-Akt (Fig. 7C). In control
experiments, DC and LC exposed to HPV16L1L2 VLP that were heated for 10 min at 95
°C to disrupt the VLP’s structure resulted in levels of activated signaling molecules
similar to the levels of untreated DC or LC (Fig. 7D), indicating the importance of an
intact viral structure for PI3K signaling activation in LC. These data when taken together,
indicate that LC activate PI3K and suppress Akt early after encounter with HPV16L1L2
VLP, then at later time points activate Akt. However, DC induce the activation of both
PI3K and Akt after 45 min and sustain this level of activation for 24 h.
Additionally, a number of molecules, other than Akt, mediate effector functions
of PI3K activation. Therefore, we also examined if other such molecules downstream of
PI3K are affected in LC after exposure to HPV16L1L2 VLP. We specifically examined
the activation of phosphoinositide-dependent kinase 1 (PDK1), glycogen synthase kinase
(GSK)-3β, and serum- and glucocorticoid-regulated kinase (SGK). We found that the
activated levels of each of these molecules are unchanged in LC after exposure to
HPV16L1L2 VLP (data not shown), indicating that a specific signal is transduced by
PI3K in LC after stimulation with HPV16L1L2 VLP, which is mediating the down-
regulation of activated Akt.

39

Figure 7. After exposure to an intact HPV16L1L2 VLP, LC activate PI3K but
down-regulate p-Akt. Western blot analysis of elements of the PI3K pathway in cellular
lysates from DC and LC either untreated or treated with LPS or HPV16L1L2 VLP for A,
15 min, B, 45 min, or C, 24 h. D. DC and LC were incubated for 15 min with LPS,
HPV16L1L2 VLP, or HPV16L1L2 VLP that were heated for 10 min at 95 °C to disrupt
the viral capsid structure. Cellular extracts were subjected to western blot analysis. The
numbers below the blots represents the average density of the band relative to the band in
untreated APC. The experiments were repeated three times and yielded similar results.

40
Figure 7. Continued

41
Since DC and LC exposed to HPV16L1L2 VLP differ in their activation of PI3K
and PI3K activation has been implicated in the control of immune responses, we
determined whether the activation of PI3K by HPV16L1L2 VLP-exposed LC plays a role
in the lack of surface marker up-regulation. For these experiments we used LY294002, a
potent, specific inhibitor of PI3K (Vlahos et al., 1994). We incubated DC and LC with or
without LY294002, then with or without HPV16L1L2 VLP. DC treated with both the
inhibitor and HPV16L1L2 VLP showed a higher fold increase in marker expression than
DC exposed to HPV16L1L2 VLP alone as assessed by flow cytometry (Fig. 8). LC
exposed to only HPV16L1L2 VLP did not increase marker expression, however after
treatment with LY294002 and HPV16L1L2 VLP, LC increased marker expression (Fig.
8). These data indicate that the activation of PI3K suppresses the activation of signaling
cascades required for surface marker up-regulation.

Figure 8. Activation of PI3K by HPV16L1L2 VLP-exposed LC inhibits surface
marker expression. DC and LC were either untreated (-) or treated (+LY) with the PI3K
inhibitor LY294002 and subsequently incubated with or without HPV16L1L2 VLP. Fold
change in expression of MHC class I, CD80, or CD86 as determined by flow cytometry
of DC and LC after stimulation with HPV VLP relative to untreated DC and LC,
respectively. The data are expressed as the mean of three experiments ± SEM.

42

Because HPV16L1L2 VLP-exposed LC up-regulated activation markers only
after incubation with the PI3K inhibitor, we next sought to determine if these LC could
then initiate a CD8
+
epitope-specific immune response by performing an in vitro
immunization procedure followed by IFN-γ ELISPOT analysis. The HPV16L1L2 cVLP
used in these experiments harbor HPV16-E7, which contains a well-characterized human
HLA-A*0201-restricted epitope E7 amino acids (aa) 86-93 recognized by human CD8
+
T
cells (Ressing et al., 1995), fused to the L2 minor capsid protein. DC and LC generated
from HLA-A*0201 positive donor PBL are capable of initiating an epitope-specific
immune response to the E7
86-93
peptide after the in vitro immunization procedure (Fausch
et al., 2002). Previously it was shown that DC induce a potent response against E7
86-93
after incubation with HPV16L1L2 cVLP, whereas LC require an additional activation
stimulus such as CD40L (Fausch et al., 2002). In the experiments presented here DC and
LC were treated with or without LY294002 and with or without HPV16L1L2 cVLP.
Then we incubated the cells with autologous naïve CD8
+
T cells and the cultures were
restimulated twice with their respective treated DC or LC. Seven days after the last
restimulation, the cells from each culture were collected and tested for a specific response
to the HLA-A*0201-restricted HPV16-E7-derived peptide aa 86-93 by IFN-γ ELISPOT.
As expected, DC exposed to HPV16L1L2 cVLP initiated an epitope-specific response
while LC did not (Fig. 9). However after treatment with the PI3K inhibitor and
HPV16L1L2 cVLP LC induced a response (Fig. 9), indicating that the lack of the LC’s
ability to generate a response after treatment with HPV16L1L2 cVLP alone could be

43
overcome by the inhibition of PI3K. Overall the data indicate that the activation of PI3K
by HPV16L1L2 VLP in LC suppresses the ability of LC to induce an immune response.

Figure 9. Inhibition of PI3K allows HPV16L1L2 VLP-stimulated LC to induce an
HPV-specific CD8
+
T cell response. ELISPOT analysis of HPV16L1L2 cVLP exposed
DC and LC treated with or without the PI3K inhibitor against the known E7-derived
HLA-A*201-restricted CTL epitope [E7 peptide aa 86-93, TLGIVCPI, (Ressing et al.,
1995)]. Responder cells were analyzed in triplicate for IFN-γ production in an ELISPOT
assay. The number of spots in each well was counted and averaged. These data are
expressed as the mean ± SEM (*P < .005 by students T-Test assuming homoscedastic,
one-tailed distribution). The experiment was repeated three times yielding similar results.

Discussion
The high prevalence of HPV-associated diseases in the world indicates that HPV
must have evolved mechanisms to evade host immune recognition resulting in increased
viral transmission and increased incidence of disease. In this study we present data
identifying the targeting of LC, the APC found at the sites of primary HPV infection, as a
mechanism HPV16 utilizes to evade host immunity. After LC encounter HPV16L1L2
VLP, PI3K is activated which in turn leads to the down-regulation of Akt, MAPK, and
NF-κB activation through an unknown phosphatase. This downstream phosphatase target
of PI3K needs to be further explored and identified. One potential target is protein

44
phosphatase 2A (PP2A), which may mediate the down-regulation of Akt, MAPK, and
NF-κB. PP2A is a multimeric serine/threonine phosphatase, which has previously been
shown to inhibit the activities of Akt, PKC, MAPK, and the IκB kinases (Milward et al.,
1999), thereby regulating multiple signaling cascades involved in the control of immune
responses. Additionally, PKC may also play a role in the inhibition of Akt activity
because it has been shown to inhibit Akt activation independent of PP2A (Doornbos et
al., 1999). However, seeing as PKC has also been demonstrated to positively regulate
MAPK and NF-κB signaling pathways there is likely an additional protein beyond PKC
that is involved in mediating the down-regulation of the signaling cascades (Cohen et al.,
2006; Frey et al., 2006; Lee et al., 2006; Yang et al., 2007). Therefore, we propose a
potential mechanism that drives the immune escape of HPV16; upon binding and
internalization of the virus, LC activate PI3K, which subsequently activates PP2A,
inducing the suppression of MAPK, NF-kB, and Akt activity. PKC is also likely to be
activated down-stream of PI3K, aiding in the inhibition of Akt activation (Fig. 10).
Previous data has shown that HPV16L1L2 VLP are endocytosed, processed, and
presented in the context of MHC class I by LC, however they do so in the absence of co-
stimulation (Fausch et al., 2003; Fausch et al., 2002). After inhibition of PI3K and
exposure to HPV16L1L2 VLP, LC up-regulate surface activation markers and are
capable of initiating a potent HPV16L1L2 VLP-specific immune response. This indicates
that the activation of PI3K by HPV16L1L2 VLP in LC defines a mechanism of immune
escape utilized by HPV16. The targeting of LC by HPV16 is a function of the intact viral

45
protein capsid, as we show that HPV16L1L2 VLP with a disrupted structure do not
induce PI3K activation.

Figure 10. Potential signal transduction pathways initiated in LC after exposure to
HPV16L1L2 VLP. Upon exposure of HPV16L1L2 VLP, LC induce the activation of
PI3K and subsequently the activation of PP2A, which inhibits the activity of Akt, MAPK,
and NF-kB, leading to no transcription from immune response genes. PKC could also
directly mediate the inhibition of Akt activity. Inhibition of PI3K would relieve the
suppressive mechanisms in LC, thereby allowing transcription from immune response
genes.

After encounter with HPV16L1L2 VLP, DC initiate the MAPK, NF-κB, and
PI3K signaling cascades. The activation of MAPK and NF-κB cascades in DC results in
maturation and affords these cells the ability to initiate an immune response. PI3K, also

46
activated by DC in response to HPV16L1L2 VLP, but not as profound as in LC, partially
negatively regulates marker expression, although not enough to hinder the DC’s ability to
induce an immune response. Overall the data indicate that DC and LC respond differently
to HPV16L1L2 VLP and that the activation characteristics observed are specific to
HPV16 viral particles.
Previously it was shown that HPV-associated lesions displayed an increased PI3K
activity and a decreased activation of Akt (Zhang and Steinberg, 2002). The authors
concluded that the results obtained were due to the increased expression of phosphatase
and tensin homolog (PTEN), a negative regulator of Akt activation. However, we did not
observe any change in PTEN levels in our DC and LC extracts (data not shown) therefore
PTEN does not appear to be mediating the inactivation of Akt in LC. Another stimulus
may be causing the increased PTEN expression after lesion formation. When taken
together, these data indicate that the activation of PI3K is involved in many steps in
lesion development and the escape of immunity. Also this suggests that in addition to a
newly identified immune escape mechanism utilized by HPV16, PI3K may serve as an
effective clinical target for inhibition in order to enhance HPV16 immunity.

47
Chapter 3. A Major Role for the Minor Capsid Protein of HPV16 in
Immune Escape
2

Introduction
As previously mentioned, the life cycle of HPV is dependent on the
differentiation of basal cells into keratinocytes, in turn it is difficult to produce large
quantities of HPV virions in vitro. For that reason, HPV VLP have been developed and
are used for immunological analysis of HPV. When the major capsid protein L1 is
expressed, it can self-assemble into a L1 VLP and if both L1 and the minor capsid protein
L2 are concurrently expressed, the proteins assemble into L1L2 VLP (Kirnbauer et al.,
1992; Kirnbauer et al., 1993; Zhou et al., 1991).
While the HPV minor capsid protein L2 is not required for VLP formation, it has
been shown to possess a variety of functions critical to the viral life cycle. In contrast to
the type specific L1 sequence, L2 has several highly conserved sequences across human
and animal genotypes (Gambhira et al., 2007; Kawana et al., 1999; Kawana et al., 2001;
Kondo et al., 2007), which suggests an evolutionary pressure to maintain the functions of
L2. L2 binds directly to L1 through a primarily hydrophobic interaction (Finnen et al.,
2003) and is integral in the encapsidation of viral DNA (Zhao et al., 1998; Zhou et al.,
1994) implying a significant role for L2 in the formation of the virion (Ishii et al., 2005).
Additionally, L2 has been shown to facilitate HPV infection (Kawana et al., 2001; Yang

2
This work is currently under review by The Journal of Immunology, The American
Association of Immunologists, Inc. I have received permission to use this manuscript as
part of my dissertation.

48
et al., 2003). HPV virions that are deficient in L2 have a 100-fold reduction in infectivity
(Holmgren et al., 2005). Further functions of L2 include binding of the virion to the
cytoskeleton, transport within the cytoplasm (Florin et al., 2006; Yang et al., 2003), and
facilitation of endosomal escape of the viral genome after infection (Kämper N et al.,
2006).
It has been found in various studies that HPVL1 VLP and HPVL1L2 VLP can
bind to and activate human dendritic cells (DC) (de Witte et al., 2008; Lenz et al., 2001;
Rudolf et al., 2001), providing evidence that the structural surface components of HPV
can induce the maturation of APC. However, we have previously defined an HPV16
immune escape mechanism that targets LC, suppressing LC function (Fausch et al., 2003;
Fausch et al., 2002), and this HPV16 immune escape mechanism is due to the
deregulation of the PI3K-Akt pathway [chapter 2, (Fausch et al., 2005)]. Thus, even
though DC and LC are both potent APC, they respond differently to HPV.
In apparent contradiction to our studies, human LC exposed to HPV L1 VLP were
shown to prime naïve CD8
+
T cells and generate effector T cells in vitro (Yan et al.,
2004). Additionally, HPV L1 VLP were shown to be taken up by human LC through
either a clathrin mediated (Bousarghin et al., 2005) or caveolae-dependent mechanism
(Yan et al., 2004), while we previously demonstrated that HPV16L1L2 VLP were taken
up by human LC through a clathrin-, caveolae-, actin-independent pathway (Fausch et al.,
2003). These contrasting studies highlight differences in the interaction between human
LC and HPV L1 VLP versus HPV L1L2 VLP, and point to the potentially important

49
presence of L2. Therefore, we sought to elucidate if the minor capsid protein L2 is
responsible for the induction of immune escape of HPV16.
In this study we compared the effects of HPV16L1 VLP on human LC to those of
HPV16L1L2 VLP. We demonstrate that human LC exposed to HPV16L1 VLP are
phenotypically and functionally activated while human LC exposed to HPV16L1L2 VLP
appear to be tolerizing-like. These results strongly imply a newly identified role of L2 in
the induction of immune escape and put L2 at the forefront of the dynamic interplay
between HPV16 and the human immune system. This novel function is the first immune
modulating interaction attributed to the L2 protein. By further defining the immune
escape mechanism of HPV16, these results identify L2 as a new target for future HPV
therapeutics.

Results
LC acquire a mature phenotype when exposed to HPV16L1 VLP but not when exposed to
HPV16L1L2 VLP
The mechanism by which HPV16 manipulates LC has yet to be fully defined. We
sought to determine if L2 is responsible for initiating immune escape of HPV16 in LC. In
order to determine the effects of L2 on the phenotypic maturation of LC, we assessed the
expression of cell surface activation markers on LC after exposure to either HPV16L1
VLP or HPV16L1L2 VLP. Maturation of LC was compared with that of LPS treated LC.
LPS is a known TLR4 activating agent commonly used in vitro for its ability to activate
APC. Immature LC were cultured with media alone, treated with LPS, exposed to

50
HPV16L1 VLP, or exposed to HPV16L1L2 VLP for 48h. After the incubation, the cells
were analyzed for the up-regulation of MHC class II molecules, CD80, and CD86 by
flow cytometry. After exposure to HPV16L1 VLP, LC up-regulated MHC class II
molecules, CD80, and CD86 in comparison to untreated LC. On the contrary, LC
exposed to HPV16L1L2 VLP had only a minor up-regulation of MHC class II molecules,
CD80, and CD86 when compared to untreated LC (Fig. 11). Most notably, there was a
statistically significant difference in expression levels of MHC class II molecules, CD80,
and CD86 on LC exposed to HPV16L1L2 VLP in comparison to LC exposed to
HPV16L1 VLP, indicating that the presence of L2 blocked the up-regulation of key
surface activation markers on LC.

Figure 11. Differential expression of surface markers on LC after exposure to
HPV16 VLP. HPV16L1 VLP induce the up-regulation of surface markers on LC
however these surface markers are not increased on LC exposed to HPV16L1L2 VLP.
LC were treated as indicated in the activation assay. Cells were analyzed by flow
cytometry. These data are represented as the fold change in expression of the surface
markers, relative to untreated LC. The mean of eleven separate experiments ± SEM is
presented (*P < .05 and **P< .01, determined by a two-tailed, paired t-test, as compared
to LC exposed to HPV16L1 VLP).

51
Differential expression of cytokines and chemokines by LC exposed to HPV16L1 VLP or
HPV16L1L2 VLP
One functional hallmark of LC activation is the secretion of cytokines and
chemokines. Therefore, we analyzed the types of cytokines and chemokines that are
secreted by LC upon exposure to HPV16L1 VLP or HPV16L1L2 VLP. LC were
incubated with media alone, stimulated with LPS, or exposed to either HPV16L1 VLP or
HPV16L1L2 VLP. The supernatants were collected after 48 h and evaluated using a
human cytokine LINCOplex assay. LC exposed to HPV16L1 VLP highly secreted pro-
inflammatory cytokines and chemokines indicative of a Th1 cell-mediated immune
response, specifically, TNF-α, IL-12p70, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and
RANTES (Fig. 12). The secretion of these cytokines and chemokines was similar to that
of the positive control, LPS stimulated LC. LC incubated with HPV16L1L2 VLP
secreted comparable levels of pro-inflammatory cytokines and chemokines produced by
untreated LC (Fig. 12). Markedly, LC exposed to HPV16L1L2 VLP secreted statistically
significantly less Th1 associated cytokines and chemokines than LC exposed to
HPV16L1 VLP, implying that L2 drives the repression of LC functional maturation.
LC increase migration when exposed to HPV16L1 VLP but not HPV16L1L2 VLP
In order to initiate adaptive T cell immunity, circulating T cells must have the
opportunity to interact with and become activated by mature APC that have migrated to
the LN. To assess the migratory capacity of LC incubated with either HPV16L1 VLP or
HPV16L1L2 VLP, we determined the expression level of CCR7 by flow cytometry and
performed a transwell migration assay using a CCR7 ligand, CCL21. As a positive

52

Figure 12. Differential secretion of cytokines and chemokines by LC exposed to
HPV16 VLP. LC incubated with HPV16L1 VLP secrete Th1-associated cytokines and
chemokines while LC incubated with HPV16L1L2 VLP do not secrete these cytokines
and chemokines. Supernatants collected from LC left untreated, treated with LPS,
incubated with HPV16L1 VLP or incubated with HPV16L1L2 VLP for 48h at 37°C were
analyzed in triplicate for the presence of cytokines and chemokines. Cytokine and
chemokine levels were quantified using a human cytokine LINCOplex assay. These data
are expressed as the mean concentration ± SEM (**P < .01, ***P< .001 as determined by
a two-tailed, unpaired t-test, as compared to LC exposed to HPV16L1 VLP). The
experiment was repeated three times and yielded similar results.

53
Figure 12. Continued

54

control, we analyzed LC incubated with LPS. Phenotypically, LC exposed to HPV16 L1
VLP induced an up-regulation of CCR7, while LC exposed to HPV16 L1L2 VLP had
similar CCR7 levels to LC left untreated (data not shown). Functionally, exposure to
HPV16 L1 VLP induced increased LC migration towards CCL21 compared to that of
untreated LC and LC exposed to HPV16 L1L2 VLP (Fig. 13). Notably, LC exposed to
HPV16L1L2 VLP migrated statistically significantly less than LC exposed to HPV16L1
VLP. These findings demonstrate that L1 functionally activates LC cells to migrate
towards regional LN, but that L2 suppresses this maturation and migration.

Figure 13. HPV16 VLP induce LC migration. LC exposed to HPV16L1 VLP migrate
towards CCL21, however LC exposed to HPV16L1L2 VLP do not migrate towards
CCL21. LC were left untreated, treated with LPS, incubated with HPV16L1 VLP or
incubated with HPV16L1L2 VLP for 48h at 37°C and used in a migration assay. CCL21-
directed migration was calculated as the ratio of cells that migrated with CCL21 to cells
that spontaneously migrated. (**P< .01 as determined by a two-tailed, paired t-test, as
compared to LC exposed to HPV16L1 VLP). The mean of three separate experiments ±
SEM is presented.

55

LC exposed to HPV16L1L2 VLP fail to induce an HPV-specific CD8
+
T cell response in
contrast to the strong response induced by human LC exposed to HPV16L1 VLP
The establishment of an adaptive CD8
+
T cell mediated immune response is
implicated in the clearance of HPV induced cervical lesions. Therefore, we investigated
whether LC exposed to HPV16L1 VLP or HPV16L1L2 VLP would lead to differential
induction of HPV16-specific CD8
+
T cell responses by performing in vitro immunization
assays. LC exposed to HPV16L1L2 VLP failed to induce an HPV16L1-specific CD8
+
T
cell response. In contrast, LC exposed to HPV16L1 VLP induced a robust HPV16L1-
specific CD8
+
T cell response (Fig. 14). These results are of major impact because they
demonstrate that while LC are able to effectively become activated by and present
epitopes derived from HPV16 VLP comprised of L1 alone, the mere presence of L2 in
the VLP silences the ability of LC to activate effector T cells thereby crippling the HPV
specific immune response.

56

Figure 14. LC exposed to HPV16 VLP induce differential activation of HPV16-
specific CD8
+
T cells. LC exposed to HPV16 L1 VLP induce an HPV16 L1-specific
CD8
+
T cells response yet LC exposed to HPV16 L1L2 VLP do not induce an HPV16
L1-specific immune response. LC were incubated with either media alone, HPV16 L1
VLP or HPV16 L1L2 VLP for 48h at 37°C. The LC were irradiated and subsequently
incubated with autologous CD8
+
lymphocytes for 7 days. Restimulations were done with
the respective treatments. After two restimulations, responder cells were analyzed for
IFN-γ production in an ELISPOT assay against a L1 peptide [(aa 323-331,
ICWGNQLFV), (Kaufmann et al., 2001)]. The number of spots in each well was counted
and averaged over five wells, and background values (no peptide stimulation in the
ELISPOT) were subtracted. These data are expressed as the mean of three separate
experiments ± SEM (*P< .05 as determined by a two-tailed, unpaired t-test, as compared
to LC exposed to HPV16 L1 VLP).

LC activate PI3K but down-regulate Akt after exposure to HPV16L1L2 VLP but not after
exposure to HPV16L1 VLP
We examined if L2 plays a role in immune escape of HPV16 through the
deregulation of the PI3K pathway in LC, a mechanism implicated in our earlier studies.
LC exposed to HPV16L1 VLP did not induce the phosphorylation of PI3K, while

57
exposure to HPV16L1L2 VLP highly induced the phosphorylation of PI3K in LC
compared to that detected in untreated LC (Fig. 15). Additionally, we demonstrate that
HPV16L1L2 VLP down-regulated Akt activation, as shown by a marked decrease in the
phosphorylation of Akt when compared to untreated LC. HPV16L1 VLP did not
effectively inhibit the activation of Akt in LC (Fig. 15). These results combined with our
earlier study [chapter 2, (Fausch et al., 2005) ] suggest that L2 is actively suppressing the
phenotypic and functional activation of LC by the deregulation of the PI3K-Akt pathway.

Figure 15. HPV16L1L2 VLP induce an immune suppressive signal transduction
cascade in LC. LC were left untreated, treated with LPS, incubated with HPV16L1 VLP
or incubated with HPV16L1L2 VLP for 15min at 37°C. Cellular lysates were isolated
and subjected to western blot analysis. HPV16L1L2 VLP induce the activation of PI3K
but down-regulates p-Akt in LC while LC exposed to HPV16L1 VLP do not up-regulate
PI3K activity and maintain a baseline level of p-Akt. One representative experiment of
three is shown.

58
Discussion
By comparing the effects of HPV16L1 VLP and HPV16L1L2 VLP on human LC,
we investigated the role of the minor capsid protein L2 in the induction of immune
escape. HPV16L1 VLP cause a maturation of LC as shown through the up-regulation of
surface markers, the increased production of pro-inflammatory cytokines and
chemokines, the increased migration of LC, and the induction of an HPV16-specific
CD8
+
T cell response. In contrast, HPV16L1L2 VLP do not cause maturation of LC and
therefore inhibit the effective generation of an HPV-specific immune response. This lack
of immune response by LC is associated with the up-regulation of activated PI3K and the
down-regulation of phosphorylated Akt. Collectively, our results strongly suggest a novel
role for L2 in initiation of HPV16 immune escape mechanism through LC.
Limited research has been done assessing the effects of HPV on the maturation of
human LC. A previous study conducted by Yan et al. found that LC exposed to
HPV6bL1 VLP were functionally activated, as assessed by the generation of effector
CD8
+
T cells (Yan et al., 2004). In that study, Yan et al. questioned our previous results
and pointed to differences in the way CTL assays were performed as an explanation for
why their results showed HPV6bL1 VLP exposed LC were able to generate effector
CD8
+
T cells while our data demonstrated that HPV16L1L2 VLP exposed LC did not
generate effector CD8
+
T cells (Fausch et al., 2002). To resolve this apparent
discrepancy, in this current study we demonstrate that LC exposed to HPV16L1 VLP are
both phenotypically and functionally activated, while LC exposed to HPV16L1L2 VLP
are not, implying that L2 is mediating this immune escape of HPV16. Therefore, it is

59
plausible that the differing results are due to the lack of L2 proteins in the HPV6bL1 VLP
used to carry out the Yan et al. study. Notably, HPV6b used by Yan et al. is a low-risk
genotype, while HPV16 used in this study is a high-risk genotype. It remains to be
addressed whether L2 mediates immune evasion across all genotypes or whether it is a
genotype specific response. However, it is likely that L2’s role is analogous among
varied genotypes due to its highly conserved sequence across distantly related human and
animal papillomavirus types and the identification of cross-neutralizing antibodies
directed against the amino terminus of L2 (Gambhira et al., 2007; Kawana et al., 1999;
Kawana et al., 2001; Kondo et al., 2007).
Research regarding the uptake of HPV by human LC has been at times confusing
and contradictory. We previously demonstrated that the mode of uptake of HPV16L1L2
VLP by LC is clathrin-independent, caveolae-independent, and actin-independent
(Fausch et al., 2003). In a study by Yan et al., LC were shown to take up HPV6bL1 VLP
through a caveolae-dependent pathway (Yan et al., 2004). Meanwhile, Bousarghin et al.
demonstrated that HPV16L1 VLP entered LC through a clathrin-dependent pathway
(Bousarghin et al., 2005). Although these studies came to separate conclusions, they are
very likely due to the use of HPVL1 VLP versus HPVL1L2 VLP, thereby indicating the
possible presence of a specific L2 receptor and uptake mechanism.
Our results define a novel role for L2 as a key regulator of an immune escape
mechanism of HPV. Through an active and specific signal transduction cascade, L2
inhibits the up-regulation of surface activation markers, blocks secretion of Th1-
associated cytokines and chemokines, limits migration, and hinders the generation of an

60
HPV16-specific CD8
+
T cells response. Normally, in order to generate an effective T cell
response, T cells must receive three signals- signal one through the T cell receptor, signal
two through co-stimulatory molecules, and signal three through cytokines and
chemokines. It has been demonstrated that APC displaying signal one in the absence of
signals two and three have the ability to both anergize T cells (Tan et al., 1993) and
generate regulatory T cells (Tregs) (Jonuleit et al., 2000). Therefore, by silencing
maturation but continuing to present peptides, LC exposed to HPV16L1L2 VLP are
likely to become tolerizing APC that possess the ability to anergize HPV16-specific T
cells and/or induce HPV16-specific Tregs. In context of an HPV16 infection, this
dampened LC response directed by the presence of L2 is a potent immune escape
mechanism that allows the virus to remain active and infectious by selectively
eliminating beneficial T cells and actively suppressing HPV16-specific immune
responses. This study is of major impact because it identifies L2 as a critical protein,
which drives this immune escape of HPV16 through interaction with human LC, thereby
highlighting L2 as a target for future immune based therapies against high-risk HPV
infections.

61
Chapter 4. Annexin A2 is a Candidate Receptor for HPV16 on LC

Introduction
Virus-receptor interactions are not only conduits for viral entry but they also
initiate signaling cascades to induce a cellular state more permissive for infection.
Considering our previous studies demonstrate that HPV16L1L2 VLP are manipulating
LC through the activation of the PI3K pathway [chapter 2, (Fausch et al., 2005)] it is
highly plausible that there exists a specific receptor(s) on LC that HPV16 is binding to
and activating, which in turn initiates the immune suppressive signaling cascade
identified. However, the receptor(s) to which HPV16 binds to and enters LC through is
presently unknown. Identifying the HPV16 receptor(s) on LC would not only further
elucidate the immune escape mechanism of HPV16 but would also define a potential
therapeutic target for treatment of HPV16-induced infections.
Many studies have been carried out in hopes of identifying the receptor(s) for
HPV on various cell types. The quest has been long, difficult, and controversial. The
α6β4 integrin complex was initially identified as a receptor for HPV (Evander et al.,
1997; McMillan et al., 1999), while heparan sulfate proteoglycans have been shown to be
necessary for attachment of virions (Giroglou et al., 2001; Shafti-Keramat et al., 2003).
However, it was demonstrated that HPV11 and HPV31 are able to infect α6β4 integrin or
heparan sulfate negative cells, respectively (Joyce et al., 1999; Patterson et al., 2005).
Additional proteins have also been suggested to play a role in the binding and activation
of APC. Heparan sulfates (sydencan-3) and DC-SIGN were found to be involved in the

62
attachment of HPV16L1 VLP to DC (de Witte et al., 2008; Garcia-Pineres et al., 2006)
while the binding of HPV16L1 VLP to LC was found to be independent of heparan
sulfates (Bousarghin et al., 2005). Meanwhile, HPV6bL1 VLP were shown to bind to
TLR4 and activate DC (Yan et al., 2005), yet TLR4 does not appear to be involved in the
binding of HPV16L1L2 VLP to LC or DC (unpublished data). Taken together, these
conflicting studies imply that there is more than one receptor for HPV attachment and
internalization and the receptors may be genotype and/or cell dependent. There may also
exist specific receptors for both the capsid proteins, L1 and L2, which may in part explain
the contradictory results.
The concept of an L2 receptor is supported by several studies examining the
interaction of L2 with cell surfaces and L2 mediated infectivity (Kawana et al., 2001;
Yang et al., 2003). In a study by Kawana et al. it was clearly demonstrated that
HPV16L1L2 VLP have a higher infectivity than HPV16L1 VLP. HPV16L1 VLP are less
than half as infectious as HPV16L1L2 VLP, suggesting that the cell surface receptor(s)
for L2 differs from those for L1. Additionally, in the same study it was demonstrated that
preincubation of COS-1 cells with a HPV16 L2 peptide comprised of aa 108-120
decreased infectivity of HPV16 pseudovirions. Substitutions at the N-terminus of L2, aa
108-111, inhibited binding of the mutated HPV16 psuedovirions to HeLa cells and
reduced HPV infectivity of COS-1 cells (Kawana et al., 2001). In a separate study by
Yang et al. it was suggested that HPV16L1L2 VLP binds to the cell surface of HeLa cells
via heparan sulfate proteoglycans, which causes a conformational change in the capsid
structure, leading to the exposure of aa 13-31 of the N-terminal L2 protein on the virion

63
surface that subsequently interacts with a secondary receptor and facilitates infection
(Yang et al., 2003).
Thus, the existence of an L2-specific receptor becomes highly conceivable when
these studies are viewed in the context of our results presented in chapter 3, which
demonstrate that the tolerizing-like phenotype, functional suppression, and signaling
deregulation of LC is mediated by the minor capsid protein L2. Together, these findings
demonstrate that L2 plays a critical role in HPV16 infection through the interaction
between the N-terminal region of the protein and an unidentified cellular surface protein.
In this study, we demonstrate that the N-terminus of L2 plays a role in mediating the
binding of HPV16L1L2 VLP to LC and we identify a candidate HPV16 L2 receptor on
human LC, annexin A2. Furthermore, we show that the internalization of HPV16L1L2
VLP is mediated by annexin A2 in an L2 specific manner. Our findings are of major
impact because this is the first study that has identified a potential L2 receptor.

Results
The HPV16

L2
108-120
peptide inhibits binding of HPV16L1L2 VLP to LC
We sought to determine if the N-terminus of L2 plays a critical role in facilitating
the attachment of HPV16L1L2 VLP to LC as it has been demonstrated in COS and HeLa
cells. To examine the role of the N-terminal L2
108-120
region, LC were incubated with
increasing concentrations of the L2
108-120
peptide [aa 108-120, LVEETSFIDAGAP,
(Kawana et al., 2001)]

and subsequently exposed to HPV16L1L2 VLP. Flow cytometric
analysis was used to assess bound HPV16L1L2 VLP on the surface of LC. We found that

64
as LC were exposed to increasing concentrations of the L2
108-120
peptide, the number of
HPV16L1L2 VLP bound to treated LC decreased (Fig. 16), suggesting that the N-
terminus of L2 facilitates HPV16 binding to LC.

Figure 16. The HPV16 L2
108-126
peptide inhibits binding of HPV16L1L2 VLP to LC.
LC were incubated with increasing concentrations of the L2
108-126
peptide for 1 h at 4°C
and subsequently incubated with HPV16L1L2 VLP for 1 h at 4°C. HPV16L1L2 VLP
remaining on the surface of LC were detected using a L1 specific conformational
antibody (H16.V5). Binding was assessed by flow cytometry. This graph represents one
of three experiments.

(6x)His L2
108-120
binds to a specific surface protein on LC
Next, we wanted to identify which cell surface protein(s) the L2
108-120
peptide was
binding to on LC, which is inhibiting HPV16L1L2 VLP from binding to the cells. LC
were either incubated with or without the (6x)His-L2
108-120
peptide and subsequently
exposed to a cross-linking agent, DTSSP. After cross-linking the peptide to the cell
surface protein(s) it is closely interacting with, LC were lysed and the lysates were mixed
with the Ni-NTA agarose slurry. The proteins attached to the Ni-NTA agarose were

65
eluted over 10 fractions. Each fraction (non-reduced) was separated by electrophoresis
and probed for using a monoclonal antibody specific for polyHistidine. Consistently we
have observed a unique band, just above 39 kDa, in the fractions that were isolated using
the (6x)His-L2
108-120
peptide. The specific band began to become apparent in fraction 5
and was fully eluted within fraction 6. Notably, this unique band is not present in the
negative control, no peptide (Fig. 17).

Figure 17. PolyHistidine immunoblot analysis of eluates isolated from a pulldown
assay. The immunoblot shows one (6x)His-L2
108-120
peptide specific band in elutions 5
and 6. LC were incubated with either no peptide or 50 ug/0.5x10
6
cells of (6x)His-L2
108-
120
peptide and subsequently cross-linked with DTSSP. Cells were then lysed and mixed
with a Ni-NTA agarose slurry overnight and eluted. Non-reduced eluates were
electrophoresed, transferred to nitrocellulose and probed with an anti-polyHistidine
antibody. This is a representative figure.

Subsequently, eluates 5 and 6 that contain the unique band were reduced,
separated by electrophoresis, and silver stained. Once again, a distinct band was observed
by silver stain, just above ~39 kDa. The band was only present in the eluates that were
isolated with the (6x)His-L2
108-120
peptide. The negative control did not have a
corresponding band (Fig. 18). The unique band was excised and analyzed by mass
spectrometry at the USC Proteomics Core. The majority of the protein in the band was
predicted to be annexin A2.

66

Figure 18. Silver stain analysis of eluates isolated from a pulldown assay. LC were
incubated with either no peptide or 50 ug/0.5x10
6
cells of (6x)His-L2
108-120
peptide and
subsequently cross-linked with DTSSP. Cells were lysed and mixed with a Ni-NTA
agarose slurry and eluted. Reduced eluate #6 was electrophoresed and silver stained. The
unique band right above ~39kDa was isolated and analyzed by mass spectrometry. One
representative experiment of two is shown.

Annexin A2 associates with the L2
108-120
peptide on the surface of LC
Knowing that annexin A2 is highly likely to interact with the L2
108-120
peptide as
identified by mass spectrometry we wanted to confirm the presence of annexin A2 in our
L2
108-120
peptide pulldown eluates. Through immunoblot analysis we found that annexin
A2 was only present in L2
108-120
peptide pulldown eluates and not in our negative control
eluates (Fig 19). Additionally, annexin A2 was found primarily in eluates 5 and 6, which
corresponds to the elution fractions that the (6x)His-L2
108-120
peptide was present in.

67
Moreover, the immunoblots showed that annexin A2 was found at the same molecular
weight that our specific band was observed at, in the immunoblots probed with anti-
polyHistidine and in our silver stained gels. Thus, these findings confirm that annexin A2
is interacting with the (6x)His-L2
108-120
peptide on the surface of LC.

Figure 19. Annexin A2 immunoblot analysis of eluates isolated from a pulldown
assay. LC were incubated with either no peptide or 50 ug/0.5x10
6
cells of (6x)His-L2
peptide (aa 108-120) and subsequently cross-linked with DTSSP. Cells were then lysed
and mixed with a Ni-NTA agarose slurry overnight and eluted. Non-reduced eluates were
electrophoresed, transferred to nitrocellulose and probed with an anti-annexin A2
antibody. This is a representative figure.

SLPI Blocks the Uptake of HPV16L1L2 VLP by LC
It has been demonstrated that secretory leukocyte protease inhibitor (SLPI)
interacts with the extracellular surface of annexin A2, which in turn inhibits HIV-1
infection of macrophages (Ma et al., 2004). Therefore we used SLPI to investigate if
annexin A2 functions in the internalization of HPV16L1L2 VLP by LC. LC were
pretreated with increasing concentrations of SLPI and then exposed to carboxyfluorescein
diacetate, succinimidyl ester (CFDA-SE) labeled-HPV16L1L2 VLP for 15 min.

68
Following the 15 min incubation, LC were fixed with 2% paraformaldehyde and uptake
was assessed by flow cytometry. CFDA-SE attaches to proteins, via amines, and upon
uptake it is cleaved by intracellular esterases, resulting in the generation of a fluorescent
signal detectable by flow cytometry. Therefore, HPV16L1L2 VLP that have been
internalized by LC will fluoresce while HPV16L1L2 VLP bound to the cell surface will
not be detected. Previously, we have shown that CFDA-SE labeling does not interfere
with the initial binding interaction between VLP and APC and LC can internalize CFDA-
SE labeled HPV16L1L2 VLP as quickly as 10 min (Fausch et al., 2002). In the current
experiments, we found that as LC were exposed to increasing concentrations of SLPI, LC
internalized decreasing amounts of HPV16L1L2 VLP. The inhibition was greatest when
LC were exposed to 30 µg/ml of SLPI, which resulted in a 50 percent decrease of
HPV16L1L2 VLP uptake in comparison to untreated LC exposed to HPV16L1L2 VLP
(Fig. 20A).
Knowing that annexin A2 mediates the uptake of HPV16L1L2 VLP by LC, we
next sought to confirm that annexin A2 is a specific receptor for the L2 protein. To do so,
LC were pretreated with 30 µg/ml of SLPI and subsequently exposed to CFDA-SE
labeled-HPV16L1 VLP for 15 min. Following the 15 min incubation LC were fixed with
2% paraformaldehyde and uptake was assessed by flow cytometry. Notably, untreated
LC and SLPI treated LC internalized similar amounts of HPV16L1 VLP (Fig. 20B),
indicating that SLPI did not inhibit HPV16L1 VLP uptake. Taken together, these results
strongly imply that annexin A2 interacts with the L2 protein and is critically involved
with the internalization of HPV16L1L2 VLP by LC.

69

Figure 20. SLPI inhibits uptake of HPV16L1L2 VLP by LC but not uptake of
HPV16L1 VLP. A. LC were incubated with increasing concentrations of SLPI for 1 h at
4°C, then incubated with CFDA-SE labeled HPV16L1L2 VLP for 15 min at 37°C, and
fixed with 2% paraformaldehyde The mean percentage of uptake ± SEM of three separate
experiments is presented (*P < .05, determined by a two-tailed, paired t-test, as compared
to the negative control). B. LC were incubated with SLPI (30 µg/ml) for 1 h at 4°C, then
incubated with CFDA-SE labeled HPV16L1 VLP for 15 min at 37°C, and fixed with 2%
paraformaldehyde. Uptake of CFDA-SE labeled HPV16 VLP by LC was assessed by
flow cytometry. The mean percentage of uptake ± SEM of three separate experiments is
presented.

*

70
Discussion
The N-terminal amino acid sequence of L2 is highly conserved in mucosal HPV
types (Kawana et al., 1999; Kawana et al., 2001). This strongly suggests that L2 plays an
important role in mucosal HPV infection and may even determine cellular tropism of
mucosal HPV by binding to a specific receptor. The region aa 108-120 of L2 has been
shown to be vital in the binding and infectivity of many cell types (Kawana et al., 2001)
but until now it has never been shown to be critical in the binding and infectivity of LC.
In this study we demonstrate, through binding and pull-down assays, that HPV16 L2 (aa
108-120) is critical in the binding of HPV16 to LC and specifically interacts with annexin
A2 on the surface of LC. Additionally, through uptake assays we show that the
internalization of HPV16L1L2 VLP by LC is mediated by annexin A2. Our data suggest
that annexin A2 is a candidate receptor for HPV16 on LC.
Annexin A2 (annexin II) is a member of the annexin family of proteins. It is
found in the cytoplasm as a 36kDa monomer and in the plasma membrane at the cell
surface as a heterotetramer, which consists of two annexin A2 monomers bridged non-
covalently by a S100A10 dimer (Glenney, 1986). S100A10 is also known as p11 or
annexin A2 light chain and it is a member of the S100 family of proteins (Rescher and
Gerke, 2008). Notably, S100A10 has been demonstrated to be expressed in LC (Rust et
al., 2006). Therefore, annexin A2 heterotetramers can form and be presented on the
surface of LC.
Annexin A2 translocation from the cytoplasm to the cell membrane is dependent
on both the expression of S100A10 and tyrosine phosphorylation of annexin A2 (Deora

71
et al., 2004). Annexin A2 heterotetramer has been implicated in exocytosis, endocytosis,
membrane fusion, and membrane trafficking (Gerke et al., 2005). It has also been shown
to facilitate binding of both plasminogen and plasmin to endothelial cells, macrophages,
and human peripheral monocytes (Falcone et al., 2001; Hajjar et al., 1994; Kassam et al.,
1998; Laumonnier et al., 2006). It has been demonstrated that when plasmin binds to
monocytes it initiates the release of pro-inflammatory lipid mediators (Weide et al.,
1996), chemotaxis (Syrovets et al., 1997), and expression of TNF-α, IL-1, MCP-1, and
CD40 (Burysek et al., 2002; Syrovets et al., 2001). Collectively, these studies
demonstrate that annexin A2 heterotetramers located on immune cells can play a role in
the induction of an immune response. Furthermore, annexin A2 has been associated with
CMV binding, fusion, and infection (Derry et al., 2007; Raynor et al., 1999; Wright et al.,
1994), identified as a co-factor for HIV infection in macrophages (Ma et al., 2004), and
identified as a receptor for respiratory syncytial virus (Malhotra et al., 2003). Until now
annexin A2 has never been associated with HPV as a receptor. Notably, annexin A2 has
been shown to be up-regulated in squamous cervical cancer patients (Bae et al., 2005).
This observation is of interest, however it is not known why annexin A2 is up-regulated
in cervical cancer lesions.
Currently, it is unclear if annexin A2 can transduce signaling cascades. However
there is experimental evidence suggesting that annexin A2 is involved in initiating
multiple signal transduction cascades. It is well documented that annexin A2
heterotetramers have a preference for negatively charged plasma membranes (Rescher
and Gerke, 2008). At this preferential site, annexin A2 heterotetramers interact with

72
phosphatidylinositol (4,5) bisphosphate (PI(4,5)P
2
) (Hayes et al., 2004; Rescher et al.,
2004), which can be phosphorylated by PI3K. Therefore it is possible that annexin A2
heterotetramers are associated with PI3K signal transduction cascades. Additionally,
upon plasmin binding to monocytes, the signaling cascades of NF-κB, p38K, and janus
kinase (JAK)/signal transduction and transcription (STAT) are activated (Burysek et al.,
2002; Syrovets et al., 2001), suggesting that annexin A2 mediates the induction of these
pathways. It was also demonstrated that after engagement of annexin A2 heterotetramers,
the complex dissociates and annexin A2 is cleaved. It has been proposed that this
truncated form of annexin A2 is responsible for initiating down-stream signaling
(Laumonnier et al., 2006). Moreover, it has been shown that annexin A2 can be
phosphorylated at Serine 25 and Tyrosine 23, which are likely targets of Src kinase and
PKC, respectively (Deora et al., 2004; Glenney Jr, 1985; Gould et al., 1986). When taken
together these findings make it highly conceivable that annexin A2 is a receptor that
initiates signal transduction cascades. However, the specific signaling cascades have yet
to be elucidated, so we need to investigate further whether the immune suppressive signal
transduction pathway initiated by HPV16L1L2 VLP in LC corresponds to that of the
signaling cascades transduced upon engagement and activation of annexin A2.
Furthermore, annexin A2 is detectable in various cells including epithelial cells of
the skin (Waisman, 1995). Interestingly, an immunohistochemical study investigating the
differential expression of annexins found that annexin A2 was expressed on basal cells of
the cervix but not on the cells of the intermediate epithelial layer of the cervix (Dreier et
al., 1998). Thus, it is plausible that HPV16 not only uses annexin A2 as a receptor on LC

73
but also on epithelial basal cells, which are the host cells for a productive HPV infection.
In the future, it would be interesting to determine if mucosal epithelial basal cells also
internalize HPV16 through annexin A2.
In the context of the current literature, it is highly likely that we have identified
the HPV16 L2 receptor on LC. Nonetheless, there are still confirmatory experiments that
need to be carried out. Currently, we are verifying the interaction between the (6x) His-
L2
108-120
peptide and annexin A2 via co-immunoprecipitation assays, using an anti-
annexin A2 antibody. In addition to the co-immunoprecipitation assays, we are
generating lentivirus specific for annexin A2 to confirm the function of annexin A2 in the
internalization of HPV16L1L2 VLP by LC. If the experiments we are working on yield
results that align with and confirm our current data, then the study will be of great
significance because it will not only identify a novel role for annexin A2 as the HPV16
L2 receptor on LC but it will also have implications for a future therapeutic target, as
discussed in chapter 6.

74
Chapter 5. Reversal of HPV-Specific T cell Immune Suppression Through
Toll-like Receptor Agonist 8 Treatment of LC Exposed to HPV16
3

Introduction
The first prophylactic HPV vaccine, Gardasil, has been approved, however it will
not aid in treating millions of women that are currently infected with high-risk HPV.
Considering, HPV actively escapes immune detection [chapter 1, (Kanodia et al., 2007)],
immunotherapeutic treatments are necessary to treat existing high-risk HPV infections.
Currently no therapeutics exist, therefore there is a global need to develop effective
immuno-based therapies to mediate the clearance of high-risk HPV induced lesions.
As we demonstrate here, LC express Toll-like receptor (TLR)7 and TLR8 thus
one potential therapeutic approach to treat HPV16 induced lesions would be to activate
HPV16 infected LC using synthetic imidazoquinolines (imiquimod, resiquimod, 3M-002
and 3M-031). Imidazoquinolines are TLR7 and/or TLR8 agonists and therefore are
potent innate immune modulators [(Table 3, (Schon and Schon, 2008)]. TLR7 and TLR8
are localized to endosomal membranes and naturally recognize ssRNA (Barton, 2007;
Schon and Schon, 2008). Once TLR7 and/or TLR8 are engaged, NF-κB and other
transcription factors are activated, leading to the transcription of many immune response
related genes, including genes coding for cytokines, chemokines, co-stimulatory markers,
and adhesion molecules (Gorden et al., 2005; Medzhitov et al., 1997; Schon and Schon,

3
This work has been published in The Journal of Immunology (Vol. 182, pp.2919-2928,
2009) and I have received permission to use this manuscript as part of my dissertation.
Copyright 2009. The American Association of Immunologists, Inc.

75
2008). Specifically, it has been shown that DC and macrophages treated with
imidazoquinolines secrete cytokines and chemokines, such as TNF-α, IL-6, IL-8, IL-12,
MCP-1, and IP-10, leading to a cell mediated adaptive immune response (Gibson S.J. et
al., 2002; Gorden et al., 2005; Sauder, 2003; Wagner et al., 1997; Weeks and Gibson,
1994). Imidazoquinolines demonstrate antiviral and antitumor activity primarily through
endogenous cytokines and chemokines produced by APC (Gibson et al., 1995; Reiter et
al., 1994; Sidky et al., 1992; Weeks and Gibson, 1994).
Confirming that TLR7 and TLR8 are expressed on LC we hypothesized that
synthetic imidazoquinolines would activate LC previously exposed to HPV16, leading to
the induction of an HPV16 specific immune response. Our results indicate that select
imidazoquinolines, TLR8 dominant agonists, are promising therapeutic drugs that could
potentially be used as a treatment for HPV-induced cervical lesions by inducing an anti-
HPV specific cell-mediated immune response via the activation of HPV exposed LC.

Imidazoquinoline Agonist Receptor(s)
3M-006 Inactive Analog (TLR7/8)
3M-002 TLR8
Imiquimod TLR7
Resiquimod TLR8/7
3M-031 TLR7/8

Table 3. Synthetic imidazoquinolines and the respective receptor(s).
Results

76
Results
LC express TLR7 and TLR8
In this study we are examining TLR7 and/or TLR8 agonists as a means to initiate
the activation of HPV16L1L2 VLP exposed LC, thereby inducing an effective cell-
mediated immune response against HPV16. First, we analyzed the expression of both
TLR7 and TLR8 in immature LC and HPV16L1L2 VLP exposed LC by flow cytometry.
Our results clearly demonstrate that TLR7 and TLR8 are expressed at similar levels in
immature LC and LC exposed to HPV16L1L2 VLP (Fig. 21).

Figure 21. Human monocyte-derived LC express TLR7 and TLR8. Monocyte-
derived LC were left untreated or exposed to HPV16 VLP and then permeabilized, fixed,
and stained with either anti-TLR7 or anti-TLR8 antibodies (black histograms) or isotype
matched negative controls (grey histograms). The cells were analyzed by flow cytometry.
Immature LC and LC exposed to HPV16L1L2 VLP express similar levels of TLR7 and
TLR8. One representative experiment of three is shown.

77
3M-002 and resiquimod up-regulate surface markers on LC
Knowing that TLR7 and TLR8 are expressed in immature LC and LC exposed to
HPV16L1L2 VLP we sought to determine if selected synthetic imidazoquinolines
phenotypically activate LC exposed to HPV16L1L2 VLP. We assessed phenotypic
activation by the expression of surface markers, MHC class I, MHC class II, CD80 and
CD86, on LC that have previously encountered HPV16L1L2 VLP and have been treated
with each of the imidazoquinolines. DC were used as a positive control to test for the
activity and to determine the optimal concentration of each imidazoquinoline because it
has been well established that DC are activated by imidazoquinoline compounds (Philibin
and Levy, 2007; Sauder, 2003; Stanley, 2002). As expected, DC treated with 3M-002,
imiquimod, resiquimod, and 3M-031 induced the up-regulation of surface markers, most
notably MHC class II and CD86, relative to untreated or 3M-006 treated DC (Fig. 22A).
3M-006 is an inactive small molecule TLR7/8 analog that is produced in a similar
manner as the other imidazoquinolines and used as a negative control. The optimal
concentration for each imidazoquinoline to activate APC was determined by assessing a
range of concentrations (0.1µM-60µM) for each agonist. The concentration of each
agonist that resulted in the maximum expression of surface makers on DC, as determined
by flow cytometry analysis, was used as the optimal concentration (data not shown).
Since we confirmed the agonists are active and knowing the optimal
concentrations needed to activate APC we investigated if each agonist has the ability to
reverse the phenotype of LC exposed to HPV16L1L2 VLP. LC were left untreated,
stimulated with LPS, exposed to HPV16L1L2 VLP, treated with each of the

78
imidazoquinolines, or exposed to HPV16L1L2 VLP and subsequently treated with each
of the imidazoquinolines. Subsequently each population of cells was analyzed by flow
cytometry for the expression of surface markers. Consistent with our previously reported
data (Fausch et al., 2002), LC exposed to HPV16L1L2 VLP did not increase the
expression of surface markers when compared to untreated LC and 3M-006 treated LC
(Fig. 22B). LC treated with either 3M-002 or resiquimod significantly induced the up-
regulation of surface marker, as seen with the positive control, LPS stimulation.
Surprisingly, imiquimod and 3M-031 treated LC induced only a minor up-regulation of
surface markers above that of the negative controls, untreated LC, LC exposed to
HPV16L1L2 VLP and 3M-006 treated LC (Fig. 22B). It should be noted that imiquimod
could not be used at any higher dose because it was found to be toxic to the cells at two
fold higher concentrations than used in our assays. Consequently, when LC were exposed
to HPV16L1L2 VLP and subsequently treated with each of the imidazoquinolines, only
3M-002 and resiquimod significantly induced the up-regulation of the surface markers,
while imiquimod and 3M-031 moderately increased the expression of surface markers on
LC exposed to HPV16L1L2 VLP, relative to the negative controls (Fig. 22B). Of note, it
appears that TLR7 and TLR8 agonists induced a slightly greater up-regulation of surface
markers on LC that have previously been exposed to HPV16L1L2 VLP than on untreated
LC, however these differences in expression are not statistically significant. Thus, these
phenotypic data begin to suggest that imidazoquinolines have different effects on DC and
LC. Specifically, 3M-002 and resiquimod appear to be far more potent agonists for LC
than imiquimod and 3M-031.

79
Figure 22. Differential expression of surface markers on DC and LC stimulated with
imidazoquinolines. A. DC were left untreated, treated with LPS, or treated with each of
the imidazoquinolines. The cells were analyzed by flow cytometry for the expression of
MHC class I and II molecules, CD80, and CD86. Surface markers are up-regulated when
treated with 3M-002, imiquimod, resiquimod, and 3M-031. These data are represented by
fold increase in surface marker expression, which are based on mean fluorescence
intensity. The mean ± SEM of four separate experiments is presented (*P < .05,
determined by a two-tailed, paired t-test, as compared to the negative control). B. LC
were left untreated, stimulated with LPS, exposed to HPV16L1L2 VLP, treated with each
of the imidazoquinolines, or exposed to HPV16L1L2 VLP and subsequently treated with
each of the imidazoquinolines. After the final incubation the cells were analyzed by flow
cytometry for the expression of MHC class I and II molecules, CD80, and CD86. 3M-002
and resiquimod induced the up-regulation of surface markers on LC and LC exposed to
HPV16L1L2 VLP. These data are represented by fold increase in surface marker
expression, which are based on mean fluorescence intensity. The mean ± SEM of four
separate experiments is presented (*P < .05, **P < .01, ***P< .001, determined by a
one-way ANOVA and Tukey’s Multiple Comparison Test as compared to the negative
controls).

80
Figure 22. Continued.

81
Differential production of cytokines and chemokines from LC stimulated with
imidazoquinolines
Imidazoquinolines stimulate both an innate and an adaptive immune response.
The innate immune response induced by imidazoquinolines drives the adaptive immune
response into a Th1 cell-mediated response via the local cytokine and chemokine milieu
generated primarily by activated macrophages and DC. Thus, we wanted to determine if
selected imidazoquinolines could stimulate LC exposed to HPV16L1L2 VLP to produce
a pro-inflammatory cytokine and chemokine profile similar to the cytokine milieu known
to be generated by imidazoquinoline activated DC. Cytokines and chemokines produced
by untreated LC, LC exposed to HPV16L1L2 VLP, LC treated with each of the
imidazoquinoline compounds, and LC exposed to HPV16L1L2 VLP and treated with the
imidazoquinoline compounds were evaluated. Supernatant from each treatment was
collected and analyzed using a human cytokine LINCOplex assay. IL-12 p70, TNF-α, IL-
6, IL-8, and MIP-1β concentrations were statistically significantly elevated when LC
were stimulated with 3M-002, resiquimod, or when LC were exposed to HPV16L1L2
VLP and then stimulated with either 3M-002 or resiquimod in comparison to the negative
controls, untreated LC, LC exposed to HPV16L1L2 VLP, 3M-006 treated LC, and LC
exposed to HPV16L1L2 VLP and treated with 3M-006 (Fig. 23). LC treated with 3M-
031 or LC exposed to HPV16L1L2 VLP and subsequently stimulated with 3M-031 only
slightly induced the production of these cytokines and chemokines above that of the
negative controls (Fig. 23). IP-10, MCP-1 and RANTES were also found to be highly
secreted by LC treated with 3M-002, resiquimod, or 3M-031 and LC exposed to HPV16

82
VLP and then stimulated with either 3M-002, resiquimod, or 3M-031 (data not shown).
Markedly, imiquimod stimulated LC and LC exposed to HPV16L1L2 VLP and
subsequently treated with imiquimod secreted comparable amounts of TNF-α, IL-12 p70,
IL-6, IL-8, MIP-1β (Fig. 23), IP-10, RANTES or MCP-1 (data not shown) as that
observed in the negative controls. The cytokine and chemokine analyses demonstrate that
3M-002 and resiquimod are more efficient activators of HPV16L1L2 VLP exposed LC in
comparison to 3M-031 and imiquimod. The cytokine and chemokine profiles produced
by both 3M-002 and resiquimod activated LC are similar to that of imidazoquinoline
stimulated DC (Sauder, 2003; Stanley, 2002). Thus, like DC, LC activated by either 3M-
002 or resiquimod likely induce a Th1 cell mediated response via the production of
cytokines and chemokines.
3M-002 and resiquimod induce the up-regulation of CCR7 and migration of LC exposed
to HPV16L1L2 VLP towards CCL21
CCR7 mediates the migration of LC to T cell zones of the draining LN by binding
to either secondary lymphoid tissue chemokine (SLC/CCL21)

or MIP-3β (CCL19).
Therefore, we investigated whether the imidazoquinoline compounds can induce the up-
regulation of CCR7 and CCL21-directed migration of LC exposed to HPV16 VLP.
Untreated LC, LPS stimulated LC, LC exposed to HPV16L1L2 VLP, and LC exposed to
HPV16L1L2 VLP and subsequently treated with each of the imidazoquinolines were
analyzed for the expression of CCR7 by flow cytometry. LC exposed to HPV16L1L2

83

Figure 23. 3M-002 and resiquimod highly induce the secretion of Th1 associated
cytokines and chemokines by LC previously incubated with or without HPV16L1L2
VLP. Supernatants collected from untreated LC, LC exposed to HPV16L1L2 VLP, LC
treated with each of the imidaziquinolines, or LC exposed to HPV16L1L2 VLP and then
treated with imidaziquinolines were analyzed in triplicate for the presence of cytokines
and chemokines. Cytokine and chemokine levels were quantified using a human cytokine
LINCOplex assay. These data are expressed as the mean concentration with error bars
representing the SD (*P < .05, ***P< .001, determined by a one-way ANOVA and
Tukey’s Multiple Comparison Test as compared to the negative controls). The
experiment was repeated three times and yielded similar results.

84

Figure 23. Continued

85

VLP stimulated with either 3M-002 or resiquimod induced the up-regulation of CCR7
similar to the positive control, LPS-treated LC (Fig. 24A). In contrast, imiquimod and
3M-031 did not induce the expression of CCR7 on LC previously exposed to
HPV16L1L2 VLP (Fig. 24A). Next, we examined whether the expression of CCR7
functionally corresponded to enhanced migration of LC towards CCL21 by a transwell
migration assay. We observed that 3M-002 and resiquimod significantly induced the
migration of LC exposed to HPV16L1L2 VLP towards CCL21, as seen similarly in the
positive control, while imiquimod and 3M-031 did not enhance CCL21-directed
migration of LC exposed to HPV16L1L2 VLP (Fig. 23B). Collectively, these
experiments demonstrate that 3M-002 and resiquimod are providing LC exposed to
HPV16L1L2 VLP with a potent stimulus to acquire the potential to migrate effectively
towards the LN derived chemokine, CCL21.

86

Figure 24. 3M-002 and resiquimod induce the up-regulation of CCR7 and migration
of LC exposed to HPV16L1L2 VLP towards CCL21. LC were left untreated,
stimulated by LPS, exposed to HPV16L1L2 VLP, or exposed to HPV16 VLP and
subsequently treated with each of the imidaziquinolines. After the final incubation LC
were either, A. harvested and analyzed for the expression of CCR7 (black line) by flow
cytometry (grey line is the isotype control antibody), or B. used in a migration assay. The
mean ± SEM of three separate experiments is presented (***P< .001, determined by a
one-way ANOVA using repeated measures and Tukey’s Multiple Comparison Test as
compared to the negative controls).

87
Induction of an epitope-specific CD8
+
T cell response by LC exposed to HPV16L1L2
cVLP and stimulated with either 3M-002 or resiquimod
Thus far we have demonstrated that 3M-002 and resiquimod can effectively
activate LC previously exposed to HPV16L1L2 VLP, unlike imiquimod and 3M-031, so
we next sought to determine if LC exposed to HPV16L1L2 cVLP and stimulated with
each of the imidazoquinolines could induce an HPV16 E7-specific, MHC class I-
restricted T cell response by performing in vitro immunization assays followed by IFN-γ
ELISPOT analysis. LC generated from HLA-A*0201 positive monocytes were exposed
to HPV16L1L2 cVLP and treated with each of the imidazoquinolines. We then incubated
the cells with autologous naïve CD8
+
T cells and the cultures were stimulated twice with
their respective treated LC. Seven days after the last restimulation, the cells from each
culture were collected and analyzed for a specific CD8
+
T cell response to the HLA-
A*0201-restricted HPV16-E7
86-93
peptide (Ressing et al., 1995) by an INF-γ ELISPOT.
Notably, LC exposed to HPV16L1L2 cVLP and stimulated with either 3M-002 or
resiquimod initiated a statistically significant HPV16 epitope-specific response when
compared to untreated LC and LC exposed to HPV16L1L2 cVLP, while LC exposed to
HPV16L1L2 cVLP and stimulated with either imiquimod or 3M-031 did not induce a
significant HPV16 epitope specific immune response (Fig. 25). These experiments
demonstrate that both 3M-002 and resiquimod can initiate an HPV16 specific cell-
mediated immune response through the activation of LC.

88

Figure 25. 3M-002 and resiquimod induce an HPV16 epitope-specific CD8
+
T cell
immune response through the activation of LC exposed to HPV16L1L2 cVLP. LC
were incubated with media alone or with HPV16L1L2 cVLP and each of the
imidazoquinolines. The treated LC were incubated with autologous CD8
+
lymphocytes
and restimulated twice. Responder cells were analyzed in triplicate for IFN-γ production
in an ELISPOT assay against the E7
86-93
peptide. The number of spots in each well was
counted and averaged. These data are expressed as the mean ± SEM (*P < .05 and **P<
.01, determined by a two-tailed, unpaired t-test, as compared to the negative control). The
experiment was repeated three times using two independent HLA-A* 0201 positive
donors and yielded similar results.

Discussion
In this study, we investigated synthetic imidazoquinolines as potential activators
of LC previously exposed to HPV16L1L2 VLP, which could lead to further exploration
of specific imidazoquinolines as therapeutic compounds for treating existing HPV16-
induced infections. Our data clearly demonstrate that 3M-002 and resiquimod can induce
the phenotypic maturation of naïve LC and LC previously exposed to HPV16 VLP via
the up-regulation of surface markers (MHC class I, MHC class II, CD80 and CD86).

89
Moreover, 3M-002 and resiquimod induce functional activation of LC exposed to
HPV16L1L2 VLP as demonstrated by the production of Th1 associated cytokines and
chemokines, CCL21-directed migration, and the induction of an HPV16-specific CD8
+
T
cell response. However, imiquimod does not phenotypically or functionally activate LC
while 3M-031 partially induces the activation of LC. Collectively, our data strongly
suggest that 3M-002 and resiquimod can reverse the phenotype and function of LC
exposed to HPV16, unlike imiquimod and 3M-031. Therefore, our results support
exploring 3M-002 and resiquimod as therapeutic small-molecule compounds for treating
high-risk HPV infections and HPV-induced cervical lesions.
It should be noted that imiquimod is a FDA approved drug (Aldara) to treat
external anogenital warts (condyloma accuminatum) caused by low-risk HPV infection.
More recently, imiquimod has been shown to be successful in treating high-risk HPV
induced vulvar intraepithelial neoplasia (VIN) (van Poelgeest et al., 2005; van Seters et
al., 2008). However, imiquimod has yet to be reported as an effective therapeutic
treatment for HPV-induced CIN. The reason why there is a difference in response
initiated by imiquimod against different types of HPV induced lesions (genital warts,
VIN lesions and CIN lesions) is unclear. This disparity in response could be due to the
difference in cellular composition and structure of the external genitalia and the cervix.
Considering we demonstrate that imiquimod does not activate LC, an effective immune
response against anogenital warts and VIN lesions is likely due to the activation of APC
other than LC, such as DC and macrophages.

90
The effects of synthetic imidazoquinolines on LC had not been well studied until
now. Previously, it was shown that imiquimod and resiquimod do not phenotypically but
functionally activate LC (Burns et al., 2000; Suzuki et al., 2000). Past studies assessed
phenotypic activation of LC by the expression of surface markers. The results from these
studies are in accordance with our results for imiquimod, however, we found that
resiquimod does phenotypically activate LC. The reason for this discrepancy between the
present and past studies, concerning the effects of resiquimod on LC, could be explained
because Burns et al. examined phenotypic activation 6 h after LC were treated with
resiquimod (Burns Jr. et al., 2000), while we assessed the maturation of LC 24 h post
treatment with resiquimod. Furthermore, the functional activation of LC was examined in
the previous study in multiple ways, one of which was by the level of messenger RNA
(mRNA) encoding pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12 p40.
The results from this study showed that both imiquimod and resiquimod enhanced the
transcription of the genes for these specific cytokines (Burns Jr. et al., 2000). We also
assessed cytokine levels as a means of evaluating functional activation, however, we did
so at the more relevant level of protein production. We observed pro-inflammatory
cytokine and chemokine secretion by LC treated with either 3M-002 or resiquimod, and
to a modest extent with 3M-031, however we did not observe this with imiquimod. Our
results are an improvement on previous reports because we assayed for a different end
product, namely protein, and mRNA transcripts do not always translate to protein
expression. Additionally, our results are consistent with recent findings showing that

91
TLR8 agonists are more effective than TLR7 agonists at inducing pro-inflammatory
cytokines and chemokines by monocyte-derived DC (GM-CSF/IL-4/TGF-β) (Gorden et
al., 2005).
Previous data has demonstrated that LC exposed to HPV16L1L2 VLP cannot up-
regulate CCR7, migrate, or induce an HPV16-specific CD8
+
T cell response (Fausch et
al., 2002). To explore the effects of synthetic imidazoquinolines on the migration of LC
exposed to HPV16L1L2 VLP we assessed the expression of CCR7 and the ability of LC
previously exposed to HPV16L1L2 VLP to migrate towards CCL21. Our results clearly
show CCR7 is up-regulated on LC exposed to HPV16L1L2 VLP that are treated with
either 3M-002 or resiquimod, but not when treated with imiquimod or 3M-031.
Furthermore, we demonstrate that the expression of CCR7 correlates to the migratory
ability of LC exposed to HPV16L1L2 VLP. Our data illustrate that only 3M-002 and
resiquimod treated LC previously exposed to HPV16L1L2 VLP are able to migrate in
response to CCL21. However, in a contrasting study it was shown that imiquimod
functionally activates LC by demonstrating that imiquimod induces the migration of LC,
yet this study was performed using a mouse model and it did not confirm that the
migrating LC were effective in inducing an epitope specific adaptive immune response
(Suzuki et al., 2000). Nevertheless, we sought to determine if LC exposed to
HPV16L1L2 VLP that are treated with imidazoquinolines have the ability to induce an
HPV16 epitope specific CD8
+
T cell response. Our results show that 3M-002 and
resiquimod can effectively overcome the phenotype and function of LC exposed to
HPV16L1L2 VLP and can induce an HPV16-specific CD8
+
T cells response, which is

92
critical in mediating the clearance of HPV16 infections and HPV16-induced cervical
lesions. In addition to our findings, Burns et al. investigated the functional activation of
LC after treatment with either imiquimod or resiquimod by assessing the allostimulatory
capacity of the treated LC. They found that imiquimod only modestly induced T cell
proliferation in an allogenic MLR assay while resiquimod highly increased the
allostimulatory capacity of LC (Burns et al., 2000). Their results from this functional
assay are in line with our functional data, which is further support that resiquimod is
more potent than imiquimod in activating LC.
Collectively, our findings imply that strong TLR8 agonists, such as 3M-002 and
resiquimod, are more effective in inducing LC activation and overcoming the tolerizing-
like phenotype and function of LC exposed to HPV16L1L2 VLP, in comparison to TLR7
agonists, such as imiquimod. It has been shown that TLR7 and TLR8 agonists differ in
their target cell selectivity (Gorden et al., 2005). Notably, resiquimod and 3M-031 are
both TLR7 and TLR8 agonists, however, resiquimod is much more effective in activating
LC. This may occur because the agonists differ in their target cell selectivity and
preferentially activate one TLR over the other; resiquimod is known to preferentially act
through TLR8 (Schon and Schon, 2008), while it has yet to be reported which receptor
3M-031 preferentially acts through. This explanation is plausible considering that
functional differences have been observed between TLR7 and TLR8 (Gorden et al., 2005;
Wang et al., 2006). It was demonstrated that TLR7 activation primarily leads to the
production of IFN-α and IFN-regulated cytokines, which is similar to TLR9 activation,
while TLR8 is functionally associated with the production of pro-inflammatory

93
cytokines, such as TNF- α (Gorden et al., 2005). One explanation for the functional
distinction between TLR7 and TLR8 is the difference in the signal transduction pathways
initiated by each of the receptors. TLR8-mediated activation of NF-κB and JNK are
dependent on MAPK/extracellular signal-regulated kinase kinase kinase 3 (MEKK3)
(Qin et al., 2006), while TLR7–mediated activation of NF-κB is transforming growth
factor–β-activated kinase 1 (TAK-1) dependent (Agrawal and Kandimalla, 2007). Bruton
tyrosine kinase (Btk) has also been shown to directly interact with the intracellular
domain of TLR8 and plays an important role in the signal transduction of TLR8; however
Btk has yet to be demonstrated to be associated with TLR7 (Jefferies et al., 2003;
Sochorová et al., 2007). Alternatively, another explanation of our findings may be that
TLR8 is inhibiting TLR7 function. In HEK293 cells it was demonstrated that the co-
expression of TLR8 and TLR7 results in inhibition of TLR7 to respond to its agonist
(Wang et al., 2006). Therefore, TLR8 may inhibit LC from responding to agonists that
preferentially bind TLR7, which explains why TLR8 dominant agonists (such as 3M-002
and resiquimod) are more effective than TLR7 dominant agonists (such as imiquimod
and potentially 3M-031) in activating LC and in driving a strong cell-mediated immune
response.
Since LC are critical in controlling the induction of an immune response in the
epithelium and they are targeted by HPV16 to escape immune detection, LC are highly
attractive targets for immunotherapy of HPV16-induced cervical lesions. In addition, LC
have recently been shown to be able to directly kill cervical epithelial cells that express
HPV16 E6 and E7, thereby generating a source of antigen that could be processed and

94
presented by APC to T cells. LC cytotoxicity is mediated in part by tumor necrosis-
related apoptosis-inducing ligand (TRAIL) expression, which can be up-regulated by the
presence of IFN-γ (Le Poole et al., 2008). Furthermore, it has been demonstrated that
TLR7/8 stimulated DC-like cells have cytotoxic activity, which is mediated by the
expression of TRAIL and the secretion of perforin and granzyme B (Stary et al., 2007).
Thus it is conceivable that TLR8 agonists stimulate LC not only to induce an HPV-
specific Th1 mediated cellular immune response but may also enhance LC cytotoxicity
towards HPV16-infected epithelial cells, further augmenting antiviral and antineoplastic
activity. In conclusion, TLR8 agonists, 3M-002 and resiquimod, are promising
therapeutic compounds for the treatment of high-risk HPV infections.

95
Chapter 6. Discussion and Future Directions

It has been established that the primary risk factor in the development of cervical
cancer is persistent high-risk HPV infection. HPV persistence occurs because the virus
has coevolved with its host and subsequently developed immune evasion mechanisms.
HPV escapes immune detection and suppresses immune function through many different
immune evasion tactics, one of which is the manipulation of LC function (Fausch et al.,
2003; Fausch et al., 2002). In the present studies we aimed to elucidate the mechanism of
how HPV16 manipulates LC. We found that HPV16L1L2 VLP induce the activation of
PI3K in LC, suppressing the ability of LC to initiate an immune response. Additionally,
we identified the minor capsid protein L2 as the HPV16 protein responsible for inducing
the immune suppressive signaling cascade. Experimental evidence from these studies
also suggest that annexin A2 is the L2 receptor on LC, indicating that annexin A2 likely
plays a role in the immune escape of HPV16 through the manipulation of LC.
Considering that LC are responsible for inducing an immune response against HPV and
having identified an HPV16 immune escape mechanism that suppresses LC function, our
studies have highlighted LC as an attractive target for immunotherapy. Therefore, we
sought to investigate immune modulating compounds, TLR7 and/or TLR8 agonists, as
potential immunotherapeutics targeting LC. We demonstrated that strong TLR8 agonists,
such as 3M-002 and resiquimod, are effective in inducing LC maturation and overcoming
the tolerizing-like phenotype and function of LC exposed to HPV16L1L2 VLP,
suggesting that they are promising therapeutic compounds for the treatment of high-risk

96
HPV-induced cervical infections. The studies presented in this work are highly
significant as we have identified a novel immune evasion tactic of HPV16 and have
directed the field towards various immunotherapeutic targets and potential immune-based
therapies that have clinical implications.
Importantly, it should be addressed that the findings presented are based upon a
model system that mimics the interaction between HPV16 and LC in the human
epidermis. Due to limitations of working with human material, these studies were not
conducted using human LC isolated from mucosal epidermal sheets. The mere process of
isolating human LC from epidermal sheets induces the maturation of LC (Klechevsky et
al., 2008), thus making it difficult to carry out our study since activation status is an
endpoint. Nonetheless, monocyte-derived LC are an appropriate alternative model,
because they express MHC class II molecules, Langerin, E-cadherin, CD1a, and Birbeck
granules [chapter2, (Fausch et al., 2002)], which classically define human LC located in
the epidermis (Merad et al., 2008). Recently the status of LC as the only APC in the
epithelium that express langerin was challenged. It was reported that dermal langerin
+
DC
exist in mice and may play a role in the immune surveillance of the skin (Ginhoux et al.,
2007; Poulin et al., 2007). However, Klechevsky et al. demonstrated that while two
different subsets exist of human dermal DC, neither of these subsets express langerin,
highlighting a difference in human and murine APC populations located in the epithelium
(Klechevsky et al., 2008). In addition, the studies presented herein were carried out using
HPV VLP. Due to the limitations in producing large quantities of live virions in vitro,
HPV VLP have been developed as an alternative for structural and immunological

97
analysis. Thus, due to the facts that human LC are the only APC at the site of infection,
that monocyte-derived LC have been shown to be phenotypically equivalent to human
epidermal LC, and that VLP are an accepted alternative to purified virions for
immunological analysis of HPV, these studies use the most appropriate model to
critically examine the interaction of HPV16 and human LC.
Currently, patients with advanced cervical cancer have poor prognoses despite the
conventional therapies of radical hysterectomy and surgical debulking in combination
with chemotherapy or radiotherapy (Chuang et al., 2009). Thus, the development of
innovative therapeutics remains a global priority. Much research is being carried out in
hopes of developing an effective therapeutic HPV vaccine. The majority of therapeutic
vaccines aim to generate T cell-mediated immune responses against early proteins, E6
and E7. They are ideal candidates because they are expressed during a productive
infection and in cervical cancer lesions. Also, they are only expressed in viral infected
cells. However, development has been challenging and various strategies have been
investigated and applied clinically including: peptide- (Muderspach et al., 2000; Ressing
et al., 2000; Welters et al., 2008) and protein-based (Frazer et al., 2004; Roman et al.,
2007), live-vector-based (García-Hernández et al., 2006; Kaufmann et al., 2002), nucleic
acid-based (Garcia et al., 2004), and cell-based vaccines (Santin et al., 2008). Each
approach has demonstrated strengths and weaknesses (Hung et al., 2008), which has led
to the development of combinatorial vaccination strategies such as prime-boost regimens,
that can circumvent the limitations that arise during a single vaccination approach
(Davidson et al., 2004; Fiander et al., 2006). Additionally, considering the complexity of

98
numerous molecular mechanisms that drive the immune evasion of HPV and the
drawbacks to therapeutic vaccination strategies, it is highly conceivable that an effective
therapy against high-risk HPV-induced lesions will require a combination of a
therapeutic vaccine and agents that are capable of overcoming immune suppressive
mechanisms utilized by HPV. Further understanding of HPV immune escape
mechanisms will aid in identifying novel molecular targets that can be altered or blocked
in order to enhance the therapeutic effect of vaccines.
Our data illuminate a novel molecular mechanism mediating the immune evasion
of HPV16 and identify potential molecular immunotherapeutic targets, such as PI3K and
annexin A2. These molecular targets could be impeded or manipulated in combination
with therapeutic vaccination strategies. PI3K pathways have been demonstrated to play a
critical role in the regulation of immune cells, such as DC, macrophages, and T cells
(Deane and Fruman, 2004; Fukao and Koyasu, 2003; Katso et al., 2001). Specifically, it
has been demonstrated that in APC, PI3K suppression can induce a Th1 dominant
immune response. For example, when DC were treated with a PI3K inhibitor,
wortmannin, IL-12 synthesis was increased (Fukao et al., 2002). Additionally, it was
shown that PI3K
-/-
mice show enhanced Th1 responses against Leishmania major
infection, unlike wild-type mice. This enhanced Th1 response was attributed to the
increased production of IL-12 generated by splenic and bone-marrow derived DC (Fukao
et al., 2002). Notably, many PI3K inhibitors have been developed because PI3K
pathways are frequently over-activated in human cancer. PI3K inhibitors are promising
targeted therapies for cancer treatment. Some inhibitors have actually been shown to be

99
effective in mediating the clearance of cancers. For instance, the use of LY294002 as a
topical treatment for melanoma tumors in mice resulted in decreased tumor size and
vasculature (Bedogni et al., 2004). Taking these findings into consideration, in
combination with vaccination strategies suppressing PI3K in LC could be a practical and
effective approach to initiate the clearance of HPV-induced lesions and cervical cancer.
However modulation of PI3K activity is not without potential side effects seeing as PI3K
pathways modulate critical cellular response such as cell growth, survival, and
chemotaxis (Deane and Fruman, 2004; Katso et al., 2001).
In addition to PI3K, annexin A2 could also be a potential molecular target for
immunotherapeutics. Our findings indicate that annexin A2 is likely the HPV16 L2
receptor on LC that mediates the uptake of HPV16L1L2 VLP and plays a role in the
immune suppression of LC. Therefore, a plausible immunotherapeutic approach to treat
high-risk HPV infections would be to block annexin A2, either physically or functionally,
thereby removing HPV16 L2 mediated uptake and suppression. Meanwhile HPV16 could
still be internalized via an L1 mediated pathway, which we have demonstrated leads to
activation of LC (chapter 3), resulting in the induction of an HPV-specific adaptive
immune response and clearance. It should be noted that, our studies thus far have yet to
validate annexin A2 as the HPV16 L2 receptor inducing HPV16 immune evasion through
LC. Currently we are carrying out experiments to confirm that annexin A2 is the L2
receptor. If these future results support our current findings, annexin A2 will be a future
topic to further explore as a therapeutic target. Furthermore, future studies should also
focus on further elucidating immune suppressive signal transduction cascades initiated in

100
LC upon HPV16L1L2 VLP binding and uptake, which could not only lead to the
identification of additional potential immunotherapeutic targets but it could also clarify
the role of annexin A2 in initiating signaling pathways.
The studies presented in this work not only identify potential therapeutic targets
but also identify potential therapeutic small molecules that could be applied clinically to
mediate the clearance of high-risk HPV-induced cervical lesions. We demonstrated that
TLR8 agonists, 3M-002 and resiquimod, can overcome the HPV16L1L2 VLP induced
phenotype and function of LC, thereby inducing an HPV-specific cell-mediated immune
response. Interestingly, TLR8 agonists have also recently been shown to reverse Treg
function (Peng et al., 2005). It was demonstrated by Peng et al. that adoptive transfer of
TLR8 treated Tregs resulted in enhanced antitumor immunity in tumor bearing mice,
indicating that TLR8 agonists might be useful in immunotherapy (Peng et al., 2005). As
stated previously, HPV-specific CD4
+
Tregs have been identified in CIN and cervical
cancer patients and the frequency of Tregs correlates with persistence of HPV16 induced
lesions (Adurthi et al., 2008; Molling et al., 2007; van der Burg et al., 2007; Visser et al.,
2007). Tregs are another immune suppressive tactic of HPV immune evasion. Not only
are they induced during persistent infections but it was also demonstrated that vaccination
strategies can lead to the generation of Tregs (Welters et al., 2008). Collectively, these
studies indicate that Tregs are another hurdle to overcome in therapeutic immunization
strategies against high-risk HPV infections. A recent study evaluated the benefits of
depleting Tregs prior to HPV DNA vaccination in mice (Chuang et al., 2009). They
found that there was an increased generation of E7-specific CD8
+
T cells when

101
CD4
+
CD25
+
Tregs were depleted using an anti-CD25 antibody (PC61) prior to
vaccination with E7/Hsp70 DNA in comparison to the control, mice vaccinated without
Treg depletion. The enhanced HPV-specific CD8
+
T cell response corresponded to
significantly improved therapeutic and long-term protective antitumor effects (Chuang et
al., 2009). Therefore, reversing Treg suppression is critical when generating an effective
immune response to mediate the clearance of persistent HPV infections. If TLR8 agonists
were used as a treatment for HPV-induced lesions, they would not only reverse Treg
function but would also activate LC, thereby initiating an HPV-specific adaptive immune
response. These studies along with our findings presented in chapter 5, propose TLR8
agonists as a highly attractive immunotherapeutic that could be used either alone or in
conjunction with vaccination strategies for treatment of persistent high-risk HPV-induced
lesions.
Currently it is unclear which cell(s) is responsible for the induction of HPV-
specific CD4
+
Tregs generated in CIN and cervical cancer patients. As mentioned
previously, inducible Tregs are generated in the periphery from naïve CD4
+
and CD8
+
T
cells after encounter with APC that have sub-optimal activation status, presenting
peptides in the absence of costimulatory molecules (Dhodapkar et al., 2001; Jonuleit et
al., 2000). Interestingly, we found that LC exposed to HPV16L1L2 VLP posses a sub-
optimal phenotype thus it is plausible that LC exposed to HPV16 are responsible for
inducing HPV16-specific CD4
+
Tregs. This is an interesting and novel question that
should be addressed in future studies. Additionally, future efforts should also be directed
towards determining how C8
+
T cells and CD4
+
T cells respond to LC exposed to

102
HPV16L1L2 VLP. In our studies it was demonstrated that CD8
+
T cells co-cultured with
LC exposed to HPV16L1L2 VLP do not initiate an HPV16-specific CD8
+
T cell response
however, it remains to be determined if these cells are tolerized by LC, becoming either
anergic or regulatory in nature. Furthermore, it should be addressed whether CD4
+
T cells
become effector Th1 cells, effector Th2 cells, anergic, or Tregs after interacting with LC
exposed to HPV16L1L2 VLP.
In addition to the T cell response generated by LC exposed to HPV16L1L2 VLP,
future studies should further explore the extent of HPV immune evasion through the
manipulation of LC. The majority of studies investigating immune escape mechanisms
focus on high-risk HPV types; however, immune escape mechanisms have also been
defined for low-risk types as well. Like high-risk types, low-risk HPV6 and HPV11 have
been shown to negatively modulate antigen presentation. Similar to HPV18 E7, HPV6b
E7 represses TAP1/LMP2 promoter activity. E7 from both HPV6 and HPV11 has also
been shown to co-precipitate along with TAP1 and calreticulin. HPV11 E7 has also been
demonstrated to inhibit ATP-dependent transport, resulting in poor presentation of
antigens. Moreover, like high-risk types, E6/E7 proteins from low-risk types suppress the
production of immune mediators such as MIP-3α by infected cells, aiding in viral
infection persistence (Guess and McCance, 2005). Even though immune escape
mechanisms of low-risk types have been identified and are similar to those mediated by
high-risk HPV types, there are some immune evasion mechanisms that appear to be high-
risk type specific. For instance, HPV16 and HPV18 have evolved intricate mechanisms to
suppress the action of IFNs, thus far this has yet to be demonstrated in low risk types. A

103
study conducted by Schneider et al. found that cells isolated from HPV16 or HPV18
infected lesions show decreased responses to IFN-α than cells infected with HPV6 or
HPV11 (Schneider et al., 1987). Therefore an interesting question remains: is the immune
escape mechanism we have defined in the present studies HPV16 specific, high-risk type
specific, or do both low-risk and high-risk types suppress LC maturation to escape
immune detection? Considering that the N-terminus of L2 is highly conserved across
mucosal genotypes and this is the region that is exposed at the virion surface, mediating
the induction of the immune escape through LC, it is highly plausible that both high-risk
and low-risk mucosal HPV types will manipulate LC to escape immune detection.
Furthermore, it has yet to be described whether LC can transcribe and translate
HPV encoded genes into proteins. Because LC can internalize HPV it is plausible that
they may also express HPV encoded proteins. This question is of interest and should be
explored because it could have great implications. If LC do express HPV encoded
proteins it is likely that the proteins are further aiding in the immune escape of HPV
through LC, seeing as immune evasion mechanisms utilized by HPV are primarily
mediated via viral-encoded proteins, E6 and E7 (as described in chapter 1). It is important
to note however that HPV encodes one DNA replication enzyme, E1, one viral
transcription factor, E2, and no translational factors, therefore replication of the viral
genome and protein expression are both dependent on the host’s cellular machinery. To
date this process is still poorly understood. Taking this into consideration, LC expression
of HPV encoded proteins is dependent on the location of the viral genome and the
expression of transcription and translational factors within LC. If the viral genome can

104
translocate from the cytoplasm to the nucleus in LC and if LC express the necessary
machinery to transcribe and translate HPV genes into proteins then there is possibility
that viral gene expression occurs in LC, as it does in keratinocytes. Thus, it is an
interesting question to address and further elucidation of HPV mediated immune escape
mechanisms will only advance the field towards an optimal therapeutic for treatment of
high-risk HPV-induced infections.
Our findings presented herein are novel and are of great significance. We are the
first to describe an immune evasion mechanism of HPV16 mediated by L2, which
initiates the suppression of LC function. We are also the first to isolate a candidate L2
receptor on LC and to identify TLR8 agonists as a potential immunotherapeutic strategy
to treat high-risk HPV infections. Our studies are of major impact because they further
define mechanisms responsible for establishing and maintaining a persistent HPV
infection, they aid in designing more optimal therapeutic strategies, and they have clinical
implications; all of which address a global plea to eradicate cervical cancer.

105
Chapter 7. Materials and Methods

Antibodies and Agonists
The antibodies recognizing conformational HPV16 L1 epitopes (H16.V5, and
H16.E70), BPV L1 (B1.A1), or linear HPV16 L1 epitopes (Camvir-1, H16.D9, and
H16.H5) and anti-BPV-L1 were gifts from Neil Christensen (Penn. State, Hershey, PA)
except Camvir-1, which was purchased from BD Biosciences (San Jose, CA). Polyclonal
serum (DK44214) recognizing HPV16L2 was a gift from John Schiller (National
Institutes of Health, Bethesda, Maryland). The human antibodies, CD197 (CCR7) PE;
CD1a PE; CD80 FITC; CD86 FITC; HLA-DR, DQ, DP FITC; HLA-A, B, C FITC;
isotype controls, biotinylated anti-rabbit IgG; biotinylated anti-mouse-IgG2b; anti-
mouse-IgG-HRP streptavidin-PE; streptavidin-FITC; and streptavidin-HRP were
purchased from BD Biosciences. The antibody to human CD207 (langerin) was
purchased from immunotech (Marseille, France) while the anti-human E-cadherin
antibody was purchased from Millipore (Temecula, CA). Anti-human TLR7 and TLR8
PE antibodies were purchased from Abcam (Cambridge, MA). The antibodies specific
for p-ERK1/2 (Tyr 204), ERK1/2, p-MKK4 (Thr 261), MKK4, p-ATF2 (Thr 71), ATF2,
IκB-alpha, p-PI3K (Tyr 508), PI3K, p-Akt (Ser 473), Akt, p-GSK3β (Tyr 216), p-SGK
(Thr 256), and PTEN were purchased from Santa Cruz Biotechnology (Santa Cruz,
California) while p-PDK1 (Ser 241) and p-PTEN (Ser 380), were purchased from Cell
Signaling (Beverly, MA). The antibody specific for annexin A2 was purchased from BD
Biosciences and the antibody specific for GAPDH was purchased from Chemicon

106
(Temecula, California). Anti-goat IgG-HRP was purchased from Vector Laboratories
(Burlingame, California) and anti-mouse-IgG-HRP was purchased from Promega (San
Luis Obispo, CA), while goat anti-mouse-IgG-FITC was purchased from Biosource
(Carlsbad, CA). Goat-anti-mouse-IR Dye 800 was purchase from Rockland
(Gilbertsville, PA) and goat-anti-rabbit-Alexa Fluor 680 was purchased from Molecular
Probes (Eugene, OR). Anti-human IFN-γ and biotinylated anti-human IFN-γ antibody
were purchased from Mabtech (Cincinnati, OH). TLR7, 8, and 7/8 agonists (3M-006,
3M-002, 3M-005, 3M-007, 3M-031) were gifts from 3M Pharmaceuticals (St. Paul, MN).

Donor Material
Peripheral blood leukocytes from healthy donors were obtained by leukapheresis.
Leukocytes were purified using Lymphocyte Separation Media (Mediatech, Inc,
Herndon, Virginia) by gradient centrifugation and stored in liquid nitrogen. HPV
serology analysis of all donors showed negative results. All studies using human samples
were approved by the USC’s IRB and informed consent was obtained from all donors.

DC and LC Generation
Frozen PBL were thawed, washed once with RPMI1640, containing 2mM
Glutamax (GIBCO, Carlsbad, CA), 10 mM sodium pyruvate (GIBCO), 10mM non-
essential amino acids (GIBCO), 100 µg/ml Kanamycin (Sigma-Aldrich, St. Louis, MO)
and 10% FBS (Omega Scientific, Tarzana, CA) (complete media). For DC, plastic
adherent cells were selected by plating 200 x 10
6
cells in a 175 cm
2
tissue culture flask for

107
2 h at 37°C. Non-adherent cells were washed away and the remaining adherent cells were
cultured for 7 days in complete media containing 1000 U/ml rhu-GM-CSF (Berlex,
Seattle, WA) and 1000 U/ml r-IL-4 (Biosource) of which 100% was replenished on day
three and 50% was replenished on day 6. For LC, adherent cells were cultured for 7 days
in complete media containing 1000 U/ml r-GM-CSF, 1000 U/ml r-IL-4, and 10 ng/ml
rhu-TGF-β
1
(Biosource) of which 100% was replenished on day three and 50% of rhu-
GM-CSF and rhu-IL-4 was replenished on day 6 while 100% of rhu-TGF-β
1
was
replenished on day 3 and 6.

VLP Production
Recombinant baculovirus stocks for HPV16L1 VLP, HPV16L1L2 VLP, and
HPV16L1L2-E7 cVLP were a gift from Dr. John Schiller. All recombinant baculoviruses
were propagated in Sf9 insect cells (Da Silvaet al. et al., 2001). HPV16L1 VLP,
HPV16L1L2 VLP, and HPV16L1L2-E7 cVLP were produced in HI-Five insect cells by
infection with the appropriate recombinant baculovirus (Greenstone et al., 1998).
Specifically, HI-Five cells were cultured in one L suspension vessels and grown to a
concentration of 8 x 10
5
cells/ml. One hundred million HI-Five cells were infected at a
multiplicity of infection of ~5 to 10 with recombinant baculovirus. After 72 h at 27°C
cells were harvested and washed once with sterile PBS. The pellet was snap frozen in
liquid nitrogen and stored at -80°C or processed immediately. For VLP isolation, pelleted
cells were resuspended in extraction buffer (5 mM MgCl
2
, 5 mM CaCl
2
, 150 mM NaCl,
0.01% Triton X-100, 20 mM Hepes, pH 7.5) With freshly added protease inhibitors (150

108
µg/ml phenylmethylsulfonyl fluoride, 0.7 µg/ml pepstatin, 10 µg/ml E-64 and lysed by
short-pulse sonication on ice with a sonic dismembrator (Fisher Scientific). After low-
speed clarification, cleared lysates were loaded on top of 40% (w/v) sucrose PBS
cushions and centrifuged at 25,000 rpm for 2.5 h in a SW-28 rotor (Beckman, Fullerton,
CA). The resulting pellet was resuspended in 5 ml of 27% (w/v) cesium chloride
(CsCl)/PBS and sonicated briefly a second time. VLP were subjected to two subsequent
centrifugations (24 h/50,000 rpm/20°C) to equilibrium in 27% CsCl/PBS. Visible bands
with a density of ~1.3 g/ml were extracted from tubes with a 22-gauge needle and syringe
and were pooled. Purified VLP were dialyzed against 4 changes of 1 liter of 0.5 M
NaCl/PBS at 4°C, and tested by ELISA, western blot, and electron microscopy for
immunoreactivity and morphology. L1 protein was quantified by gel code blue staining
(Thermo Scientific, Rockford, IL) of proteins after sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) through 10% Bis-Tris gels (Invitrogen,
Carlsbad, CA) by comparison with a bovine serum albumin (BSA) standard. For all VLP
preparations, the L1 protein represented >95% of the total protein. An E-toxate kit
(Sigma-Aldrich) was used to detect and semi-quantitate endotoxin in the preparations.
The endotoxin level in the preparations was found to be less than 0.06 EU/ml and this
level does not activate LC. Baculovirus DNA used in VLP production procedure does not
activate LC (Fausch et al., 2002).

109
VLP ELISA
To test VLP preparations for the presence of intact particles, VLP were subjected
to an ELISA with antibodies that recognize conformationally-dependent surface epitopes
or linear epitopes. Purified wild-type or chimeric VLP (500 ng/well) were used to coat
96-well Maxisorp ELISA plates (Nunc, Naperville, IL) overnight at 4°C. Plates were
blocked with 3% BSA/PBS/0.05% Tween-20 for 2 h at room temperature. Antibodies
recognizing conformational epitopes [HPV16.V5 and H16.E70 (for HPV), B1.A1 (for
BPV) or linear epitopes [Camvir-1 (for HPV), anti-BPVL1 mAb] were incubated with
coated VLP for 2 h. Plates were washed and further incubated with HRP-labeled goat
anti-mouse IgG antibody or HRP-labeled goat anti-rabbit IgG antibody, followed by
addition of o-phenylenediamine substrate in 0.05 M phosphate citrate buffer. Optical
density (O.D.) values were read in an ELISA plate reader (Chameleon, Bioscan,
Washington D. C.) at 490 nm.

VLP Western Blot Analysis
SDS-PAGE and western blotting was performed using the NuPAGE
Electrophoresis System (Invitrogen) according to manufacturer’s instructions. VLP were
denatured by heating samples in protein loading buffer supplemented with 10x sample
reducing agent (Invitrogen) at 100°C for 10 min. Samples were separated through a 10%
Bis-Tris resolving gel, transferred to nitrocellulose, and blots were blocked overnight
with StartingBlock Blocking Buffer (Invitrogen). Membranes were incubated overnight
at 4°C with antibodies for either HPV16-L1, HPV16-L2, or HPV16-E7 depending on the

110
type of VLP being generated. Blots were subsequently incubated with HRP-labeled anti-
mouse or anti-rabbit antibodies, and developed using the Supersignal West Pico
chemiluminescent substrate (Thermo Scientific).

VLP Electron Microscopy
Transmission electron microscopy was performed by absorbing 20 µl VLP
samples to carbon-coated copper grids and staining with 2% uranyl acetate. Specimens
were examined with a Zeiss EM 900 electron microscope at 75 kV.

Activation Assay
Multiple activation assays were done in the studies presented herein. They are as
follows. In chapter 2, DC and LC were collected, washed twice with PBS, and either left
untreated or treated with 10 µM of the MAPK inhibitor SB203580 (SIGMA), 10 µM of
the NF-κB inhibitor BAY11-7082 (BIOMOL Research Laboratories Inc., Plymouth
Meeting, PA), or 20 µM of the PI3K inhibitor LY294002 (EMD Biosciences, Darmstadt,
Germany) for 30 min at 37 °C. DC and LC were subsequently incubated with or without
10 µg LPS (Escherichia coli 026:B6) (Sigma-Aldrich) or 10 µg HPV16L1L2 VLP/10
6

cells for 1 h at 37 °C. Cells were then incubated for 48 h in 20 ml complete medium
containing 1000 U/ml rhu-GM-CSF. In chapter 3, LC were harvested, washed twice with
PBS, and either left untreated or exposed to HPV16 L1 VLP or HPV16 L1L2 VLP at a
concentration of 10 µg/10
6
cells. As a positive control human LC were treated with 10 µg
of LPS. The cells were then incubated for 1 h at 37°C and mixed occasionally. Following

111
the incubation, the cells were placed at 37°C for 48h in 20 ml complete media containing
1000 U/ml rhu-GM-CSF. In chapter 5, DC and LC were harvested and washed twice with
PBS. DC were left untreated or treated with 30 µM 3M-006, 5 µM 3M-002, 30 µM
imiquimod, 30 µM resiquimod, 5 µM 3M-031, or with 10 µg LPS. The cells were
incubated for 1h at 37 °C, mixed occasionally, and finally placed at 37°C for 24h in
complete media containing 1000 U/ml rhu-GM-CSF. LC were left untreated or exposed
to 10 µg HPV16L1L2 VLP/10
6
cells. The cells were incubated for 1 h at 37°C, mixed
occasionally, and placed at 37°C for 24 h in complete media containing 1000 U/ml rGM-
CSF. Next, the cells were left untreated or treated with 30 µM 3M-006, 5 µM 3M-002,
30 µM imiquimod, 30 µM resiquimod, 5 µM 3M-031 or with 10 µg LPS and incubated
for an additional 24h at 37°C.

Flow Cytometry
DC and/or LC were collected from each activation culture following the last
incubation and washed with FACS buffer (PBS/0.5% FCS/0.1% sodium azide). Single or
double staining was performed on ice using fluorochrome-conjugated antibodies or non-
conjugated antibodies against human antigens or the appropriate isotype control for 1 h.
If secondary and/or tertiary staining was required to detect the primary non-conjugated
antibody, the cells were incubated with the appropriate secondary antibody or
streptavidin-PE for an additional 1 h. Each antibody was used at a concentration
suggested by the manufacturer. Cells were washed 3 times and fluorescence was
determined on a FACSCalibur using CellQuest software (Becton Dickinson, Mansfield,

112
MA) by analyzing at least 15,000 acquired events. Cultures contained >90% DC or LC,
as assessed by MHC class II expression.

Cytokine and Chemokine Analysis
Supernatants (500 µl) were collected from LC stimulated in the activation assays
as described for chapter 3 and chapter 5. The concentrations of cytokines and chemokines
in the supernatants were analyzed using Human Cytokine LINCOplex Kits (LINCO
Research, St. Charles, MO) and the Bio-Plex Suspension Array System (Bio-Rad,
Hercules, CA). The assays were completed as stated by the manufacturer’s protocol.

Migration Assay
Chemokine directed migration of LC was carried out using 24-well Transwell
plates with 5 µm-pore-size polycarbonate filters (Corning Costar, Cambridge, MA).
Briefly, 600 µl of medium was added to the lower chamber containing either 250 ng/ml
rhu-6Ckine/CCL21 (R&D Systems, Minneapolis, MN) or complete media alone, as a
control for spontaneous migration. We added 2 x 10
5
untreated LC, LPS stimulated LC,
HPV16L1 VLP exposed LC, HPV16L1L2 VLP exposed LC, or HPV16L1L2 VLP
exposed LC treated with each of the imidazoquinolines, using the same concentrations of
VLP and imidazoquinolines as stated in the activation assays, to the upper chambers. The
plates were incubated for 3 h at 37°C. Finally, cells that migrated to the lower chamber
were counted, and CCL21-dependent migration was calculated as the ratio of cells that
migrated with/without CCL21.

113
In Vitro Immunization Assay
In these studies multiple in vitro immunization assays were carried out. The
experimental treatments being tested in each assay differ slightly. The treatments are as
follows. In chapter 2, 1.2 x 10
6
DC or LC incubated with or without 20 µM of
LY294002. Subsequently the cells were either left untreated or exposed to 10 µg of
HPV16L1L2 cVLP/10
6
cells, for 1 h at 37°C in PBS. In chapter 3, 1.2 x 10
6
LC were
untreated or exposed to either 10µg of HPV16L1 VLP/10
6
cells or 10µg of HPV16L1L2
VLP/10
6
cells for 1 h at 37°C in PBS. Subsequently, the cells were incubated for 48 h in
20 ml of 37°C complete RPMI 1640 media supplemented with 1000 U/ml of rhu-GM-
CSF. In chapter 5, 1.2 x 10
6
LC were left untreated or exposed to 10 µg of HPV16L1L2
cVLP/10
6
cells for 1 h at 37°C in PBS. Subsequently, the cells were incubated for 4 h in
complete media supplemented with 1000 U/ml of rhu-GM-CSF at 37°C. Then the cells
were treated with or without each of the imidazoquinolines, using the same
concentrations as described in the activation assay, and incubated for 20 h at 37°C.
Following DC or LC treatment, the cells were washed with PBS, irradiated (25Gy), and
mixed with 25 x 10
6
autologous CD8
+
T cells. Autologous CD8
+
T cells derived from an
HLA-A*0201 positive donor were isolated from PBL by positive magnetic selection
using a MACS MulitSort CD8
+
isolation kit and a magnetic cell separator (Miltenyi
Biotech, Auburn, CA). Cells were cultured in 48-well plates (Corning Costar) at 0.5 x 10
6

T cells per well and 2.5 x 10
4
DC or LC per well in complete medium for 7 days at 37 °C.
Restimulations after 7 and 14 days were done with 0.5 x 10
6
cells per well of DC or LC
treated and loaded as in the first stimulation. For restimulations, the medium was

114
supplemented with IL-2 at 50 U/ml at 2 and 4 days after restimulation. After 28 days
effector cells were pooled and tested for IFN-γ production by ELISPOT.

ELISPOT Assay
To test for HPV16-E7 or HPV16-L1 specific CD8
+
T cell responses, IFN-γ
ELISPOT assays were performed. 96-well multiscreen-hemagglutinin plates (Millipore,
Bedford, MA) were coated with 10 µg/ml anti-human IFN-γ in PBS overnight, washed
with PBS/0.5% Tween-20, and blocked for 4 h with complete medium at 37°C, 5%CO
2
.
2.5 x 10
5
cells per well from the in vitro immunizations were incubated in the presence or
absence of HLA-A*0201 restricted HPV16-E7-derived peptide [aa 86-93, TLGIVCPI,
(Ressing et al., 1995)] or HLA-A*0201 restricted HPV16-L1-derived peptide [(aa 323-
331, ICWGNQLF), (Kaufmann et al., 2001)] for 16-18 h at 37°C. The wells were washed
six times with PBS/0.5% Tween-20 and then the plates were incubated for 1h with
streptavidin-HRP conjugate diluted in PBS/0.5% BSA solution. The plates were
developed with 3-amino-9-ethyl-carbazole (AEC) substrate, which is composed of AEC,
dimethyl formamide, H
2
O
2
(30%), and 0.05M Sodium Acetate buffer (Sigma-Aldrich).
Spots were imaged and counted using the automated computer-assisted video-imaging
KS ELISPOT analysis system (Carl Zeiss, Thornwood, NY).

Western Blot Analysis
DC and LC were collected, washed twice with PBS, and were incubated with or
without 10 µg LPS, 10 µg of HPV16L1 VLP/10
6
, 10 µg of HPV16L1L2 VLP/10
6
cells,
or 10 µg of heated (95°C, 10 min) HPV16L1L2 VLP/10
6
cells at 37 °C for the times

115
indicated. Cellular extracts were prepared using the Mammalian Protein Extraction
Reagent containing Halt Protease Inhibitor Cocktail (100x) (Thermo Scientific). Protein-
normalized aliquots of cell lysates were electrophoresed on 10% NuPage Novex Bis-Tris
gels, transferred to nitrocellulose membranes, and blocked with StartingBlock Blocking
Buffer. Immunoblotting of cellular extracts was performed using antibodies specific to p-
ERK1/2, ERK1/2, p-MKK4, MKK4, p-ATF2, ATF2, IκB-alpha, p-PI3K, PI3K, p-Akt,
Akt, p-GSK-3β, p-SGK, PTEN, p-PDK1, p-PTEN, or GAPDH. Secondary antibodies
were anti-goat IgG-HRP, anti-mouse-IgG-HRP, anti-rabbit-IgG-HRP, goat-anti-mouse-
IR Dye 800, and goat-anti-rabbit-Alexa Fluor 680. Depending on which secondary
antibody was used, detection was performed using either the Supersignal West Pico
chemiluminescent or the Odyssey (LI-COR Bioscience, Lincoln, NE). Nuclear extracts
were isolated using the Nuclear and Cytoplasmic extraction reagents (Thermo Scientific)
containing Halt Protease Inhibitor Cocktail and the assessment of CREB-1, NF-κB p50,
and NF-κB p65 binding activity was performed using the BD Mercury Transfactor
Profiling Kit (BD Biosciences) and analyzed at an absorbance of 450nm.

HPV16L1L2 VLP Binding Assay
LC were incubated with the HPV16 L2

peptide [(aa 108-120,
LVEETSFIDAGAP), (Kawana et al., 2001)] at varying concentrations (1-100 µg)/0.5
x10
6
cells for 1 h at 4°C. Subsequently, the LC were incubated with 0.25µg of
HPV16L1L2 VLP/treatment for 1h at 4°C and then incubated with anti-L1 (HPV16.V5)
antibody at a concentration of 1:25,000 for 30 min at 4°C. The cells were then incubated

116
with biotinylated anti-mouse-IgG2b at a concentration of 1:50 for 30 min at 4°C. Next,
the HPV16L1L2 VLP/anti-L1/biotin treated cells were stained with strepavidin-FITC at a
concentration of 1:50 for 30 min. The cells were washed between each incubation with
135 µl FACS buffer. In control experiments, cells were left untreated or probed with
either peptide/anti-L1/biotin-strepavidin-FITC or VLP/anti-L1/biotin–strepavidin-FITC.
Finally, HPV16L1L2 VLP binding to LC was assessed by flow cytometry.

L2
108-120
Peptide Pulldown Assay
LC were harvested, washed with PBS, and aliquoted into 1.5 ml microcentrifuge
tubes in PBS. Then (6x)His-L2
108-120
peptide was added to the LC, at a concentration of
50 µg/0.5 x10
6
cells, and incubated for 1h. In control experiments, LC were left untreated
with peptide but exposed to each condition thereafter. Following the incubation, DTSSP
(final concentration of 1.5 mM) was added to the LC and incubated for 2 h to cross-link
the peptide to the receptor. After the cross-linking reaction was quench with 1 M Tris, LC
were washed with PBS and resuspended in and incubated with a bursting solution [10
mM Hepes, 2 mM MgCl, 10 mM KCl
2
, 0.05% Tween-20, and Halt Protease Inhibitor
Cocktail] for 20 min. Next, the cells were centrifuged for 30 min, at 13, 000 g. The
supernatants were decanted and LC were resuspended in lysis buffer [50 mM NaH
2
PO
4
,
300 mM NaCl, 50 mM imidazole, 0.05% Tween-20, and Halt Protease Inhibitor Cocktail,
pH 8.0]. The cells were snap frozen, allowed to thaw, incubated on ice for 30 min, and
sonicated for 10 s. Subsequently the lysates were centrifuged for 30 min, at 10, 000 g.
The lysate supernatants were decanted, mixed with 50% Ni-NTA agarose slurry (Qiagen,

117
Valencia, CA), and incubated overnight. The following day a column (Thermo Scientific)
was assembled to elute the proteins from the Ni-NTA agarose slurry. Once the column
was assembled the lysate-Ni-NTA agarose slurry was washed twice with wash buffer (50
mM NaH
2
PO
4
, 300 mM NaCl, 50 mM imidazole, and 0.05% Tween-20, pH 8.0). The
proteins associated with the Ni-NTA agarose were eluted over 10 fractions. The first
fraction was eluted using elution buffer #1 (50 mM NaH
2
PO
4
, 300 mM NaCl, 100 mM
imidazole, and 0.05% Tween-20, pH 8.0). Fractions 2-10 were elated using elution buffer
#2 (50 mM NaH
2
PO
4
, 300 mM NaCl, 250 mM imidazole, and 0.05% Tween-20, pH 8.0).
The eluates were collected and analyzed by non-reducing immunoblots and reduced
silver stain gels. It should be noted that each step of the pulldown assay was performed at
4°C. For the immunoblots, non-reduced eluates were run on 10% Bis-Tris gels using the
NuPAGE Electrophoresis System according to manufacturer’s instructions. The protein
was then transferred to nitrocellulose, blocked with StartingBlock Blocking Buffer, and
the membranes were probed with either an anti-His antibody, to detect the (6x)His-L2
peptide, or an anti-annexin A2 antibody. Blots were subsequently incubated with either
an HRP-labeled anti-mouse antibody or an HRP-labeled anti-rabbit antibody,
respectively, and developed using the Supersignal West Pico chemiluminescent substrate.
10% Tris-HCL gels (Bio-Rad) were used to separate out reduced eluates for silver stain
analysis.

118
Silver Staining Assay
The gel was fixed overnight in 250 ml of fixing solution (50% MetOH, 12%
Acetic Acid, and 0.05% formaldehyde). After fixation, the gel was washed with 35%
EtOH for 20 min, three times and washed twice with double distilled water. Following
the washing, the gel was sensitized using 250 ml of sensitization buffer (100 mM
Na
2
S
2
O
3
, 30 mM FeK
3
(CN)
6
for 2 min. Next, the gel was washed four times with double
distilled water and stained for 20 min with 200 ml of staining solution (0.2% AgNO
3
,
0.076% formaldehyde). Once the gel was stained it was washed again, two times with
double distilled water, and developed to desired darkness with 250 ml of developing
solution (6% Na
2
CO
3
, 0.05% formaldehyde, 0.0004% Na
2
S
2
O
3
). When the gel was
developed to the desired darkness it was stopped using 50%MetOH/12% Acetic acid for
min. Specific bands were excised and analyzed by mass spectrometry (Thermo LTQ-
ETD mass spectrometer, Thermo Scientific) at the USC proteomics core.

HPV16 VLP Uptake Assay
HPV16L1L2 VLP and HPV16L1 VLP were labeled with CFDA-SE using
Vybrant CFDA-SE cell tracer kit (Invitrogen) as directed by the manufacturer’s
instructions. Following the 4 h incubation the HPV16 VLP were dialyzed against 4 L of
cold PBS/0.5 M NaCl to remove all the excess free dye. LC were harvested, washed with
PBS, and aliquoted at a concentration of 1.0x 10
6
cells/200 µl cold PBS into 1.5 ml amber
tubes. Subsequently the cells were either left untreated or incubated with increasing
concentrations (5-30 µg/ml) of rhu-SLPI (R & D systems) for 1 h at 4°C. Following the

119
incubation the cells were washed with 500 µl cold PBS and spun down at 800 g for 5 min
at 4°C. The supernatant was removed and the LC were resuspended in 400 µl of room
temperature FACS buffer. Next, CFDA-SE labeled HPV16L1L2 VLP or HPV16L1 VLP
(1 µg/1 x10
6
) were incubated with the LC at 37°C. After 15 min LC were harvested and
fixed in 2% paraformaldehyde. Finally, HPV16 VLP uptake by LC was assessed via flow
cytometry.

Statistical Analysis
All statistical analyses were performed using GraphPad Prism (GraphPad
Software Inc., San Diego, CA).

120
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Abstract (if available)

Abstract High-risk human papillomaviruses (HPV) infect the epithelial layer of cervical mucosa and are causally associated with the generation of cervical cancer. Although most women infected with HPV clear their lesions, the long latency period from infection to resolution indicates that HPV has evolved immune escape mechanisms. Langerhans cells (LC) are the resident antigen-presenting cells at the site of infection and therefore are responsible for initiating an immune response against HPV. However, LC exposed to HPV16L1L2 virus-like particles do not induce an HPV-specific T cell response, suggesting that LC are targeted by HPV to evade immune detection. Herein we describe a novel immune escape mechanism of HPV16 that targets LC function and identify therapeutic compounds to overcome the HPV16 induced suppression of LC. We demonstrate that LC incubated with HPV16L1L2 virus-like particles up-regulate phosphoinositide 3-kinase activation while down-regulating mitogen-activated protein kinase and nuclear factor-κB pathways. When phosphoinositide 3-kinase activation is inhibited and LC are subsequently exposed to HPV16L1L2 virus-like particles, LC initiate a potent HPV-specific response, revealing that phosphoinositide 3-kinase activation in LC is an escape mechanism utilized by HPV16. Importantly, we also show that the minor capsid protein L2 is responsible for the induction of this immune escape of HPV16 through the manipulation of LC. Additionally, using pulldown assays we demonstrate that the N-terminus of L2 associates with annexin A2. Inhibiting the interaction between HPV16 L2 and annexin A2 disrupts the internalization of HPV16L1L2 virus-like particles by LC, indicating that annexin A2 is the L2 receptor for HPV16, which likely initiates the immune escape mechanism of HPV16 through LC.

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Creator Fahey, Laura Marie (author)

Core Title Targeting Langerhans cells: a human papillomavirus type 16 immune evasion mechanism

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Molecular Microbiology

Publication Date 04/28/2010

Defense Date 03/20/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cell mediated immunity,HPV,immune evasion,Langerhans cells,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Kast, W. Martin (committee chair), Epstein, Alan L. (committee member), Schonthal, Axel (committee member)

Creator Email laura.fahey@gmail.com,lfahey@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2141

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Dmrecord 222283

Document Type Dissertation

Rights Fahey, Laura Marie

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

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