University of Southern California Dissertations and Theses

The silenced sentinel: a human papillomavirus type 16 immune evasion mechanism targeting Langerhans cells

(USC Thesis Other)

The silenced sentinel: a human papillomavirus type 16 immune evasion mechanism targeting Langerhans cells

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

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

by

Adam Benjamin Raff

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(SYSTEMS BIOLOGY AND DISEASE)

August 2010

Copyright 2010 Adam Benjamin Raff
ii

Success is sweet and sweeter if long delayed and gotten through many struggles and
defeats.
- Amos Bronson Alcott
iii
Dedication

I would like to dedicate this thesis to my Mom and Dad. You truly have made me the
man I am today. Your inspiration, guidance and love over the years have supported me
through all the mountains and valleys of my life. I owe you both a tremendous debt of
gratitude.
iv
Acknowledgements

To Laura Fahey, my fiancée and research partner for life: I could not have reached this
milestone without your love and support. This thesis is truly a testament to the triumphs
that can be accomplished when two minds deeply connect. You guided me through the
early days, taught me to be a productive scientist, shielded me during the rough times and
inspired me to achieve new heights. I look forward to a lifetime of love, learning and
laughter with you by my side. I love you now and forever.

To my family and friends: A PhD is not merely the sum of papers accrued during a
graduate career, but the grand total of knowledge gained at and away from the lab bench.
It is only with the support of my family and friends that I have accomplished my goals. I
am eternally grateful to all of you who continue to fill my life with countless moments of
light and love.

To the members of the Kast laboratory: Every scientist stands on the shoulders of the
men and women who preceded them. Today, I stand on your shoulders. Your teachings
and guidance have given me the skills to proceed confidently through my future scientific
career. Thank you all for years of encouragement and help.

To W. Martin Kast, my mentor: I tell every student who asks advice on a PhD mentor
that communication is the key to success. Our conversation began when I first
interviewed with you nearly 6 years ago and it continues today. It is that very same ever-
v
lengthening conversation that ties my entire graduate career together, weaving between
and through all the projects, papers, failures and successes. Thank you for sharing your
endless enthusiasm for science with me – it has filled me with a passion for research that
will never fade. I look forward to continuing this conversation we started as I journey on
new paths of discovery.
vi
Table of Contents
Epigraph ii
Dedication iii
Acknowledgements iv
List of Tables viii
List of Figures ix
Abbreviations xi
Abstract xv
Chapter 1: Introduction 1
Human Papillomavirus Taxonomy and Genome 1
HPV Life Cycle 3
HPV Virus-like Particles 5
HPV Capsid Structure 6
HPV Cell Surface Binding 8
HPV Internalization 13
HPV Vesicular Trafficking and Egress from Endosomes 15
HPV Nuclear Transport 16
HPV Pathogenesis and Cervical Cancer 16
Antigen Presenting Cells and Adaptive Immune Responses 19
Natural HPV Immunity 23
General HPV Immune Escape Mechanisms 25
LC-Mediated HPV Immune Escape 26
Reversal of LC-Mediated HPV Immune Escape 30
Project Goals and Significance 32

Chapter 2: Results 35

Chapter 3: Discussion and Future Directions 61

Chapter 4: Materials and Methods 86
Antibodies 86
Donor Material 86
DC and LC Production 87
VLP Production 87
VLP ELISA 89
VLP Western Blot Analysis 89
VLP Electron Microscopy 90
vii
Activation Assay 90
Flow Cytometry 91
Cytokine and Chemokine Analysis 91
Migration Assay 91
In Vitro Immunization Assay 92
ELISPOT Assay 93
Western Blot After LC Activation 93
HPV16 VLP Uptake Assay 94
HPV16 VLP Uptake Assay with Confocal Microscopy 94
HPV16L1L2 VLP Binding Assay 95
L2
108-120
Peptide Pulldown Assay 96
Silver Staining Assay 97
HPV16 VLP Uptake Assay with SLPI 98
siRNA Inhibition of ANXA2 in LC and HPV16 VLP Uptake Assay 98
Uptake Model Figures 99
Statistical Analysis 99

Bibliography 100
viii
List of Tables

Table 1. Classification of Human Papillomaviruses 2
Table 2. Synthetic imidazoquinolines and their respective receptor(s) 31
ix
List of Figures

Figure 1. HPV viral particles 1
Figure 2. HPV16 genome and proteins 3
Figure 3. Infection with HPV 5
Figure 4. Schematics of VLP 6
Figure 5. Theoretical model of HPV cell surface binding and uptake 9
Figure 6. Potential signal transduction pathways initiated in LC after 29
exposure to HPV16L1L2 VLP
Figure 7. 3M-002 and resiquimod induce an HPV16 epitope-specific CD8
+
32
T cell immune response through the activation of LC exposed to HPV16L1L2
chimeric VLP
Figure 8. Human monocyte-derived LC express langerin, CD1a and E-cadherin 35
Figure 9. Differential expression of surface markers on LC after exposure to 36
HPV16 VLP
Figure 10. Differential secretion of Th1-associated cytokines and chemokines 38
by LC exposed to HPV16L1 VLP or HPV16L1L2 VLP
Figure 11. Equivalent secretion of Th1-associated cytokines and chemokines 39
by DC exposed to HPV16L1 VLP or HPV16L1L2 VLP
Figure 12. HPV16L1 VLP induce LC migration 40
Figure 13. LC exposed to HPV16 VLP induce differential activation of 41
HPV16-specific CD8
+
T cells
Figure 14. HPV16L1L2 VLP induce an immune suppressive signal 43
transduction cascade in LC
Figure 15. The suppressive effect of HPV16L1L2 VLP is not dominant over 45
potent activating signals
Figure 16. LC internalize HPV16L1L2 VLP twice as much as HPV16L1 VLP 48
x
Figure 17. HPV16L1 VLP and HPV16L1L2 VLP enter and travel through LC 49
in different cellular compartments
Figure 18. The HPV16L2
108-120
peptide inhibits binding of HPV16L1L2 VLP 50
to LC
Figure 19. Polyhistidine immunoblot analysis of eluates isolated from an 52
L2
108-120
pulldown assay
Figure 20. Silver stain analysis of eluates isolation from an L2
108-120
53
pulldown assay
Figure 21. ANXA2 immunoblot analysis of eluates isolated from an 54
L2
108-120
pulldown assay
Figure 22. SLPI inhibits the uptake of HPV16L1L2 VLP by LC 55
Figure 23. SLPI does not inhibit the uptake of HPV16L1 VLP by LC 56
Figure 24. Immunoblot demonstrating siRNA-mediated knockdown of 57
ANXA2 in LC
Figure 25. Downregulation of ANXA2 inhibits uptake of HPV16L1L2 VLP 58
but not HPV16L1 VLP
Figure 26. Unified theory of HPV uptake 75

xi
Abbreviations

aa, amino acid
ANXA2, annexin A2
APC, antigen presenting cells
ATP, adenosine triphosphate
BPV, bovine papillomavirus
CCL, c-c chemokine ligand
CCR, c-c chemokine receptor
CD, cluster of differentiation
CFDA-SE, carboxyfluorescein diacetate, succinimidyl ester
CIN, cervical intraepithelial neoplasia
CMV, cytomegalovirus
CTL, cytotoxic T lymphocytes
CyPB, cyclophilin B
DC, dendritic cells
DC-SIGN, dendritic cell-specific ICAM-grabbing non-integrin
DNA, deoxyribonucleic acid
DTSSP, 3,3’-Dithiobis-(sulfosuccinimidylpropionate)
ECM, extracellular matrix
ELISA, enzyme-linked immunosorbent assay
ELISPOT, enzyme-linked immunosorbent spot
ERK, extracellular signal-regulated protein kinase
xii
FAK, focal adhesion kinase
FDA, Food and Drug Administration
GFP, green fluorescent protein
GM-CSF, granulocyte macrophage-colony stimulating factor
HDAC, histone deacetylases
HIS, histidine
HIV, human immunodeficiency virus
HLA, human leukocyte antigen
HPV, human papillomavirus
HSPG, heparin sulfate proteoglycans
HSV, herpes simplex virus
ICAM, inter-cellular adhesion molecule
IFN, interferon
IL, interleukin
Ins(1,4,5)P
3
, inositol (1,4,5)-triphosphate
IP-10, interferon-gamma induced protein-10
ITC, isothermal titration calorimetry
JAK, janus kinase
kDa, kilodalton
LC, Langerhans cells
LMP, low molecular mass protein
LN, lymph node
LPS, lipopolysaccharide
xiii
MAPK, mitogen-activated protein kinase
MCP, monocyte chemoattractant protein
MHC, major histocompatibility complex
MIP, macrophage inflammatory protein
NF-κB, nuclear factor-κB
NK, natural killer
NMR, nuclear magnetic resonance
p38K, p38 stress-activated proteins kinases
Pap, Papanicolaou
PBS, phosphate buffered saline
PI3K, phosphoinositide 3-kinase
PI(3,4,5)P
3
, phosphatidylinositol (3,4,5)-trisphosphate
PI(4,5)P
2
, phosphatidylinositol (4,5)-bisphosphate
PIP5K, phosphatidylinositol 4-phosphate, 5-kinase
PKC, protein kinase C
PLC, phospholipase C
PP2A, protein phosphatase 2A
pRb, retinoblastoma protein
PsV, pseudovirion
RANTES, regulated upon activation, normal T cell expressed, and secreted
RFP, red fluorescent protein
RNA, ribonucleic acid
SD, standard deviation
xiv
SEM, standard error of the mean
Ser, serine
SFK, src-family kinases
SH2, src-homology domain 2
shRNA, short hairpin RNA
siRNA, small interfering RNA
SLPI, secretory leukocyte protease inhibitor
STAT, signal transduction and transcription
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
TLR, Toll-like receptor
TNF, tumor necrosis factor
T
R
1, T regulatory 1
Tregs, regulatory T cells
Tyr, tyrosine,
VLP, virus-like particle

xv
Abstract

Persistent high-risk human papillomavirus (HPV) infection is causally associated
with the generation of several cancers, including cervical cancer. While most women
infected with HPV clear their lesions, the average time to clearance is one year,
indicating that HPV has evolved specific mechanisms that allow it to circumvent the
human immune system and establish persistent infections. Currently, there is no
treatment for persistent HPV infections, and therefore our long-term goal is to elucidate
how HPV escapes immune mediated clearance in order to develop novel ways to treat
HPV infections and HPV-induced lesions. During its natural life cycle, HPV infects the
basal cells of the epithelium and interacts with Langerhans cells (LC), the resident
antigen presenting cells of the epithelium. Due to their location, LC are responsible for
initiating immune responses against pathogens entering the epithelium. However, HPV
does not activate LC, preventing the induction of an HPV specific adaptive immune
response and implicating an HPV immune escape mechanism that targets human LC. The
HPV protein responsible for inducing this immune escape has not been determined. We
demonstrate that LC exposed to the minor capsid protein L2 in HPV16L1L2 virus-like
particles (VLP) do not phenotypically or functionally mature. However, HPV16L1 VLP
significantly induce the activation of LC. Our data suggest that the L2 protein plays a
specific role in the induction of this immune escape of HPV16 through the manipulation
of LC. This novel function is the first immune modulating action attributed to the L2
protein and adds significantly to our understanding of the mechanism of HPV immune
escape. Further, we demonstrate that the N-terminus of L2 associates with the annexin
xvi
A2 (ANXA2) heterotetramer on the LC surface. Inhibiting the interaction between HPV
L2 and the ANXA2 heterotetramer or downregulating ANXA2 expression disrupts the
internalization of HPV by LC, indicating that the ANXA2 heterotetramer is the uptake
receptor for HPV. This result is highly significant because, despite decades of HPV
research, neither a specific receptor for the HPV L2 protein nor an uptake receptor for
HPV has been identified. Taken together, these studies describe a novel HPV16 immune
escape mechanism that leads to persistent HPV infection and direct the development of
future therapeutics for persistent HPV infections and HPV-induced lesions.

1
Chapter 1: Introduction

Human Papillomavirus Taxonomy and Genome
Human papillomaviruses (HPV) are members of the papillomaviridae family.
HPV are non-enveloped, icosahedral, double-stranded DNA viruses that infect the
epithelium of the 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 are mucosotropic (de Villiers, 2001). Mucosotropic HPV can be further
subdivided into low-risk types (such as HPV6 and 11), which cause benign condylomas,
and high-risk types (such as HPV16, 18, 31 and 45), which cause several cancers,
including cervical cancer (Table 1) (Munoz et al., 2003; Walboomers et al., 1999; zur
Hausen, 1991).

Figure 1. HPV viral particles. A. Illustration of an HPV virion composed of the major
capsid protein L1 and minor capsid protein L2 surrounding the double stranded circular
HPV genome. B. Image of an HPV viral particle composed of the L1 and L2 proteins
assembled into pentamers (Buck et al., 2008). C. Image of an HPV viral particle
composed of L1 and L2 proteins, depicting L2 density in red (Buck et al., 2008).
2

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

Table 1. Classification of Human Papillomaviruses.

HPV have a ~8000 base pairs long, double-stranded, closed circular genome that
encodes for early, late and long control regions. The early gene products (E1, E2, E4, E5,
E6 and E7) are critical in viral DNA replication, transcription, genome persistence and
cellular transformation (zur Hausen, 1996). The late genes encode for the major capsid
protein L1 and the minor capsid protein L2 (Kirnbauer et al., 1993, 1992; Zhou et al.,
1994). The late genes control proper capsid assembly, DNA packaging and virus
infectivity (Figure 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 is
therefore bound by cellular and viral transcription factors that augment the viral life
cycle.
3

Figure 2. HPV16 genome and proteins. The HPV16 genome is 7904 base pairs in
length and codes for early and late viral proteins. The early genes (E1, E2, E4, E5, E6 and
E7) are critical in viral DNA replication, transcription, genome persistence and cellular
transformation. The late genes encode for the major capsid protein L1 and the minor
capsid protein L2 and control proper capsid assembly, DNA packaging and virus
infectivity. The function of each viral protein and subcellular localization is indicated in
the figure.

HPV Life Cycle
The life cycle of HPV is dependent upon the differentiation of basal cells in the
epithelium into keratinocytes. The replication cycle of HPV is about 3 weeks from host
cell infection to virion release, which corresponds to the differentiation of basal cells into
keratinocytes and subsequent desquamation (Stanley et al., 2007). Basal cells are a single
4
layer of undifferentiated, proliferating cells that act as the stem cells of the epithelium.
Although basal cells are not normally in direct contact with the outside environment,
HPV are thought to interact with basal cells via a wound, foreign body or microabrasion
in the epithelium (Figure 3). After infection of the basal cells, the viral genome is
amplified to about 50-100 genomes per cell (Stanley et al., 2007). During this initial
process, early viral genes are expressed at low levels. E1 and E2 form a complex that
binds to the origin of replication, recruiting cellular polymerases and accessory proteins
to mediate DNA replication (Doorbar, 2006). The infected basal cell then exits the cell
cycle and begins differentiation, causing strong upregulation of viral gene expression and
viral DNA amplification. Genome amplification reaches at least one thousand genomes
per cell, indicating the potential for the production of one thousand virions per cell. Once
the infected cell reaches the upper layer of the epithelium, the late viral genes L1 and L2
are expressed, virion assembly begins and virion release occurs upon desquamation
(Doorbar, 2006; Stanley et al., 2007).
5

Figure 3. Infection with HPV. The right side of the figure depicts the normal maturation
of squamous epithelium. The antigen presenting cells shown are Langerhans cells, the
resident immune sentinels of the epithelium. The left side of the figure depicts an HPV
infection. HPV gains access to the basal cells of the epithelium via a foreign body or
microabrasion. The life cycle of HPV is subsequently tied to the differentiation of basal
cells into keratinocytes. The figure also demonstrates the sites of expression of HPV
proteins as the virus life cycle progresses. Figure reproduced from (Leggatt and Frazer,
2007).

HPV Virus-like Particles
Because the life cycle of HPV is dependent on the differentiation of basal cells
into keratinocytes, it is difficult to produce large quantities of HPV virions in vitro.
Therefore, as an alternative to HPV virions, HPV virus-like particles (VLP) have been
developed for structural and immunological analysis of HPV. HPV VLP phenotypically
and functionally mimic native HPV, however they do not contain any genomic material,
rendering them non-transforming. There are two types of VLP that are currently being
6
utilized by the HPV field – L1 VLP and L1L2 VLP (Fig. 4). When the major capsid
protein L1 is expressed it can self-assemble into a L1 VLP with icosahedral structure
(Kirnbauer et al., 1992). If both L1 and the minor capsid protein L2 are simultaneously
expressed, the proteins assemble into L1L2 VLP (Kirnbauer et al., 1993; Buck et al.,
2008; 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 L1 VLP are not morphologically equivalent.

Figure 4. Schematics of VLP. A. VLP composed of L1 protein. B. VLP composed of L1
and L2 proteins. C. Electron micrograph of the 55 nm icosahedral L1L2 VLP. Bar = 100
nm.

HPV Capsid Structure
As obligatory intracellular pathogens, HPV particles must gain entry into host
cells in order to deliver their genetic information to the nucleus and initiate viral
replication. The HPV viral capsid plays a key role in directing the series of consecutive
steps necessary for virion cell entry, uptake and infection (Joyce et al., 1999; Richards et
al., 2006; Evander et al., 1997). The understanding of HPV-cell interactions and uptake
7
mechanisms is critical in order to develop novel therapeutic strategies to disrupt HPV
infections and viral persistence.
The HPV virion is a 55 nm diameter, T = 7 icosahedral capsid composed of two
structural proteins, the major capsid protein L1 and the minor capsid protein L2 (Modis et
al., 2002; Buck et al., 2008). The capsid is formed by 360 molecules of L1 organized into
72 five-fold capsomeres (Baker et al., 1991). There are 72 molecules of L2 found in the
capsid, which appear to be located in the central internal cavity of the L1 pentamer (Buck
et al., 2008). The majority of L2 is hidden from the capsid surface, expect for residues 60
- 120 located within the N-terminus, which are exposed on the viral capsid surface (Liu et
al., 1997; Kondo et al., 2007).
While the HPV minor capsid protein L2 is not required for capsid formation, it
has been shown to possess a variety of critical functions. The C-terminus of L2 binds
directly to L1 primarily through hydrophobic interactions. This region of L2 is proline-
rich allowing for sharp bending of the protein that may facilitate L2 to loop through the
central cavity of the L1 pentamer (Finnen et al., 2003). It has also been demonstrated that
L2 interacts with the viral genome and is integral in the encapsidation of viral DNA
(Zhao et al., 1998). Collectively, these interactions imply a significant role for L2 in the
formation of the virion. Furthermore, L2 facilitates HPV infection through an interaction
between the N-terminus region of the L2 protein and an unknown cell surface receptor
(Kawana et al., 2001; Yang, Day, et al., 2003). Further functions of L2 include binding of
the virion to the cytoskeleton, transport within the cytoplasm (Yang, Yutzy, et al., 2003)
and facilitation of endosomal escape of the viral genome after infection (Kämper et al.,
2006).
8
HPV Cell Surface Binding
The concept of viruses binding to a single receptor and subsequently entering
cells through a single uptake mechanism has been challenged (Marsh and Helenius, 2006;
Mercer et al., 2010; Sieczkarski and Whittaker, 2005). Instead, a more complex picture is
forming where specific co-receptors and multiple attachment sites lead eventually to viral
entry by one or multiple uptake mechanisms. In fact, some viruses, including influenza
A, may actually be able to cause productive infections through multiple entry pathways,
suggesting complimentary uptake pathways (Mercer et al., 2010).
Specifically for HPV, research to date demonstrates that virus entry into host cells
is initiated upon the binding of the virion to multiple cell surface receptors, however the
secondary uptake receptor remains unknown (Figure 5). It has recently been identified
that the primary site of HPV binding may be the extracellular matrix (ECM) (Roberts et
al., 2007). In vitro studies have identified laminin-5, a ligand of α
6
β
4
integrin, as a
putative HPV receptor in the ECM (Culp, Budgeon, and Christensen, 2006; Culp,
Budgeon, Marinkovich, et al., 2006). However, while the affinity for HPV binding to
laminin-5 is higher than heparin sulfate (discussed below), it appears that its interaction
with HPV is not as critical for mediating a productive infection as heparin sulfate binding
(Culp, Budgeon, Marinkovich, et al., 2006; Selinka et al., 2007).
9
Figure 5. Theoretical model of HPV cell surface binding and uptake. HPV capsids
have been shown to bind to the ECM protein laminin-5 before binding to the host cell
surface, although this interaction may not be critical to productive infection. On the host
cell surface, it is suggested that HPV initially interacts with heparan sulfate proteoglycan
(HSPG), a widely expressed and evolutionary conserved cell surface receptor. However,
several other potential cell surface receptors/binders for HPV have been identified,
including α6β1/4 integrin, cyclophilin B (CyPB) and tetraspanins (CD63 and CD151).
All of these cell surface molecules (HSPG, α6 integrins, CyPB and tetraspanins) have
been shown to interact with each other and facilitate functionality, such as signaling.
Therefore, binding of HPV may occur via a singular molecule or a complex of molecules,
as shown in the figure. What is clear is that interaction of HPV with HSPG results in a
conformational change that results in the exposure of a furin cleavage site. This leads to
proteolytic cleavage of the L2 protein, resulting in additional conformational changes that
decreases the affinity of the capsid for the primary receptor and exposes a binding site for
the secondary cell surface receptor. This results in a hand off of the HPV capsid to the
secondary receptor, which leads to uptake of HPV through endocytosis. The specific
HPV endocytosis pathway has been shown to be clathrin-, caveolin-, dynamin-, flotillin-
and lipid-raft independent. Subsequent to endocytosis, HPV travels with the early
endosome to the late endosome and lysosome. The decreased pH in the late
endosome/lysosome triggers viral uncoating. After uncoating, the L2 protein mediates the
egress of the HPV DNA out of the endosome and both the HPV DNA and L2 protein
enter the host cell nucleus through a yet unknown mechanism.
10
Figure 5. Continued.

Preliminary studies on HPV binding to cells showed that the virus binds primarily
through interaction between L1 and a widely expressed cell surface receptor (Roden et
al., 1994; Müller et al., 1995). Heparan sulfate proteoglycans (HSPG) were proposed as
the main cell surface receptors for HPV and indeed HPV infection appears to require the
initial interaction with HSPG (Joyce et al., 1999; Giroglou et al., 2001; Combita et al.,
11
2001; Drobni et al., 2003). The binding of HPV to HSPG leads to a conformational
change in the virus capsid that exposes a highly conserved consensus furin convertase
site on the L2 N-terminus and eventual furin-mediated cleavage of L2 (Day et al., 2008;
Richards et al., 2006). However, HSPG-independent infection of cells using pre-cleaved
HPV demonstrates that the binding of HPV to HSPG is only a means to an end, with the
end being the furin cleavage of L2 (Day et al., 2008). Therefore, while this cleavage is
required for infection, it is clear that HSPG are not the receptors that mediate HPV uptake
and infection, but instead a separate secondary receptor or co-receptor is involved in the
infectious internalization of HPV into host cells (Selinka et al., 2007; Day et al., 2008). In
fact, it has been suggested that this furin cleavage or a subsequent conformational change
after cleavage may expose a binding site for a secondary cell receptor (Horvath et al.,
2010; Day et al., 2007).
A separate molecule, cyclophilin B (CyPB), has recently been implicated in
initiating conformational changes in the HPV capsid to facilitate productive infection.
CyPB can be secreted and detected on the cell surface, where it associates with HSPG
(Pakula et al., 2007). Additionally, CyPB has been shown to interact and mediate cell
signaling through β1 integrin, a molecule discussed in detail below (Melchior et al.,
2008). It was demonstrated that CyPB facilitates conformational changes in HPV16 that
expose the L2 N-terminus (Bienkowska-Haba et al., 2009). The requirement for CyPB
could be bypassed by mutation of the CyPB binding site, however, this mutant was still
sensitive to CyPB inhibition, suggesting that CyPB may be required for infection after
internalization.
12
Another proposed HPV receptor is the cell adhesion molecule α
6
integrin
(Evander et al., 1997; McMillan et al., 1999; Yoon et al., 2001). It was demonstrated that
HPV bind specifically to the α
6
integrin subunit complexed with either β
1
or β
4
integrin
(α6β1/4 integrin) (Evander et al., 1997). Although subsequent studies indicate that some
papillomavirus types, including HPV11, HPV33 and bovine papillomavirus (BPV) type
4, do not require α6 integrins for cell entry (Shafti-Keramat et al., 2003; Giroglou et al.,
2001; Sibbet et al., 2000), several interesting correlations between α6 integrins, HSPG,
tetraspanins and intracellular signaling pathways provide sufficient evidence to include
integrins in a model for HPV host cell binding and entry. Specifically, there is close
association between proteoglycans and integrins as matrix components (Horvath et al.,
2010). Furthermore, there is a tight association between tetraspanin CD151, which was
found to be important in HPV cell entry (Spoden et al., 2008), and both α6β1 and α6β4
integrins (Sterk et al., 2002). This association between tetraspanins and integrins is
thought to facilitate the coupling of signaling pathways. Notably, HPV has been shown to
activate the phosphoinositide 3-kinase (PI3K) pathway via α6β4 integrin binding
(Fothergill and McMillan, 2006). Furthermore, CyPB was also shown to signal through
both PI3K and protein kinase C (PKC). Importantly, the activation of PI3K has been
demonstrated to be critical for the immune escape of HPV through Langerhans cells
(LC), the resident antigen presenting cells (APC) at the site of infection (Fausch et al.,
2005). In addition, endocytosis of HPV requires both PI3K and PKC activation (Mercer
et al., 2010).
Based on current research, it appears that although the major capsid protein L1
dominates the initial cell surface interaction, the minor capsid protein L2 may facilitate
13
infection by binding to a secondary receptor. This concept of a specific L2 secondary
receptor is supported by several studies examining L2 mediated infectivity (Kawana et
al., 2001; Yang, Day, et al., 2003). In a study by Kawana et al. it was clearly
demonstrated that HPV16L1L2 VLP are twice as infectious as HPV16L1 VLP (Kawana
et al., 2001). Additionally, Kawana et al. demonstrated that pre-incubation of COS-1 cells
with the HPV16 L2 peptide amino acids (aa) 108-120 decreased infectivity of HPV16
pseudovirions (Kawana et al., 2001). Additionally, Yang et al. suggested that
HPV16L1L2 VLP binding to the cell surface of HeLa cells causes aa 13-31 of the L2
protein to be displayed on the virion surface, interact with a yet unknown secondary
receptor and facilitate infection (Yang, Day, et al., 2003). In the context of these studies,
the existence of an L2-specific receptor becomes highly conceivable.

HPV Internalization
Subsequent to cell surface binding, HPV must enter the cell in order to induce a
productive infection. Although many studies have been conducted, the details of the
initial steps occurring during endocytosis are yet to be fully elucidated and many results
remain contradictory. The process of endocytosis of HPV occurs very slowly and
asynchronously in epithelial cells, with an uptake half-time of up to 14 hours (Sapp and
Bienkowska-Haba, 2009). The uptake kinetics for non-epithelial cells, such as LC and
dendritic cells (DC), can be much shorter, within minutes to hours (Bousarghin et al.,
2005; Fausch et al., 2003). While the majority of viruses are internalized rapidly, the
reason for the slow kinetics of HPV is unknown. It is interesting to note that HSPG have
been shown to also have a slow rate of internalization after ligand binding (Williams and
14
Fuki, 1997). It may be that the required conformational changes, furin cleavage and
interaction with multiple disparate cell surface molecules add to the delay in
internalization (Horvath et al., 2010; Spoden et al., 2008).
For non-enveloped viruses there are two main pathways of endocytosis – clathrin-
and caveolae-mediated. Early studies on HPV endocytosis relied on biochemical
inhibitors to block internalization, however, these small molecule drugs might have
unwanted and unknown side effects on cell function (Horvath et al., 2010; Sapp et al.,
2009). These studies were also complicated by the seemingly indiscriminant use of HPV
L1 and L1L2 VLP, making conflicting results difficult to interpret. For instance, Yan et
al. demonstrated that LC uptake HPV6 L1 VLP through a caveolin-mediated uptake
pathway, while Bousarghin et al. showed that LC uptake HPV16L1 VLP through a
clathrin-mediated uptake pathway (Yan et al., 2004; Bousarghin et al., 2005). However,
when HPV16L1L2 VLP, which more closely resemble true HPV virions, were utilized, it
was demonstrated that LC internalize HPV through a clathrin- and caveolin-independent
manner (Fausch et al., 2003).
A recent study, using more advanced techniques, confirms that HPV16
endocytosis is indeed clathrin- and caveolae-independent (Spoden et al., 2008). Spoden et
al. demonstrated that inhibition of clathrin- and caveolin/raft-dependent endocytosis by
either siRNA-mediated downregulation or dominant-negative mutants, along with
inhibition of dynamin function, did not impair HPV infection. This result was confirmed
and further described as a clathrin-, caveolin-, flotillin-, lipid raft-, dynamin-independent
mechanism distinct from macropinocytosis and phagocytosis (Mercer et al., 2010). This
novel endocytosis pathway was shown to be dependent on actin polymerization, PI3K
15
and PKC activation but independent of Rho-like GTPases. Additionally, this entry
pathway may utilize tetraspanin-enriched microdomains, specifically tetraspanins CD63
and CD151, as platforms for HPV entry (Spoden et al., 2008).

HPV Vesicular Trafficking and Egress from Endosomes
Although currently there are no comprehensive studies on intracellular trafficking
of HPV using targeted gene-knockdown and dominant negative constructs, there is
consensus that HPV infection requires acidification of endocytotic vesicles as the virus
moves from early endosomes to late endosomes and eventually lysosomes (Smith et al.,
2008; Spoden et al., 2008; Mercer et al., 2010).
Upon internalization of HPV, there is disassembly of the capsid before egress
from the endosome. Early studies demonstrated that intact HPV capsids exceed the size
capacity for transport through the nuclear pore, suggesting that viral uncoating must
occur before nuclear import (Merle et al., 1999; Nelson et al., 2000). L1 protein is shed
from the viral genome during uncoating in endocytic vesicles and it cannot be detected in
the nucleus of infected cells (Spoden et al., 2008). While L2 protein may not be essential
for viral uncoating, L2 appears to mediate the escape of viral DNA from endosomes
(Kämper et al., 2006). It has been shown that furin cleavage of L2 is essential for
endosomal escape (Richards et al., 2006), which possibly mediates the release of L2 from
L1 or promotes binding of L2 to receptor that facilitates uncoating and endosomal escape
(Sapp et al., 2009).

16
HPV Nuclear Transport
The exact mechanism by which the HPV genome enters the nucleus is not well
understood. However, it is clear that both the viral genome and the L2 protein co-localize
in the nucleus, indicating that they translocate to the nucleus together (Day et al., 2004).
It was demonstrated that the L2 protein promotes viral infection by interacting with the
motor protein dynein along with the microtubule network (Florin et al., 2006).
Additionally, it appears that the L2 protein provides the nuclear import signals since it
contains two terminal peptides that function as nuclear localization signals (Becker et al.,
2003; Sun et al., 1995; Fay et al., 2004; Darshan et al., 2004). However, active nuclear
import may not be required, as suggested by a recent study demonstrating that nuclear
envelope breakdown is required for productive infection of HPV (Pyeon et al., 2009).

HPV Pathogenesis and Cervical Cancer
Mucosotropic, high-risk HPV are sexually transmitted viruses that can cause
genital warts and several cancers, including head and neck, 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 one year (Woodman et al.,
2001). However, about 15% of women that contract a high-risk HPV infection cannot
initiate an effective immune response against HPV (Stanley et al., 2007), leading to a
persistent HPV infection. The persistence of a high-risk HPV infection is the major risk
factor in the development of cervical cancer (Schlecht et al., 2001). HPV16 and HPV18
DNA are found in more than 70 percent of HPV positive biopsies obtained from cervical
17
cancer patients (Castellsagué et al., 2006), with HPV16 being the most common
mucosotropic, high-risk HPV with an overall prevalence of nearly 60 percent (Munoz et
al., 2003).
It is the specific activity of the oncoproteins E6 and E7 that induce cancer upon
infection with high-risk HPV. In the majority of HPV infections, the viral genome exists
as an episome, however in HPV-induced cancers there is an integration of a portion of the
HPV genome into a host cell chromosome, leading to an increase in genome instability in
these infected cells (Duensing and Münger, 2002). In terms of successful HPV virion
production, this integration event is not beneficial for the virus because it halts the HPV
life cycle and the production of virions. The most common integration event is the
insertion of the E2 gene into the host cell. This can lead to significantly increased
production of full length E2 protein, which actually suppresses the expression of the
oncogenic E6 and E7 proteins. However, if the E2 gene is disrupted during integration,
this can lead to an overexpression of E6 and E7 proteins (Stubenrauch and Laimins,
1999; Leggatt et al., 2007). The E6 and E7 proteins function as oncogenes by binding to
and inactivating the tumor-suppressor gene products p53 and retinoblastoma protein
(pRb), respectively (Werness et al., 1990; Dyson et al., 1989). Additionally, E6 contains a
C-terminal PDZ ligand domain, allowing it to bind to and stimulate proteasomal
degradation of proteins with PDZ motifs, such as hD1g and hScribble, both of which are
involved in the regulation of cell growth and attachment (Zeitler et al., 2004).
Furthermore, 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). However, the overexpression of the
18
oncogenes E6 and E7 is not sufficient for the development of cancer. The E6 and E7
proteins, when aberrantly expressed, drive the infected cell towards a precancerous state
that is sensitive to harmful secondary genetic changes. HPV-induced cancer most often
occurs in individuals that have persistent HPV infections along with E6 and E7 oncogene
overexpression for years to decades.
Cervical cancer is the second most common cancer among women worldwide and
is the cause of death for approximately a quarter of a million women each year (Lowndes,
2006; Parkin, 2006). Cervical cancer is a major health burden in developing countries due
to lack of resources and routine Papanicolaou (Pap) smear examinations. In fact, nearly
80 percent of cervical cancer cases occur in developing countries (Castellsagué et al.,
2006). Within the last two years, the Food and Drug Administration (FDA) have
approved two prophylactic HPV vaccines. The first was Gardasil produced by Merck,
which is based upon VLP composed of the major capsid proteins from HPV6, 11, 16 and
18. The second was Cervarix produced by GlaxoSmithKline, which is based upon VLP
composed of the major capsid protein from HPV16 and 18. In phase III clinical trials,
prophylactic HPV vaccination has been shown to be nearly 100 percent effective at
preventing HPV-induced lesions (Ault and FUTURE II Study Group, 2007; FUTURE II
Study Group, 2007). However, even with significant uptake of prophylactic HPV
vaccination programs, it is predicted that it will be decades before a quantifiable effect of
cervical cancer rates will appear in the population (Dasbach et al., 2006; Ryding et al.,
2008). Importantly, neither Gardasil nor Cervarix are therapeutic treatments. Therefore,
the implementation of prophylactic vaccines will not benefit the millions of women that
are currently infection with HPV. In fact, no therapeutic options currently exist to
19
promote HPV clearance in persistent infections due, in part, to the lack of knowledge
about natural HPV immunity and HPV immune escape mechanisms. There remains a
clear need to develop treatments to facilitate immune mediated clearance of HPV
infections and reduce the percentage of women whose lesions progress to cervical cancer.

Antigen Presenting Cells and Adaptive Immune Responses
Located in nearly all tissues, DC are APC that are responsible for initiating and
modulating immune responses (Banchereau and Steinman, 1998). Now known to be a
heterogeneous family, DC can be divided into subsets defined by their phenotype,
function and location. There are two main subdivisions of DC: plasmacytoid DC and
conventional DC (Heath et al., 2004; Villadangos and Schnorrer, 2007). Some studies use
the terms lymphoid and myeloid to refer to these DC categories, however these terms are
now considered obsolete (Villadangos and Schnorrer, 2007). Plasmacytoid DC circulate
through the blood and lymphoid tissues and are critical in the production of interferon
(IFN)-α and IFN-β, however there is not much evidence on their importance in antigen
presentation (Heath et al., 2004). Conventional DC encompass the remaining five subsets
of DC, which can be categorized into two groups: blood-derived DC and tissue-derived
DC. The blood-derived DC reside in the spleen and lymph nodes, while the tissue-
derived DC are located in the peripheral non-lymphoid organs. Blood-derived
conventional DC can be further divided based on expression of CD4 and CD8 into three
subsets: CD4
+
DC, CD8
+
DC and CD4
-
CD8
-
DC. Tissue-derived conventional DC
include two unique subsets: dermal/interstitial DC that reside in the dermis and deeper
tissues and LC that reside in the epidermis of the skin and mucosa.
20
With their projecting dendrites, LC form a continuous cellular network that
monitor the entire thickness of epidermis for foreign antigen, functioning as the first
immune sentinels between the internal and external environments (Merad et al., 2008).
Importantly, the epidermal keratinocytes that surround the LC produce transforming
growth factor-β
1
(TGF-β
1
), which is essential for the development of LC in vivo from
CD14
+
cells (Borkowski et al., 1997). It is known that steady state LC can self-renew
independently of precursor cells from the blood and bone marrow, however, during
inflammation, circulating monocytes can be recruited to the epidermis and replenish LC
(Merad et al., 2008; Ginhoux et al., 2006). In vitro, human blood-derived monocytes can
be differentiated into cells with either DC or LC phenotypic and functional characteristics
when cultured in the presence of granulocyte macrophage-colony stimulating factor
(GM-CSF) and interleukin (IL)-4 or GM-CSF, IL-4 and TGF-β
1
, respectively
(Geissmann et al., 1998; Romani et al., 1996). LC can be phenotypically differentiated
from DC because they express high levels of E-cadherin, a homotypic adhesion molecule
that anchors LC to neighboring keratinocytes; CD1a, a cell-surface protein that presents
microbial lipids; and langerin, a type II C-type lectin receptor that binds mannose and
related sugars. Additionally, LC contain rod or racket-shaped intra-cytoplasmic Birbeck
granules, which are poorly understood structures that might function in antigen
presentation (Merad et al., 2008). Furthermore, LC do not express the dendritic cell-
specific intercellular adhesion molecule (ICAM)-grabbing non-integrin (DC-SIGN)
(Klechevsky et al., 2008).
The primary functions of APC are recognition, internalization, processing and
presentation of foreign antigens to naïve T lymphocytes in regional lymph nodes (LN) in
21
order to induce an adaptive immune response (Banchereau and Steinman, 1998; Larsen et
al., 1990). Upon stimulation with certain cytokines, viruses, bacteria or necrotic cells,
APC undergo a process called “maturation”, which consists of phenotypic and functional
changes, including upregulation of co-stimulatory molecules CD80 (B7.1) and CD86
(B7.2), major histocompatibility complex (MHC) class I and II molecules, chemokine
receptors (such as c-c chemokine receptor 7 [CCR7]), increased secretion of cytokines
and chemokines and increased migration to regional LN (Albert et al., 1998; Banchereau
and Steinman, 1998; De Smedt et al., 1996; Larsen et al., 1990). Migration of APC is
directed by the secondary lymphoid tissue chemokine/c-c chemokine ligand 21
(SLC/CCL21) and macrophage inflammatory protein (MIP)-3β (also known as CCL19)
via binding to CCR7 (Hirao et al., 2000; Saeki et al., 1999). The migration of mature
APC to draining LN is critical for the presentation of antigens captured in the periphery
to naïve T cells, leading to the induction of antigen specific cell-mediated adaptive
immune responses (Banchereau and Steinman, 1998; Larsen et al., 1990). In this way,
APC residing in non-lymphoid tissues act as both sentinels in the periphery and bridges
between the innate and adaptive immune responses.
Mature APC and T cells interact through MHC molecules located on the APC
surface and T cell antigen receptors (TCR) located on the T cell surface. Peptides can be
presented on two types of MHC molecules: 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
22
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 can secrete pro-
inflammatory cytokines and directly kill a target cell presenting the corresponding
antigen (Banchereau and Steinman, 1998; Heath et al., 2004). When naïve CD4
+
T cells
recognize mature APC they become activated, their function depends on the particular
microenvironment surrounding the APC-T cell 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 activate macrophages, natural killer
(NK) cells and CD8
+
T cells, thereby inducing cell-mediated immunity. In the absence of
CD4
+
Th1 cells, CTL memory is decreased, which reduces the potential for a secondary
response. CD4
+
T cells become Th2 cells when they interact with and recognize mature
APC in the presence of IL-4. Th2 cells secrete IL-5, IL-4, IL-10 and IL-13, which cause
B cells to make specific antibody isotypes and inhibit Th1 mediated cell-mediated
immune responses (Banchereau and Steinman, 1998).
The generation of effective T cell responses requires more than just interaction
between peptide-loaded MHC molecules and TCR – typically called “signal one”. Co-
stimulatory molecules, such as CD80 and CD86, on APC bind to CD28 on the surface of
T cells along with signals from cytokines and chemokines facilitate the efficient
induction of adaptive immune responses. In the absence of co-stimulation or cytokine and
chemokines, APC presenting peptides on MHC molecules both anergize T cells and
generate regulatory T cells (Tregs) (Tan et al., 1993; Jonuleit et al., 2000).
23

Natural HPV Immunity
The immune system plays a critical role in the clearance of an HPV infection.
This is demonstrated by the increased number of persistent HPV infections and enhanced
progression of HPV-induced cervical lesions in immunodeficient patients (Laga et al.,
1992; Petry et al., 1994). Both the non-antigen specific innate and antigen specific
adaptive arms of the immune system control HPV infections during a protective immune
response. Acting as the first line of defense, the innate immune effectors – macrophages,
monocytes, polymorphic leukocytes and NK cells – detect pathogens using pattern
recognition receptors and produce a variety of immuno-regulatory molecules including
IFN-α, -β, -γ, TGF-β, tumor necrosis factor (TNF)-α, IL-1, IL-16, IL-10, IL-12 and IL-
15. These molecules can directly control virally infected cells and shape the activation of
the adaptive immune response (Stern, 2005). The importance of the innate immunity in
the clearance of HPV-induced lesions is further highlighted by the presence of
macrophages in regressing lesions that produce TNF-α in HPV-induced cervical lesions
(Coleman et al., 1994; Routes et al., 2005). TNF-α acts not only to induce inflammation
at the site of infection but it can also directly suppress viral replication. Furthermore,
TNF-α induces the activation and migration of LC (Cumberbatch et al., 2000).
Considering that LC are the resident APC at the site of HPV infection, the activation of
LC is critical to the induction of HPV-specific adaptive immune responses.
In nearly all viral infections, including HPV, the induction of activated virus-
specific CD8
+
T cells are critical in mediating immune clearance. Specifically in HPV
infections, it was demonstrated that HPV16 and HPV18 specific CTL responses were
24
present in patients with HPV16 or HPV18 induced cervical lesions but not in healthy
donors (Evans et al., 1997; Todd et al., 2004). Additionally, memory CTL responses were
identified in patients with HPV16-induced cervical lesions but were absent in healthy
donors (Ressing et al., 1996). These HPV16-specific memory CD8
+
T cells continue to
circulate for months, if not years, after an HPV16 infection has cleared (Wang et al.,
2008). These studies strongly suggest that HPV-specific CD8
+
T cells are responsible for
limiting the progression of HPV-induced lesions and eventually clearing them.
As discussed above, effective CD8
+
T cell responses require a robust CD4
+
Th1
response. CD4
+
Th1 cells play a particularly important role in mediating the expansion of
antigen-specific CD8
+
T cells. Within the context of HPV infections, there is evidence
that Th1 cells are critical for both the control and clearance of HPV infections. It was
demonstrated that within regressing HPV-induced genital warts, CD4
+
T cells made up
the majority of the infiltrating immune cells (Coleman et al., 1994). In addition to the
clearance of genital warts, HPV16- and HPV18-specific CD4
+
tumor infiltrating
lymphocytes were identified in patients with cervical cancer (Höhn et al., 1999; Piersma
et al., 2008). Interestingly, it was also demonstrated that strong memory CD4
+
Th1
responses against HPV16 E2, E6 and HPV18 E6 antigens can be detected in healthy
donors. These memory Th1 responses are most likely the result of previous effective anti-
HPV immune responses that have cleared the inciting infection (de Jong et al., 2002;
Gallagher and Man, 2007; Welters et al., 2003).

25
General HPV Immune Escape Mechanisms
While a majority of high-risk HPV infections are cleared by an HPV-specific cell-
mediated immune response, a small percentage of infections persist and 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. This immune evasion is due
to a lack of recognition of HPV viral particles and HPV infected cells caused by the
direct disruption of immune cell functions by viral proteins.
There exist a multitude of mechanisms employed by HPV to subvert the host
immune system. First, the low profile of the HPV life cycle allows minimal exposure of
HPV DNA, protein and virions to host immune cells, creates little to no inflammation
during infection and never enters a blood borne phase – all of which reduce the likelihood
of the induction of an innate and/or adaptive immune response (Frazer, 2004; Kupper and
Fuhlbrigge, 2004). Second, it has been demonstrated that HPV proteins specifically
inhibit proper antigen presentation by both MHC class I and class II leading to an
alteration of antigen processing and presentation during an HPV infection (Cromme et
al., 1994; Keating et al., 1995; Gruener et al., 2007; Zhang et al., 2003). Further, HPV
infection disrupts the IFN responses that are critical to viral clearance by downregulating
IFN response genes and increasing IFN resistance (Barnard and McMillan, 1999; Park et
al., 2000; Nees et al., 2001; Li et al., 1999). Additional alterations in the expression of
cytokines and chemokines include the downregulation of IL-18 and its downstream
receptor, suppression of monocytes chemoattractant protein (MCP)-1, macrophage
inflammatory protein (MIP)-3α and interference with nuclear factor (NF)-κB binding to
transcriptional co-activators (Cho et al., 2001; Lee et al., 2001; Huang and McCance,
26
2002; Kleine-Lowinski et al., 2003; Guess and McCance, 2005). Finally, it has been
demonstrated that CD4
+
Tregs that can inhibit HPV-specific adaptive immune responses
are found in the tumor draining lymph nodes of cervical cancer patients, suggesting that
HPV-induced lesions promote the recruitment or generation of Tregs to escape immune
clearance (Adurthi et al., 2008; Fattorossi et al., 2004; Molling et al., 2007; van der Burg
et al., 2007).
In a very recent study on patients with cervical cancer, a surprisingly broad
repertoire of HPV-specific CD4
+
and CD8
+
T cell responses were detected in tumor-
infiltrating lymphocytes and tumor-draining lymph node cells aimed at multiple E6 and
E7 epitopes (de Vos van Steenwijk et al., 2010). However, a majority of these T cells
produced only slight amounts of IFN-γ, suggesting that they were functionally inactive
within the tumor environment. This study indicates that even when a vast local T cell
population exists in a cervical cancer patient, they require proper stimulation before they
can become functionally effective. This principle could potentially be exploited for
cancer treatment by using immunotherapeutics to activate resident APC at the HPV-
induced lesion site.

LC-Mediated HPV Immune Escape
As discussed before, APC, like DC and LC, play a critical role in the initiation
and modulation of an antiviral immune response due to their ability to stimulate naïve T
cells and initiate adaptive immune responses in vivo (Banchereau et al., 2000). Because a
productive HPV infection occurs exclusively in the epidermis, LC are the only APC that
27
naturally come into contact with HPV. Consequently, LC are responsible for initiating an
immune response against an HPV infection.
In contrast to LC, DC reside below the epithelial basement membrane in the
dermis, and therefore do not normally come into contact with HPV during an infection. It
has been demonstrated that when human DC encounter HPV16L1L2 VLP they become
highly activated (Rudolf et al., 2001). These DC can subsequently generate HPV-specific
adaptive immune responses to HPV16L1L2 VLP (Rudolf et al., 2001). These results
clearly demonstrate that the structural surface components of HPV are capable of
inducing the maturation of APC. This explains the high immunogenicity and efficiency
of HPV VLP as vaccines, since the vaccines are administered by injection into the dermis
where DC are prevalent.
However, while both DC and LC equally internalize HPV16L1L2 VLP, our
laboratory has previously demonstrated that LC are not activated by HPV16L1L2 VLP
(Fausch et al., 2002; Fausch et al., 2003). Human LC exposed to HPV16L1L2 VLP
continue to present HPV-derived peptides on their MHC molecules but do not upregulate
important activation associated surface markers (including CD86, CD80 and CCR7),
secrete pro-inflammatory cytokines or generate an HPV16-specific CD8
+
T cell response
(Fausch et al., 2002; Fausch et al., 2003). Our laboratory went on to further elucidate the
molecular mechanism that mediates this immune escape through LC. It was shown that
LC incubated with HPV16L1L2 VLP upregulate PI3K, which in turn leads to the
downregulation of Akt, MAPK and NF-κB activation (Fausch et al., 2005).
To determine whether PI3K activation mediates the immune escape of HPV
through LC, our laboratory inhibited PI3K and exposed LC to HPV16L1L2 VLP. Upon
28
exposure, LC upregulated surface activation markers and initiated HPV16-specific
adaptive immune responses. Furthermore, HPV16L1L2 VLP with a disrupted structure
do not induce PI3K activation, suggesting that this targeting of LC by HPV16 is a
function of the intact viral protein capsid (Fausch et al., 2005). These studies confirm that
the activation of PI3K by HPV16 capsid proteins in LC defines a mechanism of immune
escape utilized by HPV16.
We believe the upregulation of PI3K leads to the downregulation of Akt, MAPK
and NF-κB through an unknown phosphatase. While this downstream phosphatase target
of PI3K needs to be further explored and identified, one potential target is protein
phosphatase 2A (PP2A), which may mediate the downregulation of Akt, MAPK and NF-
κB (Fausch et al., 2005; Millward et al., 1999). 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). Therefore, our laboratory proposed a
potential mechanism that drives the immune escape of HPV16 through LC: upon binding
and internalization of HPV, PI3K is activated in LC, which subsequently activates PP2A
and/or PKC, inducing the suppression of Akt, MAPK and NF-κB activity (Fig. 6).
Importantly, it has been demonstrated recently that HPV uptake is dependent on both
PI3K and PKC activation (Mercer et al., 2010), further strengthening our proposed
mechanism and suggesting that the immune escape initiated by HPV is intimately linked
to viral uptake.
29

Figure 6. 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-κB. This leads to a paucity of transcription from immune response genes. PKC
could also directly mediate the inhibition of Akt activity.

So while 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 maturation of LC, the immune sentinel at the site of infection. Although
both DC and LC quantitatively internalize HPV16L1L2 VLP equally, they do so through
different uptake mechanisms (Fausch et al., 2003). This suggests that HPV16 binds to
30
different receptors on DC and LC, which could lead to differential means of
internalization and initiation of different signal transduction pathways that could either
mediate immune response or immune escape.

Reversal of LC-mediated HPV Immune Escape
Because of this specific HPV immune escape targeting LC, the idea of
therapeutically targeting the resident APC at the site of HPV infections is of particular
interest to our laboratory. We recently demonstrated that LC express toll-like receptor
(TLR)7 and TLR8 (Fahey et al., 2009), and 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 initiate potent immune responses (Table 2)
(Schön and Schön, 2008). TLR7 and TLR8 are localized to endosomal membranes and
naturally recognize single-stranded RNA (Barton, 2007; Schön and Schön, 2008). Once
TLR7 and/or TLR8 are engaged, NF-κB and other transcription factors are activated,
leading to the transcription of many immune response genes, including genes coding for
cytokines, chemokines, co-stimulatory molecules and adhesion molecules (Gorden et al.,
2005; Medzhitov et al., 1997; Schön and Schön, 2008). Of note, imiquimod is a FDA
approved drug (Aldara) used to treat external anogenital warts caused by low-risk HPV
infection, so our laboratory was very interested to see if it would be an effective
therapeutic treatment for targeting HPV infected LC.
31

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

Table 2. Synthetic imidazoquinolines and their respective receptor(s).

Surprisingly, neither phenotypic nor functional hallmarks of activation were
observed when LC were exposed to HPV16L1L2 VLP and treated with imiquimod
(TLR7 agonist). However, we found that HPV16L1L2 VLP infected LC were activated
by 3M-002 (TLR8 agonist) and resiquimod (TLR8/7 agonist), as shown by a significant
increase in surface activation markers, proinflammatory cytokines and chemokines,
CCL21-directed migration and HPV16-specific CD8
+
T cells (Fig. 7) (Fahey et al.,
2009). Our data strongly suggest that 3M-002 and resiquimod can reverse the phenotype
and function of LC exposed to HPV16, unlike the TLR7 agonists 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.

32

Figure 7. 3M-002 and resiquimod induce an HPV16 epitope-specific CD8
+
T cell
immune response through the activation of LC exposed to HPV16L1L2 chimeric
VLP. LC were incubated with media alone or with HPV16L1L2 chimeric VLP 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
< 0.01 and ***P < 0.001, 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.

Project Goals and Significance
While a majority of women infected with HPV resolve their viral infection, there
is an average latency period of one year between infection and resolution (Woodman et
al., 2001). This indicates that HPV has evolved immune escape mechanisms in order to
avoid immune system detection and clearance. These immune evasion tactics allow for
the persistence of an HPV infection, which is the major risk factor in the development of
33
cervical cancer. Cervical cancer remains a major health concern with more than 490,000
women per year diagnosed globally (Parkin et al., 2005). Despite the recent
implementation of two prophylactic HPV vaccines, no therapeutic is available to promote
viral clearance in the approximately 24 million American women currently infected with
HPV (Dunne et al., 2007). Therefore, the development of therapeutic strategies to treat
persistent HPV infections is critical to eradicate the worldwide health burden of HPV-
induced lesions.
However, in order to develop effective immunotherapeutics for HPV-induced
lesions, the immune escape mechanisms utilized by HPV to evade viral clearance must be
fully elucidated. Our laboratory has shown that HPV16L1L2 VLP, which are
morphologically similar to live virions, suppress the activation of LC (Fausch et al.,
2002; Fausch et al., 2005). These VLP-infected LC present HPV peptides in the absence
of co-stimulation, which may lead to T cell tolerance. In apparent contradiction to our
studies, LC exposed to HPV L1 VLP were shown to generate cytolytic T cells in vitro
(Yan et al., 2004). HPV L1 VLP were also shown to be taken up by LC through either a
clathrin mediated (Bousarghin et al., 2005) or caveolae-dependent mechanism (Yan et al.,
2004), while we demonstrated that HPV16L1L2 VLP were taken up by LC through a
clathrin-, caveolae-, actin-independent pathway (Fausch et al., 2003). These contrasting
studies highlight differences in the interaction between LC and HPV L1 VLP versus HPV
L1L2 VLP, and point to the potentially important presence of the minor capsid protein
L2. Therefore, we sought to elucidate if the minor capsid protein L2 is responsible for the
induction of immune escape of HPV16.
34
Furthermore, the different uptake pathways utilized by HPV L1 VLP and HPV
L1L2 VLP and the specific signaling cascade initiated by the binding of HPV16L1L2
VLP to LC indicate the possible presence of a specific L2 receptor and uptake
mechanism. The concept of a specific L2 receptor is supported by several studies
examining L2 mediated infectivity (Kawana et al., 2001; Yang, Day, et al., 2003).
Specifically, it was demonstrated that pre-incubation of cells with HPV16 L2 peptide aa
108-120 decreased infectivity of HPV16 pseudovirions (PsV) (Kawana et al., 2001).
Therefore, we sought to identify the cell surface receptor for the L2 protein on LC.
These projects address a major medical need – the eradication of existing HPV
infections that can lead to the development of cervical cancer. The precise mechanism of
HPV immune escape is not fully defined and the identity of the uptake receptor for HPV
entry into cells has remained a challenge for decades. The knowledge from these projects
could lead to the development of strategies that inhibit binding of HPV to cells, block
downstream signaling pathways critical to HPV immune evasion at the site of infection.
In the future, the results from this mechanistic research could lead to clinical trials in
which strategies to reverse immune escape are employed.
35
Chapter 2: Results

LC acquire a mature phenotype when exposed to HPV16L1 VLP but not when
exposed to HPV16L1L2 VLP
We sought to determine if the minor capsid protein L2 is responsible for initiating
immune escape of HPV16 in LC. First, to verify the purity of the LC used in this study,
we assessed by flow cytometry the presence of surface markers commonly used to
identify LC: langerin, CD1a, and E-cadherin. Our results show that LC generated from
human monocytes are a pure population and phenotypically equivalent to LC found in the
epidermis (Fig. 8). Our LC derived from human monocytes contain Birbeck granules, as
we have previously shown (Fausch et al., 2002). Although it is possible to isolate human
LC from epidermal sheets, the isolation process induces the activation of LC (Klechevsky
et al., 2008) and therefore cannot be used. Thus, human monocyte-derived LC are the
most appropriate model to critically examine the interaction between HPV and human
LC.

Figure 8. 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.
36

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. After exposure to HPV16L1 VLP, LC upregulated CD86
(Fig. 9a, Fig. 9b), CD80, and MHC class II molecules (Fig. 9b) in comparison to
untreated LC, while LC exposed to HPV16L1L2 VLP had only a minor upregulation of
these markers (Fig. 9a, Fig. 9b). As a control, DC were exposed to HPV16L1 VLP or
HPV16L1L2 VLP and were found to be phenotypically activated by both VLP types
(Fig. 9a).

Figure 9. Differential expression of surface markers on LC after exposure to HPV16
VLP. A. HPV16L1 VLP induce the upregulation of CD86 on LC, however CD86 is not
increased on LC exposed to HPV16L1L2 VLP. Both types of HPV16 VLP induce the
upregulation of CD86 on DC. LC and DC were treated as indicated in the activation
assay and analyzed by flow cytometry. Grey lines represent isotype matched controls.
One representative experiment of eleven is shown. B. Fold change in expression of MHC
class II, CD80 and CD86 on HPV VLP exposed LC relative to untreated LC are depicted.
The mean of eleven separate experiments ± SEM is presented (*P<.05 **P<.01
determined by a two-tailed, paired t-test, as compared to LC exposed to HPV16L1 VLP).
37

Differential expression of cytokines and chemokines by LC exposed to HPV16L1
VLP or HPV16L1L2 VLP
In addition, we analyzed the types of cytokines and chemokines that are secreted
by LC upon exposure to HPV16L1 VLP or HPV16L1L2 VLP. 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, interferon-
inducible protein 10 (IP-10), MCP-1, MIP-1β and Regulated upon Activation, Normal T
cell Expressed and Secreted (RANTES) (Fig. 10). 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. 10). As a control, DC were exposed to
HPV16L1 VLP or HPV16L1L2 VLP and were found to be functionally activated by both
VLP types (Fig. 11).

38

Figure 10. Differential secretion of Th1-associated cytokines and chemokines by LC
exposed to HPV16L1 VLP or HPV16L1L2 VLP. LC exposed to HPV16L1 VLP
secrete Th1-associated cytokines and chemokines while LC exposed to HPV16L1L2
VLP do not. Supernatants collected from untreated LC (1), LPS treated LC (2),
HPV16L1 VLP-exposed LC (3), and HPV16L1L2 VLP-exposed LC (4) were analyzed in
triplicate for the presence of cytokines and chemokines. Levels of cytokines and
chemokines were quantified using a human cytokine LINCOplex assay. These data are
expressed as the mean concentration ± SEM (**P < 0.01 ***P < 0.001 determined by a
two-tailed, unpaired t-test, as compared to untreated LC). The experiment was repeated
three times and yielded similar results.

39

Figure 11. Equivalent secretion of Th1-associated cytokines and chemokines by DC
exposed to HPV16L1 VLP or HPV16L1L2 VLP. DC incubated with either HPV16L1
VLP or HPV16L1L2 VLP secrete equivalent levels of Th1-associated cytokines and
chemokines. Supernatants collected from untreated DC (1), LPS treated DC (2),
HPV16L1 VLP-exposed DC (3), and HPV16L1L2 VLP-exposed DC (4) were analyzed
in triplicate for the presence of cytokines and chemokines. Levels of cytokines and
chemokines were quantified using a human cytokine LINCOplex assay. These data are
expressed as the mean concentration ± SEM (**P < 0.01 ***P < 0.001 determined by a
two-tailed, unpaired t-test, as compared to untreated DC). The experiment was repeated
three times and yielded similar results.

LC increase migration when exposed to HPV16L1 VLP but not HPV16L1L2 VLP
In order to initiate an adaptive immune response, mature APC migrate to the
lymph node via the expression of CCR7, which binds to CCL21 (Saeki et al., 1999). To
assess the migratory capacity of LC incubated with either HPV16L1 VLP or
HPV16L1L2 VLP, we performed a transwell migration assay using CCL21. Exposure to
40
HPV16L1 VLP induced statistically significant increased LC migration towards CCL21
compared to that of untreated LC and LC exposed to HPV16L1L2 VLP (Fig. 12).

Figure 12. HPV16L1 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 treated as indicated in the activation assay, used in a migration
assay and analyzed in triplicate. (*P < 0.05 determined by a two-tailed, unpaired t-test, as
compared to LC exposed to HPV16L1 VLP). The mean number of migrating cells ±
SEM is presented. The experiment was repeated four times and yielded similar results.

LC exposed to HPV16L1L2 VLP fail to induce an HPV-specific CD8
+
T cell
response in contrast to the strong response induced by LC exposed to HPV16L1
VLP
During viral infections, APC take-up viral particles and subsequently process and
present viral peptides on MHC class I molecules to CD8
+
T cells through a process
known as cross presentation. Thus, we investigated whether LC exposed to HPV16L1
41
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. 13). These results are of major impact because they demonstrate that the
presence of L2 in the VLP silences the ability of LC to activate effector T cells thereby
crippling the HPV specific immune response.

Figure 13. LC exposed to HPV16 VLP induce differential activation of HPV16-
specific CD8
+
T cells. LC exposed to HPV16L1 VLP induce an HPV16L1-specific
CD8
+
T cells response yet LC exposed to HPV16L1L2 VLP do not. LC were treated as
indicated in the activation assay and used in an in vitro immunization assay. Responder
cells were analyzed for IFN-γ production in an ELISPOT assay against a L1 peptide. 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 < 0.05 determined by a
two-tailed, paired t-test, as compared to LC exposed to HPV16L1 VLP).
42

LC activate PI3K but downregulate Akt after exposure to HPV16L1L2 VLP but not
after exposure to HPV16L1 VLP
Furthermore, we examined if L2 plays a role in immune escape of HPV16
through deregulation of the PI3K pathway in LC, a mechanism implicated in our earlier
studies (Fausch et al., 2005). LC exposed to HPV16L1 VLP did not induce the activation,
i.e. phosphorylation of PI3K, while exposure to HPV16L1L2 VLP highly induced the
activation of PI3K in LC compared to that detected in untreated LC (Fig. 14). We also
demonstrate that HPV16L1L2 VLP downregulated Akt activation, as shown by a
decrease in the phosphorylation of Akt when compared to untreated LC, while HPV16L1
VLP maintained baseline levels of phosphorylated Akt (Fig. 14). Previously, we have
demonstrated that blocking PI3K activation during LC exposure to HPV16L1L2 VLP
allowed for LC maturation and the induction of an HPV16-specific CD8
+
T cell response,
indicating that PI3K activation by HPV16L1L2 VLP is an active immune evasion
mechanism (Fausch et al., 2005). This earlier study, combined with our current data,
suggest that the L2 protein’s mechanism of action is the deregulation of the PI3K-Akt
pathway, which leads to the suppression of LC maturation and therefore immune evasion.

43

Figure 14. 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 15 min. Cellular lysates were isolated and
subjected to western blot analysis. HPV16L1L2 VLP induce the activation of PI3K but
downregulation of p-Akt in LC while LC exposed to HPV16L1 VLP do not upregulate
PI3K activity and maintain a baseline level of p-Akt. One representative experiment of
three is shown.

Differential activation of LC exposed to ratios of HPV16L1 VLP to HPV16L1L2
VLP
To determine if the suppressing effects of HPV16L1L2 VLP are dominant over
the activating effects of HPV16L1 VLP, we exposed LC to different ratios of HPV16L1
VLP to HPV16L1L2 VLP and assessed the activation of LC. We demonstrate that
HPV16L1 VLP phenotypically (Fig. 15a) and functionally (Fig. 15b) activated LC in a
44
dose dependent manner in the presence of HPV16L1L2 VLP. As a control, we disrupted
the conformational structure of HPV16L1L2 VLP by boiling them for 10 minutes. We
exposed LC to a 1:1 ratio of HPV16L1 VLP to disrupted HPV16L1L2 VLP and
determined the activation status of LC. LC were similarly activated when exposed to
either a 1:1 ratio of VLP or a 1:1 ratio of VLP with heated HPV16L1L2 VLP, suggesting
that the conformational structure of L2 does not inhibit the activation of LC by HPV16L1
VLP. These data support our previous studies, which demonstrated that HPV16L1L2
VLP-exposed LC can be subsequently activated by many activating signals including
TLR agonists and CD40 ligand (CD40L) (Fausch et al., 2002; Fausch et al., 2003; Fahey
et al., 2009). We conclude that L2 is dominant within a HPV L1L2 VLP, however it
cannot inhibit the maturation of LC by an independent activation signal.
45
Figure 15. The suppressive effect of HPV16L1L2 VLP is not dominant over potent
activating signals. A. LC were treated as indicated in the activation assay and analyzed
by flow cytometry for the expression of CD86. Grey lines represent isotype matched
controls. One representative experiment of three is shown. B. Supernatants were collected
from each of the following treatments: untreated LC (1), LC treated with LPS (2), LC
exposed to HPV16L1 VLP (3), LC exposed to HPV16L1L2 VLP (4), LC exposed to 2:1
ratio of HPV16L1 VLP to HPV16L1L2 VLP (5), LC exposed to 1:1 ratio of HPV16L1
VLP to HPV16L1L2 VLP (6), LC exposed to 1:2 ratio of HPV16L1 VLP to HPV16L1L2
VLP (7), and LC exposed to 1:1∆ ratio of HPV16L1 VLP to heated HPV16L1L2 VLP
(8). Supernatants were analyzed in triplicate for the presence of cytokines and
chemokines. Levels were quantified using a human cytokine LINCOplex assay. These
data are expressed as the mean concentration ± SD. The experiment was repeated three
times and yielded similar results.
46
Figure 15. Continued

47
Differential internalization of HPV16L1 VLP and HPV16L1L2 VLP in LC
The differences in LC activation and signaling led us to investigate whether there
is a difference in internalization of HPV16L1 VLP versus HPV16L1L2 VLP. We decided
to assess uptake at 15 min because we have previously demonstrated that LC readily
internalize VLP by 15 min (Fausch et al., 2002) and this is the time point in which a
difference in PI3K and Akt signaling is observed (Fausch et al., 2005). LC were exposed
to carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) labeled-HPV16 VLP for
15 min, then fixed with 2% paraformaldehyde following incubation and internalization
was assessed by flow cytometry. CFDA-SE attaches to proteins, via amines, and once
internalized it generates a fluorescent signal after cleavage by intracellular esterases that
is detectable by flow cytometry. Therefore, HPV16 VLP that have been internalized by
LC will fluoresce, while HPV16 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 (Fausch et al., 2002). We found that LC
internalize over twice as much HPV16L1L2 VLP compared to HPV16L1 VLP (Fig. 16),
suggesting that there exists a specific L2 receptor and HPV16L1L2 VLP internalization
pathway.

48

Figure 16. LC internalize HPV16L1L2 VLP twice as much as HPV16L1 VLP. LC
were incubated with either CFDA-SE/PBS control, CFDA-SE labeled-HPV16L1 VLP or
CFDA-SE labeled-HPV16L1L2 VLP for 15 min. Internalization was assessed by flow
cytometry. The percent uptake is noted in the upper right quadrant. One representative
experiment of three is shown.

Differential uptake pathways of HPV16L1 and HPV16L1L2 VLP in LC
We have demonstrated that the minor capsid protein L2 plays an important role in
the interaction of HPV and LC. Additionally, the presence of L2 in the VLP appears to
alter whether LC utilize clathrin-, caveolae-, or actin-mediated uptake pathways (Fausch
et al., 2003; Yan et al., 2004). Therefore, we sought to determine whether HPV16L1 VLP
and HPV16L1L2 VLP enter LC through similar or different cellular compartments. To
assess HPV uptake, we labeled each VLP with different fluorescent dyes and incubated
LC with both VLP simultaneously. We then visualized the uptake of the VLP in LC by
confocal microscopy at various time points. We show that while both HPV16L1 VLP and
HPV16L1L2 VLP co-localize at the LC surface, they enter and travel through the LC
cytoplasm in different compartments as demonstrated by the clear separation of
fluorescent dye labeled particles (Fig. 17). This finding suggests that a specific receptor
for the L2 protein may exist on LC.
49

Figure 17. HPV16L1 VLP and HPV16L1L2 VLP enter and travel through LC in
different cellular compartments. HPV16L1 VLP and HPV16L1L2 VLP were labeled
with different fluorescent dyes and incubated with LC various periods of time. At each
given time point, the cells were visualize with confocal microscopy. One representative
experiment of three is shown.

The HPV16 L2
108-120
peptide inhibits binding of HPV16L1L2 VLP to LC
Previously it was demonstrated that the N-terminus L2
108-120
(aa 108-120,
LVEETSFIDAGAP) region plays a critical role in mediating the binding of HPV16L1L2
VLP to COS and HeLa cells (Kawana et al., 2001). To determine whether this same
region facilitated attachment in LC, we incubated LC with increasing concentrations of
the L2
108-120
peptide and subsequently exposed the cells to HPV16L1L2 VLP. We then
50
assessed the amount of bound HPV16L1L2 VLP on the surface of LC with flow
cytometric analysis. We determined that increased concentrations of the L2
108-120
peptide
resulted in significantly decreased numbers of HPV16L1L2 VLP bound to the LC surface
(Fig. 18), suggesting that the N-terminus of L2 facilitates HPV16 binding to LC.

Figure 18. The HPV16L2
108-120
peptide inhibits binding of HPV16L1L2 VLP to LC.
LC were incubated with increasing concentrations of the L2
108-120
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. These data are expressed as
the mean of three separate experiments ± SD (*P < 0.05 as determined by a two-tailed,
unpaired t-test, as compared to untreated LC).

51
(6x)His-L2
108-120
binds to a specific LC surface protein
Next, we wanted to identify which cell surface protein(s) the L2
108-120
peptide was
binding to on LC and blocking 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 membrane impermeable cross-linking agent, 3,3’-Dithiobis-
(sulfosuccinimidylpropionate) (DTSSP). After cross-linking the L2
108-120
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 eluted over 10 fractions. Each fraction was left non-reduced to maintain the
covalent bond between the L2
108-120
and cell surface protein(s) and was separated by
electrophoresis and subsequently probed using a monoclonal antibody specific for
polyhistidine. Consistently, a unique band was observed just above 39 kDa in the
fractions that were incubated with the (6x)His-L2
108-120
peptide. The specific band began
to appear in fraction 5 and was fully eluted by fraction 6 (Fig. 19). Notably, this unique
band is not present in the negative control with no peptide.

52

Figure 19. Polyhistidine immunoblot analysis of eluates isolated from an L2
108-120

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 (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. One
representative experiment of three is shown.

Subsequently, eluates 5 and 6 that contain the unique band were reduced,
separated by electrophoresis and silver stained. As observed before, a distinct band was
observed by silver stain just above 39 kDa (Fig 20). 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. The unique band at 39 kDa 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 (ANXA2).

53

Figure 20. Silver stain analysis of eluates isolated from an L2
108-120
pulldown assay.
LC were incubated with either no peptide or (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 ~39 kDa 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 ANXA2 is highly likely to interact with the L2
108-120
peptide as
identified by mass spectrometry, we wanted to confirm the presence of ANXA2 in our
L2
108-120
peptide pulldown eluates. Through immunoblot analysis, we found that ANXA2
was only present in L2
108-120
peptide pulldown eluates and not in our negative control
eluates (Fig. 21). Additionally, ANXA2 was found primarily in eluates 5 and 6, which
corresponds to the same elution fractions that the (6x)His-L2
108-120
peptide was present in
our anti-His immunoblot (Fig. 19). Moreover, the anti-ANXA2 immunoblots
demonstrated that ANXA2 is located at the same molecular weight as the observed band
54
in the anti-polyhistidine immunoblot and our silver stained gel. Thus, these findings
confirm that ANXA2 is interacting with the L2
108-120
peptide on the surface of LC.

Figure 21. ANXA2 immunoblot analysis of eluates isolated from an L2
108-120

pulldown assay. LC were incubated with either no peptide or (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 then
electrophoresed, transferred to nitrocellulose and probed with an anti-annexin A2
antibody. One representative experiment of two is shown.

Secretory leukocyte protease inhibitor blocks the uptake of HPV16L1L2 VLP by
LC
It has previously been demonstrated that secretory leukocyte protease inhibitor
(SLPI) interacts with ANXA2, inhibiting the infection of macrophages by HIV-1 (Ma et
al., 2004). Therefore, we used SLPI to investigate whether ANXA2 functions in the
internalization of HPV16L1L2 VLP by LC. LC were pretreated with increasing
concentrations of SLPI and then exposed to CFDA-SE labeled-HPV16L1L2 VLP.
Following the incubation, we found that as LC were exposed to increasing concentrations
of SLPI, LC internalized decreasing amounts of HPV16L1L2 VLP (Fig. 22).

55

Figure 22. SLPI inhibits the uptake of HPV16L1L2 VLP by LC. LC were incubated
with increasing concentrations of SLPI and then incubated with CFDA-SE labeled
HPV16L1L2 VLP. Uptake of CFDA-SE labeled HPV16L1L2 VLP by LC was assessed
by flow cytometry. The mean percentage of uptake ± SEM of three separate experiments
is presented (*P < 0.05 by a two-tailed, paired t-test, as compared to the negative
control).

We next sought to confirm that ANXA2 is a specific receptor for the L2 protein.
To do so, LC were pretreated with the optimal SLPI concentrations determined from the
previous experiment and subsequently exposed to CFDA-SE labeled-HPV16L1 VLP.
Notably, untreated LC and SLPI treated LC internalized similar amounts of HPV16L1
VLP (Fig 23), indicating that SLPI did not inhibit HPV16L1 VLP uptake. Taken
together, these results strongly suggest that ANXA2 interacts with the L2 protein and is
critically involved with the internalization of HPV16L1L2 VLP by LC.
56

Figure 23. SLPI does not inhibit the uptake of HPV16L1 VLP by LC. LC were
incubated with SLPI and then incubated with CFDA-SE labeled HPV16L1 VLP. Uptake
of CFDA-SE labeled HPV16 VLP by LC was assessed by flow cytometry. The mean
percentage uptake ± SEM of three separate experiments is presented.

siRNA mediated knockdown of ANXA2 in LC inhibits HPV16L1L2 VLP uptake
To confirm the role of ANXA2 in HPV uptake in LC, we knocked-down the
expression of ANXA2 in LC using small interfering (si)RNA. We transfected fully
differentiated LC with either no siRNA (untreated), control siRNA that does not
knockdown any proteins or siRNA targeting ANXA2. We determined that optimal
protein knockdown occurred 6 days post transfection. As shown, we were able to achieve
significant reduction in the expression of ANXA2 in LC treated with ANXA2 siRNA
compared to both untreated and control siRNA treated LC (Fig. 24).
57

Figure 24. Immunoblot demonstrating siRNA-mediated knockdown of ANXA2 in
LC. LC were transfected using the Amaxa Nucleofector kit without siRNA (Untreated),
with control siRNA or with ANXA2 siRNA. The cells were incubated for 6 days before
analysis of ANXA2 protein expression by immunoblot. GAPDH served as the loading
control. One representative experiment of three is shown.

To determine the effect of ANXA2 knockdown on HPV16L1L2 VLP uptake in
LC, LC were treated with siRNA and exposed to CFDA-SE labeled-HPV16L1L2 VLP.
Specific knockdown of ANXA2 in LC significantly reduced the uptake of HPV16L1L2
VLP into LC compared to both untreated LC and control siRNA treated LC (Fig. 25a).

58
Figure 25. Downregulation of ANXA2 inhibits uptake of HPV16L1L2 VLP but not
HPV16L1 VLP. LC were transfected using the Amaxa Nucleofector kit without siRNA
(Untreated), with control siRNA or with ANXA2 siRNA. A. The cells were incubated for
6 days and exposed to CFDA-SE labeled HPV16L1L2 VLP for 15 min. These data are
expressed as the mean of four separate experiments ± SD (*P < 0.05 as determined by a
two-tailed, paired t-test, as compared to untreated LC). B. The cells were incubated for 6
days and exposed to CFDA-SE labeled HPV16L1 VLP for 15 min. Uptake was assessed
by flow cytometry. The percentage of uptake of one experiment is presented.
59
Figure 25. Continued.

60
To further confirm that ANXA2 is a specific receptor for the L2 protein, LC were
treated with siRNA as described and subsequently exposed to CFDA-SE labeled-
HPV16L1 VLP. Notably, uptake of HPV16L1 VLP was equivalent in LC left untreated,
treated with control siRNA or treated with ANXA2-specific siRNA (Fig. 25b), indicating
that reduction in ANXA2 expression did not inhibit HPV16L1 VLP uptake. Importantly,
not only does this demonstrate that ANXA2 is a specific receptor for the L2 protein, but
it also acts as a control for ANXA2 knockdown. Because ANXA2 is involved in
endocytosis, we wanted to determine whether knockdown would cause a global decrease
in particle uptake or whether the decrease in HPV16L1L2 VLP was specific. Since
HPV16L1 VLP was not affected by ANXA2 knockdown, we can conclude that the
observed decrease in HPV16L1L2 VLP uptake was a specific result of the interaction
between ANXA2 and the L2 protein. Taken together, these results indicate that the
ANXA2 heterotetramer interacts with the L2 protein and is critically involved with the
internalization of HPV16L1L2 VLP by LC.
61
Chapter 3: Discussion and Future Directions

HPV has evolved to evade human immune detection in multiple ways in order to
establish an infection and maintain a persistent lifecycle within a hostile, anti-viral
environment (Kanodia et al., 2007). Persistence of an HPV infection is the greatest risk
factor in the development of cervical cancer (Schlecht et al., 2001). Currently, patients
with advanced cervical cancer have poor prognoses despite advances in conventional
therapies, which consist of radical hysterectomy in combination with chemo- or
radiotherapy (Chuang et al., 2009). While the morbidity and mortality of cervical cancer
can be reduced with effective Papanicolaou (Pap) smear screening, early detection and
treatment, none of these are readily available in developing countries, where cervical
cancer remains the second leading cause of cancer-related deaths among women (Parkin,
2006). Significantly, the burden of cervical cancer is expected to increase dramatically in
the coming decades due to changing demographics (Parkin et al., 2008). Therefore, the
development of innovative therapeutics to treat HPV-induced lesions and cancers remains
a critical global health priority. Further understanding of HPV immune escape
mechanisms will assist in identifying novel molecular targets that can be altered or
blocked in order to enhance the therapeutic efficacy of current treatments.
Previously, we have demonstrated that HPV inhibits the maturation of LC, the
APC at the site of infection, and therefore inhibits the induction of an HPV-specific
immune response (Fausch et al., 2002; Fausch et al., 2003). In the current studies, we
aimed to elucidate the mechanism of how HPV manipulates LC. By comparing the
effects of HPV16L1 VLP and HPV16L1L2 VLP on LC, we investigated the role of the
62
minor capsid protein L2 in the induction of immune escape. We demonstrate that
HPV16L1 VLP induce LC maturation as shown through the upregulation 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 induce LC maturation but instead suppress the
generation of an effective HPV-specific immune response via the deregulation of the
PI3K-Akt pathway in LC. Collectively, our results strongly suggest a novel role for L2 in
the initiation of HPV16 immune escape through LC. Furthermore, we demonstrate that a
specific, highly evolutionarily conserved region of the L2 protein binds directly to
ANXA2 on the LC surface. This specific interaction mediates the internalization of
HPV16 into LC and can be inhibited using a known ANXA2 ligand or downregulation of
ANXA2 protein expression. ANXA2 now represents a novel HPV L2 receptor and
uptake pathway that has broad implications for HPV infection and immunity. These
studies are highly significant because they further defined a novel immune evasion
mechanism of HPV and identified clinically relevant targets for HPV treatment.
It is important to understand that the findings presented here are based upon a
model system that mimics the interaction between HPV and LC in the human epidermis.
One potential criticism is that these studies were not conducted using human LC isolated
from mucosal epidermal sheets. However, the mere process of isolating human LC from
epidermal sheets induces the maturation of LC (Klechevsky et al., 2008), thus making it
difficult to conduct studies such as these with activation status as an endpoint. The
utilization of monocyte-derived LC in these studies is an appropriate alternative model
because they express MHC class II molecules, langerin, E-cadherin, CD1a and Birbeck
63
granules [Fig. 8, (Fausch et al., 2002)], all of which classically define human LC located
in the epidermis (Merad et al., 2008). It has been debated whether LC are the only APC
in the epithelium that express langerin. In mice, it was demonstrated that dermal
langerin
+
DC exist and may play a role in skin immune surveillance (Ginhoux et al.,
2007; Poulin et al., 2007). In humans however, while two different subsets of dermal DC
exist, neither of these express langerin, highlighting the difference between human and
murine APC populations in the epithelium (Klechevsky et al., 2008). Another potential
criticism is that true live HPV virions were not used to carry out these studies, but instead
HPV VLP were used. There is significant limitations in producing large quantities of live
virions in vitro, and therefore HPV VLP have been developed as an alternative for
structural and immunological analysis. VLP have been utilized in countless studies and
are accepted as valuable models of HPV. 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 HPV and human LC.
Our results clearly demonstrate that the presence of L2 in the capsid not only
leads to increased uptake but also mediates the induction of HPV immune evasion
through the suppression of LC. It was previously demonstrated that LC exposed to
HPV6bL1 VLP are activated as assessed by the generation of effector CD8
+
T cells (Yan
et al., 2004). This activation of LC by the HPV6bL1 VLP is likely due to the lack of the
L2 protein. Notably, HPV6b 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
64
across all genotypes or whether it is a genotype specific response. While the majority of
studies investigating immune escape mechanism focus on high-risk HPV types, immune
escape mechanisms have been defined for low-risk types as well. Similar to high-risk
HPV types, low-risk HPV6 and HPV11 have been shown to negatively modulate antigen
presentation (Georgopoulos et al., 2000; Vambutas et al., 2001) and suppress immune
modulating chemokines (Guess et al., 2005), aiding in viral persistence. However, despite
similarities in immune escape mechanisms between low-risk and high-risk types, there
appears to be particular mechanisms that are high-risk type specific. For example, while
HPV16 and HPV18 have evolved intricate mechanisms to suppress the action of
interferons (Barnard et al., 1999; Park et al., 2000), thus far this has yet to be
demonstrated in low-risk types (Schneider et al., 1987). Therefore, one interesting future
direction will be to determine whether apart from HPV16, other high-risk, low-risk and
wart type HPV also suppress LC maturation via an L2 mediated mechanism. This
knowledge is important as it can lead to generalized treatments for infections and lesions
caused by the many different types of HPV. Considering that our results presented here
demonstrate that the L2 protein mediates the suppression of LC maturation and that the
N-terminus of L2 is highly conserved across mucosal genotypes (Kawana et al., 2001;
Gambhira et al., 2007), it is plausible that both high-risk and low-risk mucosal HPV types
will manipulate LC in a similar fashion.
Previously, it was demonstrated that HPV-specific CD4
+
Tregs have been
identified in CIN and cervical cancer patients and that 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). CD4
+
Tregs play an important role in
65
immune homeostasis (Mills, 2004), as well as the curtailing of effective anti-tumor
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,
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 and biopsies from CIN lesions and cervical cancer (Adurthi et al., 2008;
Fattorossi et al., 2004; Molling et al., 2007; van der Burg et al., 2007; Visser et al., 2007).
Interestingly, CD4
+
Treg frequencies were found to be significantly increased in women
with persistent HPV16 infection (Molling et al., 2007). This increase was found in
patients who had detectable HPV16 E7-specific T-helper cells responses, suggesting that
HPV16-specific Tregs are generated in concert with HPV16-specific T effector cells
(Molling et al., 2007). Collectively, these studies suggest that CD4
+
Tregs are playing a
role in suppressing the immune response mounted against high-risk HPV.
Currently, it is unclear which cell(s) is responsible for the induction of HPV-
specific CD4
+
Tregs generated in CIN and cervical cancer patients. It has been
demonstrated that APC displaying peptides in the absence of both co-stimulation and pro-
inflammatory cytokines have the ability to both anergize T cells (Tan et al., 1993) and
generate Tregs (Jonuleit et al., 2000; Dhodapkar et al., 2001). In this current study, we
66
found that LC exposed to HPV16L1L2 VLP possess a sub-optimal phenotype. By
silencing maturation but continuing to present peptides, LC exposed to HPV16L1L2 VLP
are likely to become tolerizing APC that possess the ability to induce HPV16-specific
anergic T cells and/or Tregs. This L2 mediated immune escape mechanism allows the
virus to remain infectious by selectively eliminating beneficial T cells and actively
suppressing HPV16-specific immune responses. Future studies should address whether
CD4
+
HPV-specific T cells responses induced by LC exposed to HPV are regulatory,
effector or unresponsive T cells. Furthermore, it was demonstrated in the present study
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
CD8
+
T cells are tolerized by LC, becoming either anergic or regulatory in nature.
Our data indicate that the L2 protein dominates the interaction between
HPV16L1L2 VLP and LC, likely by preferentially binding to a specific L2 receptor and
initiating a L2 mediated signaling cascade, which leads to immune evasion. Our results
further suggest that, in the absence of L2, HPV16L1 VLP enter LC through a secondary
activating pathway. However, in a natural infection, HPV16L1 virions do not exist and
therefore only the effects of HPV16L1L2 virions on LC are physiologically relevant. Due
to the close interaction of the L1 and L2 proteins within the HPV capsid, we cannot rule
out the possibility that L1 and L2 together are mediating the immune evasion. Upon
binding of HPV to the cell surface, there may be a conformational change in the capsid
that exposes regions of L1 and/or L2 that lead to the suppression of LC activation.
Nonetheless, this study guides the field towards the use of HPV16L1L2 VLP when
examining the interaction between HPV16 and host cells.
67
Research regarding the uptake of HPV by human LC has been at times
contradictory. We previously demonstrated that the mode of uptake of HPV16L1L2 VLP
by LC is clathrin- and caveolae-independent (Fausch et al., 2003). In a study by Yan et
al., LC were shown to internalize 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 different conclusions, they are likely due to the use of HPV L1 VLP
versus HPV L1L2 VLP. Our identification of a specific L2-mediated immune escape
mechanism along with these studies indicate the possible presence of a specific L2
receptor and uptake mechanism. The concept of a specific L2 receptor is supported by
several studies examining L2 mediated infectivity (Kawana et al., 2001; Yang, Day, et
al., 2003). Kawana et al., demonstrated that pre-incubation of COS-1 cells with the
HPV16 L2 peptide aa 108-120 decreased infectivity of HPV16 pseudovirions (Kawana et
al., 2001). Additionally, Yang et al., suggested that HPV16L1L2 VLP binding to the cell
surface of HeLa cells causes aa 13-31 of the L2 protein to be displayed on the virion
surface, interact with a secondary receptor and facilitate infection (Yang, Day, et al.,
2003). The existence of an L2-specific receptor becomes highly conceivable when these
studies are viewed in the context of our results.
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 amino acid region of L2 108-120 has
been shown to be vital in the binding and infectivity of many cell types (Kawana et al.,
68
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 pulldown assays that HPV16
L2
108-120
is critical in the binding of HPV16 to LC and specifically interacts with the
ANXA2 heterotetramer on the surface of LC. Through uptake assays, we show that the
internalization of HPV16L1L2 VLP by LC is mediated by ANXA2 heterotetramers. The
uptake of HPV16 can be inhibited by either SLPI, a natural ligand of ANXA2, or siRNA
mediated knockdown of ANXA2 in LC. Our data strongly suggests that the ANXA2
heterotetramer is the uptake receptor for HPV16 on LC.
ANXA2 is a member of the annexin family of ~13 proteins. Annexins are Ca
2+

and phospholipid binding proteins that constitute an evolutionarily conserved multi-gene
family with expression throughout animal and plant kingdoms. Structurally, annexins
have a tightly packed, highly α-helical protein core domain, which serves as the Ca
2+
,
membrane and F-actin binding domain (Gerke and Moss, 2002). The second principle
domain on annexins is the N-terminus, which is unique to each member of the annexin
family and specifies individual properties in vivo. Structurally, annexin proteins have a
slight curvature and two opposing sides. The more convex side faces the membrane and
contains novel types of Ca
2+
binding sites (Weng et al., 1993). The more concave side
faces away from the membrane and contains both the C-terminus and N-terminus,
therefore the N-terminal domain is accessible for interactions with binding partners
(Swairjo and Seaton, 1994).
ANXA2 is found as both a 36 kDa monomer and a 90 kDa heterotetramer, which
consists of two ANXA2 monomers bridged non-covalently by a ANXA2 light chain
dimer (Glenney, 1986). ANXA2 light chain, also known as p11 or S100A10 light chain,
69
is a member of the S100 family of proteins (Rescher and Gerke, 2008). The
heterotetramer complex is the predominant form in most cells (Waisman, 1995).
Interestingly, ANXA2 light chain expression appears to be dependent upon the presence
of the ANXA2 protein (Puisieux et al., 1996; Zhang et al., 2010). Notably, it has been
demonstrated that LC express S100A10 (Rust et al., 2006). ANXA2 is found in the
cytoplasm and attached to the plasma membrane both intracellularly and extracellularly
(Waisman, 1995; Gerke et al., 2005; Deora et al., 2004).
The N-terminus of ANXA2 contains two important regulatory domains – the L
and P domains. The first 14 residues of the N-terminus make up the L domain and
contain the high-affinity binding site for the S100A10 protein (Waisman, 1995). The P
domain of ANXA2 contains the phosphorylation sites for PKC (Ser
25
) and pp60
src

(Tyr
23
). Phosphorylation of Ser
25
by PKC has been shown to regulate nuclear entry,
exocytosis, DNA synthesis and cell proliferation (Luo et al., 2008; Sarafian et al., 1991;
Liu et al., 2003). Phosphorylation of Tyr
23
by pp60
src
has been shown to regulate
translocation to the cell surface, binding to endosomes and endosomal transport (Morel
and Gruenberg, 2009; Deora et al., 2004). Translocation of ANXA2 from the inside of
the cell to the cell surface is dependent on both S100A10 and Tyr
23
phosphorylation
(Deora et al., 2004).
The ANXA2 heterotetramer has been proposed to function in exocytosis,
endocytosis, cell adhesion, membrane fusion and membrane trafficking (Gerke et al.,
2005). Extracellular ANXA2 functions as a receptor for heparin, plasminogen, tissue
plasminogen activator, plasmin and SLPI on endothelial cells, macrophages and human
peripheral monocytes (Kassam et al., 1997; Kassam et al., 1998; Falcone et al., 2001;
70
Hajjar et al., 1994; Laumonnier et al., 2006; Ma et al., 2004). Plasmin has been shown to
modulate the immune responses of monocytes upon binding, specifically releasing lipid
immune-mediators, initiating chemotaxis and leading to the expression of TNF-α, IL-1,
MCP-1 and CD40 (Weide et al., 1996; Syrovets et al., 1997; Syrovets et al., 2001;
Burysek et al., 2002). Taken together, these studies suggest that ANXA2 located on
immune cells can modulate immune responses.
Interestingly, ANXA2 plays a role in the binding and uptake of a variety of
different viruses. The first virus associated with ANXA2 was human cytomegalovirus
(CMV) (Wright et al., 1994). ANXA2 was isolated after a CMV pulldown assay and was
subsequently shown to bind directly to CMV virions (Wright et al., 1994). In a later
study, it was demonstrated that ANXA2 had the capacity to enhance CMV-membrane
fusion by functioning as a bridge between CMV and a phospholipid membrane,
suggesting that ANXA2 may regulate the fusion of CMV with the plasma membrane
(Raynor et al., 1999). In a recent study, ANXA2, specifically the tetramer form with
ANXA2 light chain, was shown to enhance CMV infection (Derry et al., 2007). In
addition to CMV, it was shown that ANXA2 is a receptor for respiratory syncytial virus
(RSV) and that this binding can be inhibited by an antagonist, suggesting a potential for
inhibitors of ANXA2 as treatments for RSV infections (Malhotra et al., 2003).
Furthermore, ANXA2 was shown to be a cofactor for human immunodeficiency virus
(HIV) on macrophages, which could be disrupted by using SLPI or other ANXA2
specific inhibitors, again suggesting ANXA2 as a potential therapeutic target (Ma et al.,
2004). Most recently, ANXA2 was shown to play a role in rabbit vesivirus attachment
71
and internalization (Gonzalez-Reyes et al., 2009). Until now, ANXA2 has never been
associated with HPV as a receptor.
Notably, in normal epithelium, ANXA2 expression is confined to the basal and
suprabasal cells and the protein cellular location is consistently observed at the cell
membrane in these cells (Pena-Alonso et al., 2008). More specifically, an
immunohistochemical study investigating the differential expression of annexins found
that ANXA2 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). Furthermore, ANXA2 is
abundantly expressed in proliferating cells but not quiescent cells (Frohlich et al., 1990).
These observations are critical because they demonstrate that the expression of ANXA2
mirrors the tropism of HPV infection in vivo, making the ANXA2 heterotetramer a strong
HPV candidate receptor not only on LC but also on basal cells in the epithelium, 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 HPV through the ANXA2
heterotetramer.
The results presented here strongly suggest that the ANXA2 heterotetramer is an
uptake receptor for HPV on LC. We have demonstrated through a variety of methods that
the ANXA2 heterotetramer mediates the internalization of HPV16 and that this
internalization is specific for the presence of the L2 protein. In the context of the current
HPV literature, we propose a putative model of HPV uptake that unifies a seemingly
disparate and fractured field of research (Fig. 26). Many researchers have laid claims on
discovering “the” HPV receptor over the past several decades, often time downplaying
the role of other groups and their respective discoveries. However, we believe that our
72
model of HPV uptake fits neatly within the current body of HPV receptor knowledge,
synergistically incorporating varied observations into a cohesive concept. The initial
binding of HPV to either the ECM or cell surface follows the sequence outlined and
detailed previously in Figure 5. Now, with the inclusion of the ANXA2 heterotetramer,
we can expand on the previously introduced model. Interestingly, the binding of HPV to
α6β1/4 integrins can be linked to the recruitment of ANXA2 heterotetramer to the cell
membrane and translocation to the cell surface. Binding and clustering of α6β1/4
integrins cause the recruitment of integrin-associated proteins such as talin and facilitate
the activation of focal adhesion kinase (FAK) (Mitra and Schlaepfer, 2006).
Independently, two groups demonstrated that talin directly interacts with
phosphatidylinositol 4-phosphate, 5-kinase (PIP5K), which causes PIP5K activation
(Ling et al., 2002; Di Paolo et al., 2002). PIP5K is a kinase that catalyzes the focal
production of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P
2
], which is a well-known
substrate for PI3K but also a second messenger on its own (van den Bout and Divecha,
2009). Interestingly, it was demonstrated that PI(4,5)P
2
actively recruits ANXA2
heterotetramers, along with F-actin, to specific regions of the cell membrane (Rescher et
al., 2004; Hayes et al., 2009). Therefore, the binding of HPV to α6β1/4 integrin can
cause a focal increase of PI(4,5)P
2
at the cell membrane where the HPV virion is bound,
which recruits ANXA2 heterotetramers to the site of HPV-integrin binding. As
mentioned above, the other consequence of integrin binding is the activation of FAK.
Integrin-stimulated FAK phosphorylation at Tyr
397
creates a high-affinity binding site for
the Src-homology 2 (SH2) domain of src-family kinases (SFK) (Schlaepfer et al., 2004;
Mitra et al., 2006). The binding of SFK to FAK leads to the conformational activation of
73
SFK (Schlaepfer et al., 2004). Importantly, it was shown that SFK regulate the
translocation of the ANXA2 heterotetramer to the cell surface through the
phosphorylation of ANXA2 at Tyr
23
both in vitro and in vivo (Deora et al., 2004). Now,
we can begin to see a signal cascade in which the binding of HPV to the cell surface
through a primary receptor complex that includes α6β1/4 integrin leads to the local
recruitment and subsequent translocation of the ANXA2 heterotetramer to the cell
surface. After undergoing conformational changes and furin cleavage, the HPV capsid’s
affinity for the primary receptor complex decreases and the affinity for the ANXA2
heterotetramer increases, leading to a hand off of the virion to the locally recruited
ANXA2 heterotetramer. Upon HPV binding to the ANXA2 heterotetramer, perhaps a
second signaling cascade is triggered that initiates clathrin- and caveolae-independent
internalization of HPV and the ANXA2 heterotetramer into an endosomal compartment.
Subsequent to internalization, the HPV virion proceeds along through the early endosome
to the late endosome and lysosome, where decreasing pH leads to viral uncoating
(Horvath et al., 2010). Although it is known that the L2 protein mediates viral egress
from the endosome, the mechanism by which this process occurs is not known (Kämper
et al., 2006). We propose one hypothetical method involving the ANXA2 heterotetramer
in our putative HPV uptake model (Fig. 26). It has been demonstrated that at low pH,
annexins bound to membranes can undergo a major conformational change and adopt a
transmembrane configuration (Langen et al., 1998). At high enough concentrations, this
transmembrane form of annexin can actually disrupt membranes and literally dissolve
vesicles (R. Langen, personal communication). If the internalization of HPV caused a
high concentration of ANXA2 molecules to accumulate within an endosome, the
74
decreased pH of the late endosome or lysosome could produce a conformational change
in ANXA2 that would cause ANXA2 to become transmembrane and rupture the vesicle,
thereby releasing the HPV DNA and associated L2 protein into the cytosol. From there,
both the HPV DNA and L2 protein enter the host cell nucleus through an unknown
mechanism.
75
Figure 26. Unified theory of HPV uptake. HPV binds to the host cell surface through
HSPG, CyPB, α6β1/4 integrins and/or tetraspanins (CD63, CD151), either singularly or
in complex. The binding of HPV to the cell surface sets off several signaling cascades
that lead to alterations in host cell function and facilitate virion internalization. The
binding of integrins by HPV recruits integrin-associated proteins such as talin and
activates FAK. Talin activates PIP5K, which synthesizes the second messenger PI(4,5)P
2

locally. This local accumulation of PI(4,5)P
2
has been shown to recruit ANXA2
heterotetramers to the membrane. The activation of FAK by integrin binding leads to the
activation of src-family kinases (SFK). Activated SFK phosphorylate Tyr
23
on ANXA2
heterotetramers, causing them to translocate to the cell surface. This proposed
mechanisms allows for ANXA2 heterotetramers to appear focally at the cell surface at
the site of HPV binding. After HPV interacts with the host cell primary receptor(s), it
undergoes a conformational change and subsequent furin cleavage of the L2 protein. This
conformational change leads to a decrease in affinity for the primary receptor(s) and a
potential increase in affinity for the ANXA2 heterotetramer. Upon binding to the
ANXA2 heterotetramer, HPV is internalized through a clathrin-, caveolin-, dynamin-,
flotillin- and lipid-raft independent endosomal pathway. As the endosome transitions into
a late endosome/lysosome, decreasing pH causes viral uncoating and a proposed
conformational change in ANXA2 allowing it to become transmembrane. The
transmembrane form of ANXA2 has the ability to disrupt membranes and may facilitate
HPV egress from endosomes.
76
Figure 26. Continued.

We have previously demonstrated that HPV binding and internalization in LC is
accompanied by a PI3K-dependent cell-signaling cascade that mediates the immune
escape of HPV. As shown in Figure 6, we believe the early upregulation of PI3K may
lead to activation of PKC, which downregulates Akt, and PP2A, which downregulates
77
Akt, MAPK and NF-κB. Importantly, independent of our studies, the activation of both
PI3K and PKC were shown to be critical to the uptake of HPV (Mercer et al., 2010).
However the cell-surface molecule responsible for initiating these cell signaling cascades
is unknown.
Currently, there is experimental evidence suggesting that ANXA2 is involved in
initiating multiple signal transduction cascades. As mentioned above, ANXA2
heterotetramers are actively recruited to the plasma membrane by PI(4,5)P
2
(Hayes et al.,
2009; Rescher et al., 2004). Although PI(4,5)P
2
acts as a second messenger on its own to
recruit ANXA2 heterotetramers to the cell surface, it is more commonly viewed as a
substrate for either PI3K, which produces phosphatidylinositol (3,4,5)-triphosphate
[PI(3,4,5)P
3
], or phospholipase C (PLC), which produces inositol (1,4,5)-triphosphate
[Ins(1,4,5)P
3
] and diacylglycerol (DAG) (van den Bout et al., 2009). Therefore, it is
possible that ANXA2 is associated with PI3K transduction cascades. Furthermore, the
binding of plasmin, an ANXA2 ligand, to monocytes causes the activation of NF-κB,
p38K and janus kinase (JAK)/signal transduction and transcription (STAT) signaling
cascades (Burysek et al., 2002; Syrovets et al., 2001). This suggests that ANXA2 may
also mediate the induction of these signaling pathways. Additionally, it was demonstrated
that ANXA2 could be dissociated and cleaved, and that this truncated form of ANXA2 is
responsible for initiating downstream signaling (Laumonnier et al., 2006). As mentioned
above, ANXA2 can be phosphorylated at Ser
25
and Tyr
23
, targets of PKC and pp60
src

respectively (Deora et al., 2004; Glenney, 1986; Gould et al., 1986), suggesting that it
plays a role in the signaling cascades of both PKC and pp60
src
. Finally, it was shown that
ANXA2 mediates endothelial cell activation via an interaction between cell surface
78
ANXA2 and β2-glycoprotein I, which leads to the induction of NF-κB (Zhang and
McCrae, 2005). When taken together, these studies suggest that ANXA2 possess the
ability to initiate signal transduction cascades. However, because ANXA2 specific
signaling cascades have not been fully elucidated, we must further evaluate the
downstream signaling consequences of HPV binding to the ANXA2 heterotetramer and
whether these correspond to the signaling cascades we have previously identified in LC
upon HPV binding.
Another potential mediator of signal transduction upon HPV binding is α6β1/4
integrin. Fothergill et al. demonstrated that the interaction of HPV L1 or L1L2 VLP with
α6β4 integrin in A431 cells (human epidermoid carcinoma) caused PI3K activation
(Fothergill et al., 2006). However our current study demonstrates that the early activation
of PI3K in primary human LC requires the presence of the L2 protein. These conflicting
results suggest that HPV may induce cell type specific signaling cascades. Clearly more
research into the cell-surface molecule(s) that mediates PI3K activation upon HPV
binding is needed.
We demonstrate in our results that SLPI, a ligand of ANXA2, can inhibit the
internalization of HPV. SLPI was originally identified as a serine protease inhibitor in
mucosal fluids (Hochstrasser et al., 1972; Ohlsson and Tegner, 1976). More recently,
however, SLPI has been shown to be associated with multiple functions, including host
defense. Epithelial and myeloid cells constitutively secrete SLPI, and cellular production
can be significantly upregulated in response to various stimuli (Williams et al., 2006).
SLPI has shown anti-bacterial activity against both gram-negative and gram-positive
bacteria (Hiemstra et al., 1996; Wiedow et al., 1998), in addition to activity against
79
several fungi, including Aspergillus fumigatus and Candida albicans (Tomee et al.,
1997). Furthermore, SLPI possesses the ability to modulate innate immune responses,
even acting as an anti-inflammatory in atherosclerosis, myocardial infarction, lung
emphysema and arthritis (Song et al., 1999; Williams et al., 2006). More specifically,
SLPI was shown to regulate the threshold for mucosal DC activation, therefore mediating
the ensuing antigen-specific immune response (Samsom et al., 2007). SLPI is also
upregulated during and plays a critical role in the optimal resolution of cutaneous wound
healing (Ashcroft et al., 2000). Finally, SLPI was identified as a selective HIV-1
inhibiting agent in human saliva and vaginal fluids (McNeely et al., 1997; Pillay et al.,
2001). In fact, increased concentrations of SLPI in vaginal fluids are associated with
reduced rates of perinatal HIV-1 transmission (Pillay et al., 2001). This anti-retroviral
defense appears to be mediated by the interference of virus entry through SLPI binding to
ANXA2 and disrupting HIV-1 from interacting with the host cell surface (Ma et al.,
2004).
Interestingly, it was recently demonstrated that exposure of human cervical
epithelial cells to herpes simplex virus (HSV) type-1 or HSV-2, but not HIV or vesicular
stomatitis virus, resulted in a significant and sustained reduction in SLPI levels (Fakioglu
et al., 2008). This result has potentially significant implications on HPV infections.
Historically, in the 1960s and 1970s, HSV-2 infection was first thought to be a possible
causative agent for cervical cancer (Rawls et al., 1968; Simon, 1976). However, the role
of HSV-2 in cervical cancer began to be questioned when HSV-2 DNA was not
consistently found in cervical cancer tissues (Park et al., 1983). Subsequently, HPV DNA
was detected definitively in the overwhelming majority of cervical cancer tissue and
80
determined to be the causative agent. Nonetheless, the link between HSV and HPV
persisted. It was proposed that HSV-2 might cause intracellular changes that promote
HPV induced lesions but that HSV-2 viral genes would not be required after a certain
stage (Galloway and McDougall, 1983). Later evidence from in vitro molecular data
suggested a possible synergy between the two infections (Jones, 1995). A more recent
study that performed a pooled analysis of seven case-control studies found that among
HPV DNA-positive women, HSV-2 seropositivity was associated with greater than twice
the risk of squamous cell carcinoma and greater than 3.3 times the risk of adeno- or
adenosquamous cell carcinoma (Smith et al., 2002). In the context of our results
demonstrating that SLPI may play a critical role in HPV infections by inhibiting virus
entry into host cells, the fact that HSV downregulates SLPI during infection may explain
the synergy between HSV and HPV. HSV infection may actually increase the likelihood
of HPV entry, infection and/or persistence by decreasing a mucosal inhibitor of the HPV
uptake receptor ANXA2. While further studies clearly need to be conducted, this is an
exciting and important connection that has the potential to cohesively link decades’ worth
of epidemiological data.
Very recently, our laboratory has begun a significant collaboration with Ralf
Langen (USC, Los Angeles, CA) on the interaction of HPV with the ANXA2
heterotetramer. A structural biologist, Dr. Langen has studied annexins for decades and
possesses an extremely unique insight and skill-set for studying protein-protein
interactions and membrane dynamics. This fledgling collaboration has already generated
some encouraging preliminary data and exciting future directions. Dr. Langen’s
laboratory has the capabilities to study protein structure and protein-protein interaction
81
using electron paramagnetic resonance (EPR), which is similar to nuclear magnetic
resonance (NMR) but it is electron spins that are excited instead of atomic nuclei spins.
With the benefit of sight directed mutagenesis of a protein of interest, EPR can describe a
variety of protein characteristics such as conformational secondary structure of a protein
around a particular amino acid, distance between amino acids within a protein, changes in
protein structure upon binding of a substrate or protein and the specific binding site of
proteins, among others. With additional techniques, EPR can determine if and how a
protein is embedded within a membrane. Utilizing this collaboration, we hope to further
explore the interaction between HPV and the ANXA2 heterotetramer, how this
interaction may result in internalization, what occurs between HPV and the ANXA2
heterotetramer after internalization, and if the ANXA2 heterotetramer plays a role in
HPV egress from endosomal compartments, as suggested above in our model for HPV
uptake. We anticipate detailing exactly where the HPV L2 protein binds to the ANXA2
heterotetramer, whether the interaction requires Ca
2+
or ANXA2 light chain, whether
ANXA2 interacts as a heterotetramer or monomer, whether furin cleavage of HPV
increases the affinity for the ANXA2 heterotetramer as proposed in our model, and what
conformational changes occur upon HPV binding to the ANXA2 heterotetramer.
Additional techniques within the Langen laboratory, including isothermal titration
calorimetry (ITC), will assist us in elucidating the dissociation constant (K
d
) and the
stoichiometry of the L2 and ANXA2 interaction. Clearly, this is only the beginning of a
long list of potential future directions that will no doubt evolve as the project moves
forward.
82
Along with some interesting EPR future directions, our collaboration has
highlighted some interesting potential future visualization techniques. Our laboratory is
planning on continuing the confocal microscopy, which demonstrated that HPV16L1 and
L1L2 VLP enter LC through separate uptake pathways (Fig. 17), by using live-cell
imaging techniques that would allow the visualization of particles being internalized via
time-lapse confocal microscopy. As a future direction, we could knockdown ANXA2 in
LC using siRNA and determine whether the divergent uptake pathways for HPV16L1
and L1L2 VLP still persist or whether both particles are internalized via the same
pathway in the absence of ANXA2. Furthermore, using immunoelectron microscopy, we
plan to label not only HPV VLP but also ANXA2 using antibodies and determine
whether the two molecules co-localize at various stages of internalization. This
information would allow us to determine the temporal nature of the interaction, the
physical association of the VLP and the ANXA2 heterotetramer inside an endocytic
vesicle and whether the ANXA2 heterotetramer remained associated with the endosome
as it progressed into a late endosome and lysosome. Finally, using their understanding of
annexin structure, function and conformational changes, the Langen laboratory has
developed several intriguing annexin-fluorochrome conjugates that emit photons only
when annexin is in a particular conformational state. For instance, an ANXA2-
fluorochrome conjugate can be created that only fluoresces when associated with the
membrane or only when in a transmembrane conformation. These tools could prove
extremely useful in studying the particular states in which ANXA2 interacts with HPV
and whether ANXA2 participates in other infectious processes such as viral egress from
endosomes. We envision introducing synthesized recombinant ANXA2-fluorochrome
83
conjugates to cells, allowing the ANXA2-fluorochrome conjugates to settle onto the cell
surface, and then challenging the cells with labeled HPV particles. We could then
simultaneously track HPV and the ANXA2 heterotetramer being internalized, noting the
appearance or disappearance of ANXA2 conformational states as internalization
progressed.
As a separate future direction, it has yet to be described whether LC can
transcribe and translate HPV encoded genes into proteins. The internalization of HPV by
LC may allow for the expression of HPV encoded proteins even if a productive infection
does not occur. Expression of HPV proteins by LC could further mediate the immune
escape of HPV, especially since many previously described HPV immune evasion
mechanisms involve the viral-encoded proteins E6 and E7. However, while HPV encodes
for E1, a DNA replication enzyme, and E2, a transcription factor, it does not encode for
any translational factors. Therefore the replication of the viral genome and protein
expression are both dependent on the host’s cellular machinery, a process that remains
poorly understood. Consequently, the expression of HPV encoded proteins by LC
depends on the location of the viral genome and the expression of transcription and
translation factors within LC. Whether the viral genome can translocate from the
cytoplasm to the nucleus in LC, whether LC express the necessary cellular machinery to
transcribe and translate HPV genes into proteins, and whether HPV gene expression
occurs in LC are all interesting questions that need to be addressed. This knowledge will
further elucidate the HPV mediated immune escape mechanism, advancing the field
towards optimal therapeutics for high-risk HPV-induced infections and lesions.
84
Finally, we need to further study the role of the ANXA2 heterotetramer in HPV
internalization in epithelial cells. While we demonstrate in this study that the ANXA2
heterotetramer plays a critical role in the uptake of HPV in LC, we are interested in
whether this ANXA2 heterotetramer mediated uptake can be generalized to the host cells
of productive HPV infection. To that end, we have already made significant progress in
developing a suitable model to test HPV infection. Over the past several months, we have
created lentiviral particles that express short hairpin (sh)RNA capable of integrating into
the host cell genome and stably knocking down ANXA2. These lentiviral particles also
provide us with a variety of useful research tools such as red fluorescent protein (RFP)
expression and puromycin resistance for selection. We have recently isolated clonal HeLa
epithelial cells that either express control shRNA and therefore have the same ANXA2
protein expression levels as untreated HeLa cells, or ANXA2 shRNA and therefore have
almost no ANXA2 protein expression. To detect HPV infection, we can use a
combination of techniques. In order to determine amount of viral particle uptake, we will
utilize the CFDA-SE labeled HPV VLP, as we have done in the current studies, because
we can quantify the amount of particles entering each cell. In order to determine whether
a productive infection has occurred, we will use HPV PsV, which are generated in
eukaryotic cells and enclose a reporter plasmid that expresses green fluorescent protein
(GFP). When internalized, if these HPV PsV enter the cell through an uptake mechanism
capable of causing a productive infection, the reporter plasmid will reach the nucleus and
the cell will begin expressing GFP. This is an “all or nothing” event, which is great for
determining whether a productive infection has occurred but not very useful when trying
to determine the number of virus particles entering a particular cell. Determining whether
85
ANXA2 plays a role in HPV uptake in epithelial cells is crucial to our understanding of
the HPV viral life cycle.
The findings of these studies are scientifically novel, broadly significant across
multiple disciplines and clinically relevant for both disease progression and treatment.
Our study on the minor capsid protein was the first to elucidate and describe the role of
L2 in mediating the HPV immune evasion mechanism that targets LC, which suppresses
their ability to induce adaptive immune responses. This novel function is the first immune
modulating action attributed to the L2 protein and adds significantly to our understanding
of the mechanism of HPV immune escape. Furthermore, we are the first to identify the
ANXA2 heterotetramer as the HPV16 L2 receptor on LC that is responsible for the
internalization of HPV. This result is highly significant because, until now, neither a
specific receptor for the HPV L2 protein nor an uptake receptor for HPV has been
identified. Furthermore, these data have broad implications because this ANXA2
heterotetramer mediated viral uptake pathway may represent a currently unknown type of
receptor mediated endocytosis, such as clathrin- or caveolae-mediated endocytosis.
Finally, these studies identify both the L2 protein and the ANXA2 heterotetramer as
potential therapeutic targets for the inhibition of HPV infection and therefore these
studies have future clinical implications on HPV therapy. Towards the fulfillment of our
long-term goal of understanding HPV in order to treat human disease, the studies
presented here further define the mechanisms underlying the establishment and
maintenance of a persistent HPV infection and identify critical therapeutic targets that
could be utilized to treat HPV-induced lesions, highlighting both the major impact and
clinical relevance of this work.
86
Chapter 4: Materials and Methods

Antibodies
The antibodies against conformational HPV16L1 epitopes (H16.V5 and H16.E70)
or linear HPV16L1 epitopes (Camvir-1, H16.D9 and H16.H5) were gifts from Neil
Christensen (Penn State, Hershey, PA), except Camvir-1 (BD Biosciences). Polyclonal
serum (DK44214) for HPV16L2 was a gift from John Schiller (National Institutes of
Health, Bethesda, MD). Additionally, the following antibodies were used in this study:
anti-CD1a-PE, CD80-FITC, CD86-FITC, HLA-DR, DQ, DP-FITC, isotype controls,
biotinylated anti-rabbit IgG, anti-mouse-IgG-HRP, streptavidin-PE, and streptavidin-
HRP (BD Biosciences); anti-CD207 (langerin) (Immunotech); anti-E-cadherin
(Millipore); anti-phosphorylated (p)-PI3K (Tyr 508), PI3K, p-Akt (Ser 473), Akt (Santa
Cruz Biotechnology); anti-GAPDH (Chemicon); goat anti-mouse-FITC and goat anti-
rabbit-HRP (Biosource); goat-anti-mouse-IR Dye 800 (Rockland), goat-anti-rabbit-Alexa
Fluor 680 (Molecular Probes); mouse-anti-annexin A2 (BD Biosciences) and anti-IFN-γ
and biotinylated anti-IFN-γ (Mabtech).

Donor Material
Peripheral blood leukocytes (PBL) from healthy donors were obtained by
leukapheresis. Leukocytes were purified using Lymphocyte Separation Media
(Mediatech) 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.
87
DC and LC Production
Frozen PBL were thawed, washed once with RPMI-1640, containing 2mM
Glutamax (GIBCO), 10 mM sodium pyruvate (GIBCO), 10mM non-essential amino
acids (GIBCO), 100 µg/ml Kanamycin (Sigma-Aldrich) and 10% FBS (Omega
Scientific) (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 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) 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-β1 (Biosource) of which 100% of rhu-
GM-CSF and rhu-IL-4 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-β1 was replenished on day 3 and
6.

VLP Production
Recombinant baculovirus stocks for HPV16L1 VLP and HPV16L1L2 VLP were a
gift from Dr. John Schiller (NIH, Bethesda, MD). All recombinant baculoviruses were
propagated in Sf9 insect cells (Da Silva et al., 2001). HPV16L1 VLP and HPV16L1L2
VLP 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
liter 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
88
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 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). 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 then tested by ELISA, western blot,
and electron microscopy for immunoreactivity and morphology. L1 protein was
quantified by gel code blue staining (Thermo Scientific) of proteins after sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) through 10% Bis-Tris gels
(Invitrogen) 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).
89

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 VLP (500 ng/well) were used to coat 96-well Maxisorp ELISA
plates (Nunc) 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 for BPV) 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) 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 or HPV16-L2 depending on the type of VLP being
generated. Blots were subsequently incubated with HRP-labeled anti- mouse or anti-
90
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
LC or DC were either left untreated, treated with 10 µg LPS (Sigma-Aldrich), 10
µg HPV16L1 VLP/10
6
cells or 10 µg HPV16L1L2 VLP/10
6
cells. For titration
experiments, LC were either left untreated, treated with 10 µg LPS, 10 µg HPV16L1
VLP/10
6
cells, 10 µg HPV16L1L2 VLP/10
6
cells, 6.6 µg HPV16L1 VLP/10
6
cells and
3.3 µg HPV16L1L2 VLP/10
6
cells (2:1), 5 µg HPV16L1 VLP/10
6
cells and 5 µg
HPV16L1L2 VLP/10
6
cells (1:1), 3.3 µg HPV16L1 VLP/10
6
cells and 6.6 µg
HPV16L1L2 VLP/10
6
cells (1:2), or with 5 µg HPV16L1 VLP/10
6
cells and 5 µg heated
(10 min, 95ºC) HPV16L1L2 VLP/10
6
cells (1:1∆). To validate results, three different
VLP preparations were used over the course of our experiments. The cells were then
incubated for 1h at 37°C, mixed occasionally, and placed at 37°C for 48 h in 20 ml
complete media containing 1000 U/ml rhu-GM-CSF. Supernatants were collected and
cells were harvested, washed, stained for surface markers or isotype controls, and
analyzed by flow cytometry. Supernatants were analyzed at the USC Beckman Immune
Monitoring Center using the Bio-Plex Suspension Array System (Bio-Rad).
91
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) 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.
The concentrations of cytokines and chemokines in the supernatants were analyzed using
Human Cytokine LINCOplex Kits (LINCO Research) and the Bio-Plex Suspension
Array System (Bio-Rad). 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 5µm-pore-size polycarbonate filters (Corning Costar). Briefly, 600 µl of
92
media was added to the lower chamber containing either 250 ng/ml human
r6Ckine/CCL21 (R&D Systems) or complete media alone to control for spontaneous
migration. We added 2 x 10
5
LC, untreated or treated as indicated in the activation assay
to the upper chamber and incubated the plates for 3 h at 37°C. The cells that migrated to
the lower chamber were counted using a hemocytometer.

In Vitro Immunization Assay
In vitro immunization assays were performed with the following treatments: 1.2 x
10
6
LC were left untreated or exposed to either 10 µg of HPV16L1 VLP/10
6
cells or 10
µg 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. Following 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 MultiSort CD8
+
isolation kit and a magnetic cell
separator (Miltenyi Biotech). 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
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 LC
treated as in the first stimulation. For restimulations, the medium was 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.

93
ELISPOT Assay
To test for HPV16-L1 specific CD8
+
T cell responses, IFN-γ ELISPOT assays
were performed. 96-well multiscreen-hemagglutinin plates (Millipore) 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
. Subsequently, 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-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 1 h 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).

Western Blot After LC Activation
LC were treated as described in the activation assay at 37 °C for 15 min. Cellular
extracts were prepared using the Mammalian Protein Extraction Reagent (Pierce)
containing Halt Protease Inhibitor Cocktail (Thermo Scientific). Normalized aliquots of
cell lysates were electrophoresed on 10% NuPage Novex Bis-Tris gels (Invitrogen),
transferred to nitrocellulose membranes and blocked with StartingBlock blocking buffer
(Thermo Scientific). Immunoblotting was performed using antibodies specific for p-PI3K
(Tyr 508), PI3K, p-Akt (Ser 473), Akt (Santa Cruz Biotechnology) or GAPDH
94
(Chemicon). The secondary antibodies used were goat-anti-mouse-IR Dye 800
(Rockland) and goat-anti-rabbit-Alexa Fluor 680 (Molecular Probes). Visualization was
performed using the Odyssey Infrared Imaging System (LI-COR Bioscience).

HPV16 VLP Uptake Assay
HPV16L1L2 VLP and HPV16L1 VLP were labeled with CFDA-SE using the
Vybrant CFDA-SE cell tracer kit (Invitrogen) as directed by the manufacturer’s
instructions. CFDA-SE labeled HPV16 VLP were dialyzed against 4 liters of cold
PBS/0.5 M NaCl to remove all the excess free dye. As a control, CFDA-SE was added to
PBS and dialyzed as described above. LC were harvested, washed with PBS and
aliquoted at a concentration of 1 x 10
6
cells/400 µl PBS into 1.5 ml amber tubes. Next,
CFDA-SE/PBS control, CFDA-SE labeled HPV16L1L2 VLP or HPV16L1 VLP (1 µg
VLP/1 x 10
6
cells) were incubated with 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. Percent uptake is calculated as the mean fluorescent intensity (MFI) of
the sample divided by the MFI of the untreated control multiplied by 100.

HPV16 VLP Uptake Assay with Confocal Microscopy
HPV16L1 VLP were labeled with the Alexa Fluor 546 Protein Labeling Kit
(Invitrogen) and HPV16L1L2 VLP were labeled with the Alexa Fluor 488 Protein
Labeling Kit (Invitrogen) as described in manufacturer’s instructions. After collection of
the labeled VLP, a Bradford assay was performed to quantify the amount of protein in
each VLP prep. LC were incubated with 0.1 µg VLP/10
6
cells of both AF546-HPV16L1
95
VLP and AF488-HPV16L1L2 VLP simultaneously in 400 µl PBS in amber 1.5 ml
microcentrifuge tubes at 37°C. At each time point (2.5, 5, 15 and 30 min), 400 µl of cold
4% paraformaldehyde was added to the LC to fix the cell and freeze cellular uptake. Cells
were subsequently spun down at 800xg for 5 min at 4°C and then resuspended in DAPI
staining buffer (Invitrogen). After 15 min incubation at room temperature, cells were
spun down and resuspended in PBS. Cells were visualize by confocal microscopy at the
USC Cell and Tissue Imaging Core (Los Angeles, CA). As a control, the fluorescent dye
was switched for the L1 and L1L2 VLP to ensure the result was not due to the particular
dye. Similar results were obtained when the dye was switched.

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)/0.5x10
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 an anti-L1 (HPV16.V5) antibody at a concentration of
1:25,000 for 30 min at 4°C. The cells were then incubated 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 streptavidin-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. Binding is calculated
as the MFI of the sample divided by the MFI of the untreated control multiplied by 100.
96

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
(no peptide added) 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 KCl2, 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
NaH2PO4, 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) 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
97
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 (BD Biosciences). 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 (Thermo Scientific). 10% Tris-HCL gels (Bio-Rad) were
used to separate out reduced eluates for silver stain analysis.

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 three times
with 35% EtOH for 20 min 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). After the gel was stained it was washed twice 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
98
darkness the stain was stopped using 50% MetOH/12% acetic acid for 5 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 with SLPI
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. After labeling the HPV16 VLP were dialyzed against 4 liters 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 1x10
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
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/1x10
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. Percent uptake is calculated as the mean fluorescent intensity (MFI) of the
sample divided by the MFI of the untreated control multiplied by 100.

siRNA Inhibition of ANXA2 in LC and HPV16 VLP Uptake Assay
ANXA2 siRNA was synthesized at the USC Genomics Core (Los Angeles, CA)
based on the sequence (CGGGAUGCUUUGAACAUUGAATT)
99
/(UUCAAUGUUCAAAGCAUCCCGTT). Control siRNA was from (Johnson et al.,
2007). ANXA2 or Control siRNA was transfected into LC using the Amaxa Human
Dendritic Cell Nucleofector kit as directed by the manufacturer’s instructions. The cells
were incubated for 6 days before they were analyzed by an anti-ANXA2 immunoblot and
used in an HPV16 VLP uptake assay. HPV VLP were labeled with CFDA-SE as
described above. CFDA-SE labeled HPV16L1L2 VLP or HPV16L1 VLP (1 µg/1x10
6
)
were incubated with the nucleofected 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. Percent uptake is calculated as the mean fluorescent intensity (MFI) of the
sample divided by the MFI of the untreated control multiplied by 100.

Uptake Model Figures
Both Figures 5 and 26 were made using Protein Builder Version 2.0.

Statistical Analysis
All statistical analyses were performed using GraphPad Prism (GraphPad
Software Inc., San Diego, CA).
100
Bibliography

Adurthi, S., Krishna, S., Mukherjee, G., Bafna, U. D., Devi, U., and Jayshree, R. S.
(2008). Regulatory T cells in a spectrum of HPV-induced cervical lesions:
cervicitis, cervical intraepithelial neoplasia and squamous cell carcinoma. Am. J.
Reprod. Immunol. 60, 55-65.

Albert, M. L., Sauter, B., and Bhardwaj, N. (1998). Dendritic cells acquire antigen from
apoptotic cells and induce class I-restricted CTLs. Nature 392, 86-89.

Ashcroft, G. S., Lei, K., Jin, W., Longenecker, G., Kulkarni, A. B., Greenwell-Wild, T.,
Hale-Donze, H., McGrady, G., Song, X. Y., and Wahl, S. M. (2000). Secretory
leukocyte protease inhibitor mediates non-redundant functions necessary for
normal wound healing. Nat. Med 6, 1147-1153.

Ault, K. A., and FUTURE II Study Group (2007). Effect of prophylactic human
papillomavirus L1 virus-like-particle vaccine on risk of cervical intraepithelial
neoplasia grade 2, grade 3, and adenocarcinoma in situ: a combined analysis of
four randomised clinical trials. Lancet 369, 1861-1868.

Baker, T. S., Newcomb, W. W., Olson, N. H., Cowsert, L. M., Olson, C., and Brown, J.
C. (1991). Structures of bovine and human papillomaviruses. Analysis by
cryoelectron microscopy and three-dimensional image reconstruction. Biophys. J
60, 1445-1456.

Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B.,
and Palucka, K. (2000). Immunobiology of dendritic cells. Annu. Rev. Immunol
18, 767-811.

Banchereau, J., and Steinman, R. M. (1998). Dendritic cells and the control of immunity.
Nature 392, 245-52.

Barnard, P., and McMillan, N. A. (1999). The human papillomavirus E7 oncoprotein
abrogates signaling mediated by interferon-alpha. Virology 259, 305-313.

Barton, G. M. (2007). Viral recognition by Toll-like receptors. Semin. Immunol 19, 33-
40.

Becker, K. A., Florin, L., Sapp, C., and Sapp, M. (2003). Dissection of human
papillomavirus type 33 L2 domains involved in nuclear domains (ND) 10 homing
and reorganization. Virology 314, 161-167.

Bienkowska-Haba, M., Patel, H. D., and Sapp, M. (2009). Target cell cyclophilins
facilitate human papillomavirus type 16 infection. PLoS Pathog 5, e1000524.
101

Borkowski, T. A., Letterio, J. J., Mackall, C. L., Saitoh, A., Wang, X. J., Roop, D. R.,
Gress, R. E., and Udey, M. C. (1997). A role for TGFbeta1 in langerhans cell
biology. Further characterization of the epidermal Langerhans cell defect in
TGFbeta1 null mice. J. Clin. Invest 100, 575-581.

Bousarghin, L., Hubert, P., Franzen, E., Jacobs, N., Boniver, J., and Delvenne, P. (2005).
Human papillomavirus 16 virus-like particles use heparan sulfates to bind
dendritic cells and colocalize with langerin in Langerhans cells. J. Gen. Virol 86,
1297-1305.

van den Bout, I., and Divecha, N. (2009). PIP5K-driven PtdIns(4,5)P2 synthesis:
regulation and cellular functions. J Cell Sci 122, 3837-3850.

Brehm, A., Nielsen, S. J., Miska, E. A., McCance, D. J., Reid, J. L., Bannister, A. J., and
Kouzarides, T. (1999). The E7 oncoprotein associates with Mi2 and histone
deacetylase activity to promote cell growth. EMBO J 18, 2449-2458.

Buck, C. B., Cheng, N., Thompson, C. D., Lowy, D. R., Steven, A. C., Schiller, J. T., and
Trus, B. L. (2008). Arrangement of L2 within the papillomavirus capsid. J. Virol
82, 5190-5197.

van der Burg, S., Piersma, S. J., de Jong, A., van der Hulst, J. M., Kwappenberg, K. M.
C., van den Hende, M., Welters, M. J. P., Van Rood, J. J., Fleuren, G. J., Melief,
C. J. M., et al. (2007). Association of cervical cancer with the presence of CD4+
regulatory T cells specific for human papillomavirus antigens. Proc. Natl. Acad.
Sci. U.S.A. 104, 12087-12092.

Burysek, L., Syrovets, T., and Simmet, T. (2002). The Serine Protease Plasmin Triggers
Expression of MCP-1 and CD40 in Human Primary Monocytes via Activation of
p38 MAPK and Janus Kinase (JAK)/STAT Signaling Pathways. Journal of
Biological Chemistry 277, 33509-33517.

Casares, N., Arribillaga, L., Sarobe, P., Dotor, J., Lopez-Diaz de Cerio, A., Melero, I.,
Prieto, J., Borrás-Cuesta, F., and Lasarte, J. J. (2003). CD4+/CD25+ regulatory
cells inhibit activation of tumor-primed CD4+ T cells with IFN-gamma-
dependent antiangiogenic activity, as well as long-lasting tumor immunity elicited
by peptide vaccination. J. Immunol 171, 5931-5939.

Castellsagué, X., Díaz, M., de Sanjosé, S., Muñoz, N., Herrero, R., Franceschi, S.,
Peeling, R. W., Ashley, R., Smith, J. S., Snijders, P. J. F., et al. (2006).
Worldwide human papillomavirus etiology of cervical adenocarcinoma and its
cofactors: implications for screening and prevention. J. Natl. Cancer Inst 98, 303-
315.

102
Cho, Y. S., Kang, J. W., Cho, M., Cho, C. W., Lee, S., Choe, Y. K., Kim, Y., Choi, I.,
Park, S. N., Kim, S., et al. (2001). Down modulation of IL-18 expression by
human papillomavirus type 16 E6 oncogene via binding to IL-18. FEBS Lett 501,
139-145.

Chuang, C., Hoory, T., Monie, A., Wu, A., Wang, M., and Hung, C. (2009). Enhancing
therapeutic HPV DNA vaccine potency through depletion of CD4+CD25+ T
regulatory cells. Vaccine 27, 684-689.

Coleman, N., Birley, H. D., Renton, A. M., Hanna, N. F., Ryait, B. K., Byrne, M.,
Taylor-Robinson, D., and Stanley, M. A. (1994). Immunological events in
regressing genital warts. Am. J. Clin. Pathol 102, 768-774.

Combita, A. L., Touzé, A., Bousarghin, L., Sizaret, P. Y., Muñoz, N., and Coursaget, P.
(2001). Gene transfer using human papillomavirus pseudovirions varies according
to virus genotype and requires cell surface heparan sulfate. FEMS Microbiol. Lett
204, 183-188.

Cromme, F. V., van Bommel, P. F., Walboomers, J. M., Gallee, M. P., Stern, P. L.,
Kenemans, P., Helmerhorst, T. J., Stukart, M. J., and Meijer, C. J. (1994).
Differences in MHC and TAP-1 expression in cervical cancer lymph node
metastases as compared with the primary tumours. Br. J. Cancer 69, 1176-1181.

Culp, T. D., Budgeon, L. R., and Christensen, N. D. (2006). Human papillomaviruses
bind a basal extracellular matrix component secreted by keratinocytes which is
distinct from a membrane-associated receptor. Virology 347, 147-159.

Culp, T. D., Budgeon, L. R., Marinkovich, M. P., Meneguzzi, G., and Christensen, N. D.
(2006). Keratinocyte-secreted laminin 5 can function as a transient receptor for
human papillomaviruses by binding virions and transferring them to adjacent
cells. J. Virol 80, 8940-8950.

Cumberbatch, M., Dearman, R. J., Griffiths, C. E., and Kimber, I. (2000). Langerhans
cell migration. Clin. Exp. Dermatol 25, 413-418.

Da Silva, D. M., Velders, M. P., Nieland, J. D., Schiller, J. T., Nickoloff, B. J., and Kast,
W. M. (2001). Physical interaction of human papillomavirus virus-like particles
with immune cells. Int. Immunol 13, 633-641.

Darshan, M. S., Lucchi, J., Harding, E., and Moroianu, J. (2004). The l2 minor capsid
protein of human papillomavirus type 16 interacts with a network of nuclear
import receptors. J. Virol 78, 12179-12188.

103
Dasbach, E. J., Elbasha, E. H., and Insinga, R. P. (2006). Mathematical models for
predicting the epidemiologic and economic impact of vaccination against human
papillomavirus infection and disease. Epidemiol Rev 28, 88-100.

Day, P. M., Baker, C. C., Lowy, D. R., and Schiller, J. T. (2004). Establishment of
papillomavirus infection is enhanced by promyelocytic leukemia protein (PML)
expression. Proc. Natl. Acad. Sci. U.S.A 101, 14252-14257.

Day, P. M., Lowy, D. R., and Schiller, J. T. (2008). Heparan sulfate-independent cell
binding and infection with furin-precleaved papillomavirus capsids. J. Virol 82,
12565-12568.

Day, P. M., Thompson, C. D., Buck, C. B., Pang, Y. S., Lowy, D. R., and Schiller, J. T.
(2007). Neutralization of human papillomavirus with monoclonal antibodies
reveals different mechanisms of inhibition. J. Virol 81, 8784-8792.

De Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P.,
Urbain, J., Leo, O., and Moser, M. (1996). Regulation of dendritic cell numbers
and maturation by lipopolysaccharide in vivo. J. Exp. Med 184, 1413-1424.

Deora, A. B., Kreitzer, G., Jacovina, A. T., and Hajjar, K. A. (2004). An annexin 2
phosphorylation switch mediates p11-dependent translocation of annexin 2 to the
cell surface. J. Biol. Chem 279, 43411-43418.

Derry, M. C., Sutherland, M. R., Restall, C. M., Waisman, D. M., and Pryzdial, E. L. G.
(2007). Annexin 2-mediated enhancement of cytomegalovirus infection opposes
inhibition by annexin 1 or annexin 5. J. Gen. Virol 88, 19-27.

Dhodapkar, M. V., Steinman, R. M., Krasovsky, J., Munz, C., and Bhardwaj, N. (2001).
Antigen-specific inhibition of effector T cell function in humans after injection of
immature dendritic cells. J. Exp. Med. 193, 233-238.

Di Paolo, G., Pellegrini, L., Letinic, K., Cestra, G., Zoncu, R., Voronov, S., Chang, S.,
Guo, J., Wenk, M. R., and De Camilli, P. (2002). Recruitment and regulation of
phosphatidylinositol phosphate kinase type 1 gamma by the FERM domain of
talin. Nature 420, 85-89.

Doorbar, J. (2006). Molecular biology of human papillomavirus infection and cervical
cancer. Clin. Sci 110, 525-541.

Doornbos, R. P., Theelen, M., van der Hoeven, P. C., van Blitterswijk, W. J., Verkleij, A.
J., and van Bergen en Henegouwen, P. M. (1999). Protein kinase Czeta is a
negative regulator of protein kinase B activity. J. Biol. Chem 274, 8589-8596.

104
Dreier, R., Schmid, K. W., Gerke, V., and Riehemann, K. (1998). Differential expression
of annexins I, II and IV in human tissues: an immunohistochemical study.
Histochem. Cell Biol 110, 137-148.

Drobni, P., Mistry, N., McMillan, N., and Evander, M. (2003). Carboxy-fluorescein
diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce
after internalization and interact with heparan sulfate for binding and entry.
Virology 310, 163-172.

Duensing, S., Duensing, A., Flores, E. R., Do, A., Lambert, P. F., and Münger, K. (2001).
Centrosome abnormalities and genomic instability by episomal expression of
human papillomavirus type 16 in raft cultures of human keratinocytes. J. Virol 75,
7712-7716.

Duensing, S., and Münger, K. (2002). The human papillomavirus type 16 E6 and E7
oncoproteins independently induce numerical and structural chromosome
instability. Cancer Res 62, 7075-7082.

Dunne, E. F., Unger, E. R., Sternberg, M., McQuillan, G., Swan, D. C., Patel, S. S., and
Markowitz, L. E. (2007). Prevalence of HPV infection among females in the
United States. JAMA 297, 813-819.

Dyson, N., Howley, P. M., Münger, K., and Harlow, E. (1989). The human papilloma
virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product.
Science 243, 934-937.

Evander, M., Frazer, I. H., Payne, E., Qi, Y. M., Hengst, K., and McMillan, N. A. (1997).
Identification of the alpha6 integrin as a candidate receptor for papillomaviruses.
J. Virol 71, 2449-2456.

Evans, E. M., Man, S., Evans, A. S., and Borysiewicz, L. K. (1997). Infiltration of
cervical cancer tissue with human papillomavirus-specific cytotoxic T-
lymphocytes. Cancer Res 57, 2943-2950.

Fahey, L. M., Raff, A. B., Da Silva, D. M., and Kast, W. M. (2009). Reversal of human
papillomavirus-specific T cell immune suppression through TLR agonist
treatment of Langerhans cells exposed to human papillomavirus type 16. J.
Immunol 182, 2919-2928.

Fakioglu, E., Wilson, S. S., Mesquita, P. M. M., Hazrati, E., Cheshenko, N., Blaho, J. A.,
and Herold, B. C. (2008). Herpes simplex virus downregulates secretory
leukocyte protease inhibitor: a novel immune evasion mechanism. J. Virol 82,
9337-9344.

105
Falcone, D. J., Borth, W., Khan, K. M., and Hajjar, K. A. (2001). Plasminogen-mediated
matrix invasion and degradation by macrophages is dependent on surface
expression of annexin II. Blood 97, 777-784.

Fattorossi, A., Battaglia, A., Ferrandina, G., Buzzonetti, A., Legge, F., Salutari, V., and
Scambia, G. (2004). Lymphocyte composition of tumor draining lymph nodes
from cervical and endometrial cancer patients. Gynecol. Oncol. 92, 106-115.

Fausch, S. C., Da Silva, D. M., and Kast, W. M. (2003). Differential uptake and cross-
presentation of human papillomavirus virus-like particles by dendritic cells and
Langerhans cells. Cancer Res 63, 3478-82.

Fausch, S. C., Da Silva, D. M., Rudolf, M. P., and Kast, W. M. (2002). Human
papillomavirus virus-like particles do not activate Langerhans cells: a possible
immune escape mechanism used by human papillomaviruses. J Immunol 169,
3242-9.

Fausch, S. C., Fahey, L. M., Da Silva, D. M., and Kast, W. M. (2005). Human
papillomavirus can escape immune recognition through Langerhans cell
phosphoinositide 3-kinase activation. J. Immunol. 174, 7172-7178.

Fay, A., Yutzy, W. H., Roden, R. B. S., and Moroianu, J. (2004). The positively charged
termini of L2 minor capsid protein required for bovine papillomavirus infection
function separately in nuclear import and DNA binding. J. Virol 78, 13447-
13454.

Finnen, R. L., Erickson, K. D., Chen, X. S., and Garcea, R. L. (2003). Interactions
between papillomavirus L1 and L2 capsid proteins. J. Virol 77, 4818-4826.

Florin, L., Becker, K. A., Lambert, C., Nowak, T., Sapp, C., Strand, D., Streeck, R. E.,
and Sapp, M. (2006). Identification of a dynein interacting domain in the
papillomavirus minor capsid protein l2. J. Virol 80, 6691-6696.

Fothergill, T., and McMillan, N. A. J. (2006). Papillomavirus virus-like particles activate
the PI3-kinase pathway via alpha-6 beta-4 integrin upon binding. Virology 352,
319-328.

Frazer, I. H. (2004). Prevention of cervical cancer through papillomavirus vaccination.
Nat Rev Immunol 4, 46-54.

Frohlich, M., Motté, P., Galvin, K., Takahashi, H., Wands, J., and Ozturk, M. (1990).
Enhanced expression of the protein kinase substrate p36 in human hepatocellular
carcinoma. Mol Cell Biol 10, 3216-3223.

106
FUTURE II Study Group (2007). Quadrivalent vaccine against human papillomavirus to
prevent high-grade cervical lesions. N. Engl. J. Med 356, 1915-1927.

Gallagher, K. M. E., and Man, S. (2007). Identification of HLA-DR1- and HLA-DR15-
restricted human papillomavirus type 16 (HPV16) and HPV18 E6 epitopes
recognized by CD4+ T cells from healthy young women. J. Gen. Virol 88, 1470-
1478.

Galloway, D. A., and McDougall, J. K. (1983). The oncogenic potential of herpes
simplex viruses: evidence for a 'hit-and-run' mechanism. Nature 302, 21-24.

Gambhira, R., Karanam, B., Jagu, S., Roberts, J. N., Buck, C. B., Bossis, I., Alphs, H.,
Culp, T., Christensen, N. D., and Roden, R. B. S. (2007). A protective and
broadly cross-neutralizing epitope of human papillomavirus L2. J. Virol 81,
13927-13931.

Geissmann, F., Prost, C., Monnet, J. P., Dy, M., Brousse, N., and Hermine, O. (1998).
Transforming growth factor beta1, in the presence of granulocyte/macrophage
colony-stimulating factor and interleukin 4, induces differentiation of human
peripheral blood monocytes into dendritic Langerhans cells. J. Exp. Med 187,
961-966.

Georgopoulos, N. T., Proffitt, J. L., and Blair, G. E. (2000). Transcriptional regulation of
the major histocompatibility complex (MHC) class I heavy chain, TAP1 and
LMP2 genes by the human papillomavirus (HPV) type 6b, 16 and 18 E7
oncoproteins. Oncogene 19, 4930-4935.

Gerke, V., Creutz, C. E., and Moss, S. E. (2005). Annexins: linking Ca2+ signalling to
membrane dynamics. Nat. Rev. Mol. Cell Biol 6, 449-461.

Gerke, V., and Moss, S. E. (2002). Annexins: From Structure to Function. Physiol. Rev.
82, 331-371.

Ginhoux, F., Collin, M. P., Bogunovic, M., Abel, M., Leboeuf, M., Helft, J., Ochando, J.,
Kissenpfennig, A., Malissen, B., Grisotto, M., et al. (2007). Blood-derived dermal
langerin+ dendritic cells survey the skin in the steady state. J. Exp. Med 204,
3133-3146.

Ginhoux, F., Tacke, F., Angeli, V., Bogunovic, M., Loubeau, M., Dai, X., Stanley, E. R.,
Randolph, G. J., and Merad, M. (2006). Langerhans cells arise from monocytes in
vivo. Nat. Immunol 7, 265-273.

Giroglou, T., Florin, L., Schafer, F., Streeck, R. E., and Sapp, M. (2001). Human
Papillomavirus Infection Requires Cell Surface Heparan Sulfate. Journal of
Virology 75, 1565-1570.
107

Glenney, J. (1986). Two related but distinct forms of the Mr 36,000 tyrosine kinase
substrate (calpactin) that interact with phospholipid and actin in a Ca2+-
dependent manner. Proc. Natl. Acad. Sci. U.S.A 83, 4258-4262.

Gonzalez-Reyes, S., Garcia-Manso, A., del Barrio, G., Dalton, K. P., Gonzalez-Molleda,
L., Arrojo-Fernandez, J., Nicieza, I., and Parra, F. (2009). Role of annexin A2 in
cellular entry of rabbit vesivirus. J Gen Virol 90, 2724-2730.

Gorden, K. B., Gorski, K. S., Gibson, S. J., Kedl, R. M., Kieper, W. C., Qiu, X., Tomai,
M. A., Alkan, S. S., and Vasilakos, J. P. (2005). Synthetic TLR agonists reveal
functional differences between human TLR7 and TLR8. J. Immunol 174, 1259-
1268.

Gould, K. L., Woodgett, J. R., Isacke, C. M., and Hunter, T. (1986). The protein-tyrosine
kinase substrate p36 is also a substrate for protein kinase C in vitro and in vivo.
Mol. Cell. Biol 6, 2738-2744.

Greenstone, H. L., Nieland, J. D., de Visser, K. E., De Bruijn, M. L., Kirnbauer, R.,
Roden, R. B., Lowy, D. R., Kast, W. M., and Schiller, J. T. (1998). Chimeric
papillomavirus virus-like particles elicit antitumor immunity against the E7
oncoprotein in an HPV16 tumor model. Proc. Natl. Acad. Sci. U.S.A 95, 1800-
1805.

Gruener, M., Bravo, I. G., Momburg, F., Alonso, A., and Tomakidi, P. (2007). The E5
protein of the human papillomavirus type 16 down-regulates HLA-I surface
expression in calnexin-expressing but not in calnexin-deficient cells. Virol. J 4,
116.

Guess, J. C., and McCance, D. J. (2005). Decreased migration of Langerhans precursor-
like cells in response to human keratinocytes expressing human papillomavirus
type 16 E6/E7 is related to reduced macrophage inflammatory protein-3alpha
production. J. Virol 79, 14852-14862.

Hajjar, K. A., Jacovina, A. T., and Chacko, J. (1994). An endothelial cell receptor for
plasminogen/tissue plasminogen activator. I. Identity with annexin II. Journal of
Biological Chemistry 269, 21191-21197.

zur Hausen, H. (1991). Human papillomaviruses in the pathogenesis of anogenital cancer.
Virology 184, 9-13.

zur Hausen, H. (1996). Papillomavirus infections--a major cause of human cancers.
Biochim. Biophys. Acta 1288, F55-78.

108
Hayes, M. J., Shao, D., Grieve, A., Levine, T., Bailly, M., and Moss, S. E. (2009).
Annexin A2 at the interface between F-actin and membranes enriched in
phosphatidylinositol 4,5,-bisphosphate. Biochim. Biophys. Acta 1793, 1086-1095.

Heath, W. R., Belz, G. T., Behrens, G. M. N., Smith, C. M., Forehan, S. P., Parish, I. A.,
Davey, G. M., Wilson, N. S., Carbone, F. R., and Villadangos, J. A. (2004).
Cross-presentation, dendritic cell subsets, and the generation of immunity to
cellular antigens. Immunological Reviews 199, 9-26.

Hiemstra, P. S., Maassen, R. J., Stolk, J., Heinzel-Wieland, R., Steffens, G. J., and
Dijkman, J. H. (1996). Antibacterial activity of antileukoprotease. Infect. Immun
64, 4520-4524.

Hirao, M., Onai, N., Hiroishi, K., Watkins, S. C., Matsushima, K., Robbins, P. D., Lotze,
M. T., and Tahara, H. (2000). CC chemokine receptor-7 on dendritic cells is
induced after interaction with apoptotic tumor cells: critical role in migration from
the tumor site to draining lymph nodes. Cancer Res 60, 2209-2217.

Hochstrasser, K., Reichert, R., Schwarz, S., and Werle, E. (1972). Isolation and
characterisation of a protease inhibitor from human bronchial secretion. Hoppe-
Seyler's Z. Physiol. Chem 353, 221-226.

Höhn, H., Pilch, H., Günzel, S., Neukirch, C., Hilmes, C., Kaufmann, A., Seliger, B., and
Maeurer, M. J. (1999). CD4+ tumor-infiltrating lymphocytes in cervical cancer
recognize HLA-DR-restricted peptides provided by human papillomavirus-E7. J.
Immunol 163, 5715-5722.

Holmgren, S. C., Patterson, N. A., Ozbun, M. A., and Lambert, P. F. (2005). The minor
capsid protein L2 contributes to two steps in the human papillomavirus type 31
life cycle. J. Virol 79, 3938-3948.

Horvath, C. A. J., Boulet, G. A. V., Renoux, V. M., Delvenne, P. O., and Bogers, J. J.
(2010). Mechanisms of cell entry by human papillomaviruses: an overview. Virol.
J 7, 11.

Huang, S., and McCance, D. J. (2002). Down regulation of the interleukin-8 promoter by
human papillomavirus type 16 E6 and E7 through effects on CREB binding
protein/p300 and P/CAF. J. Virol 76, 8710-8721.

Johnson, S. S., Zhang, C., Fromm, J., Willis, I. M., and Johnson, D. L. (2007).
Mammalian Maf1 is a negative regulator of transcription by all three nuclear
RNA polymerases. Mol. Cell 26, 367-379.

Jones, C. (1995). Cervical cancer: is herpes simplex virus type II a cofactor? Clin.
Microbiol. Rev 8, 549-556.
109

de Jong, A., van der Burg, S. H., Kwappenberg, K. M. C., van der Hulst, J. M., Franken,
K. L. M. C., Geluk, A., van Meijgaarden, K. E., Drijfhout, J. W., Kenter, G.,
Vermeij, P., et al. (2002). Frequent detection of human papillomavirus 16 E2-
specific T-helper immunity in healthy subjects. Cancer Res 62, 472-479.

Jonuleit, H., Schmitt, E., Schuler, G., Knop, J., and Enk, A. H. (2000). Induction of
interleukin 10-producing, nonproliferating CD4+ T cells with regulatory
properties by repetitive stimulation with allogeneic immature human dendritic
cells. J. Exp. Med. 192, 1213-1222.

Joyce, J. G., Tung, J. S., Przysiecki, C. T., Cook, J. C., Lehman, E. D., Sands, J. A.,
Jansen, K. U., and Keller, P. M. (1999). The L1 major capsid protein of human
papillomavirus type 11 recombinant virus-like particles interacts with heparin and
cell-surface glycosaminoglycans on human keratinocytes. J. Biol. Chem 274,
5810-5822.

Kämper, N., Day, P. M., Nowak, T., Selinka, H., Florin, L., Bolscher, J., Hilbig, L.,
Schiller, J. T., and Sapp, M. (2006). A membrane-destabilizing peptide in capsid
protein L2 is required for egress of papillomavirus genomes from endosomes. J.
Virol 80, 759-768.

Kanodia, S., Fahey, L. M., and Kast, W. M. (2007). Mechanisms used by human
papillomaviruses to escape the host immune response. Curr Cancer Drug Targets
7, 79-89.

Kassam, G., Choi, K., Ghuman, J., Kang, H., Fitzpatrick, S. L., Zackson, T., Zackson, S.,
Toba, M., Shinomiya, A., and Waisman, D. M. (1998). The Role of Annexin II
Tetramer in the Activation of Plasminogen. Journal of Biological Chemistry 273,
4790-4799.

Kassam, G., Manro, A., Braat, C. E., Louie, P., Fitzpatrick, S. L., and Waisman, D. M.
(1997). Characterization of the Heparin Binding Properties of Annexin II
Tetramer. Journal of Biological Chemistry 272, 15093-15100.

Kaufmann, A. M., Nieland, J., Schinz, M., Nonn, M., Gabelsberger, J., Meissner, H.,
Müller, R. T., Jochmus, I., Gissmann, L., Schneider, A., et al. (2001). HPV16
L1E7 chimeric virus-like particles induce specific HLA-restricted T cells in
humans after in vitro vaccination. Int. J. Cancer 92, 285-293.

Kawana, K., Yoshikawa, H., Taketani, Y., Yoshiike, K., and Kanda, T. (1999). Common
neutralization epitope in minor capsid protein L2 of human papillomavirus types
16 and 6. J. Virol 73, 6188-6190.

110
Kawana, Y., Kawana, K., Yoshikawa, H., Taketani, Y., Yoshiike, K., and Kanda, T.
(2001). Human papillomavirus type 16 minor capsid protein L2 N-terminal region
containing a common neutralization epitope binds to the cell surface and enters
the cytoplasm. J Virol 75, 2331-6.

Keating, P. J., Cromme, F. V., Duggan-Keen, M., Snijders, P. J., Walboomers, J. M.,
Hunter, R. D., Dyer, P. A., and Stern, P. L. (1995). Frequency of down-regulation
of individual HLA-A and -B alleles in cervical carcinomas in relation to TAP-1
expression. Br. J. Cancer 72, 405-411.

Kirnbauer, R., Booy, F., Cheng, N., Lowy, D. R., and Schiller, J. T. (1992).
Papillomavirus L1 major capsid protein self-assembles into virus-like particles
that are highly immunogenic. Proc Natl Acad Sci U S A 89, 12180-4.

Kirnbauer, R., Taub, J., Greenstone, H., Roden, R., Dürst, M., Gissmann, L., Lowy, D.
R., and Schiller, J. T. (1993). Efficient self-assembly of human papillomavirus
type 16 L1 and L1-L2 into virus-like particles. J. Virol 67, 6929-6936.

Klechevsky, E., Morita, R., Liu, M., Cao, Y., Coquery, S., Thompson-Snipes, L., Briere,
F., Chaussabel, D., Zurawski, G., Palucka, A. K., et al. (2008). Functional
specializations of human epidermal Langerhans cells and CD14+ dermal dendritic
cells. Immunity 29, 497-510.

Kleine-Lowinski, K., Rheinwald, J. G., Fichorova, R. N., Anderson, D. J., Basile, J.,
Münger, K., Daly, C. M., Rösl, F., and Rollins, B. J. (2003). Selective suppression
of monocyte chemoattractant protein-1 expression by human papillomavirus E6
and E7 oncoproteins in human cervical epithelial and epidermal cells. Int. J.
Cancer 107, 407-415.

Kondo, K., Ishii, Y., Ochi, H., Matsumoto, T., Yoshikawa, H., and Kanda, T. (2007).
Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by
immunizing rabbits with synthetic peptides representing segments of the HPV16
minor capsid protein L2 surface region. Virology 358, 266-272.

Kupper, T. S., and Fuhlbrigge, R. C. (2004). Immune surveillance in the skin:
mechanisms and clinical consequences. Nat. Rev. Immunol 4, 211-222.

Laga, M., Icenogle, J. P., Marsella, R., Manoka, A. T., Nzila, N., Ryder, R. W.,
Vermund, S. H., Heyward, W. L., Nelson, A., and Reeves, W. C. (1992). Genital
papillomavirus infection and cervical dysplasia--opportunistic complications of
HIV infection. Int. J. Cancer 50, 45-48.

Langen, R., Isas, J. M., Hubbell, W. L., and Haigler, H. T. (1998). A transmembrane
form of annexin XII detected by site-directed spin labeling. Proceedings of the
National Academy of Sciences of the United States of America 95, 14060-14065.
111

Larsen, C. P., Steinman, R. M., Witmer-Pack, M., Hankins, D. F., Morris, P. J., and
Austyn, J. M. (1990). Migration and maturation of Langerhans cells in skin
transplants and explants. J Exp Med 172, 1483-93.

Laumonnier, Y., Syrovets, T., Burysek, L., and Simmet, T. (2006). Identification of the
annexin A2 heterotetramer as a receptor for the plasmin-induced signaling in
human peripheral monocytes. Blood 107, 3342-3349.

Lee, S. J., Cho, Y. S., Cho, M. C., Shim, J. H., Lee, K. A., Ko, K. K., Choe, Y. K., Park,
S. N., Hoshino, T., Kim, S., et al. (2001). Both E6 and E7 oncoproteins of human
papillomavirus 16 inhibit IL-18-induced IFN-gamma production in human
peripheral blood mononuclear and NK cells. J. Immunol 167, 497-504.

Leggatt, G. R., and Frazer, I. H. (2007). HPV vaccines: the beginning of the end for
cervical cancer. Curr Opin Immunol 19, 232-8.

Li, S., Labrecque, S., Gauzzi, M. C., Cuddihy, A. R., Wong, A. H., Pellegrini, S.,
Matlashewski, G. J., and Koromilas, A. E. (1999). The human papilloma virus
(HPV)-18 E6 oncoprotein physically associates with Tyk2 and impairs Jak-STAT
activation by interferon-alpha. Oncogene 18, 5727-5737.

Ling, K., Doughman, R. L., Firestone, A. J., Bunce, M. W., and Anderson, R. A. (2002).
Type I gamma phosphatidylinositol phosphate kinase targets and regulates focal
adhesions. Nature 420, 89-93.

Liu, J., Rothermund, C. A., Ayala-Sanmartin, J., and Vishwanatha, J. K. (2003). Nuclear
annexin II negatively regulates growth of LNCaP cells and substitution of ser 11
and 25 to glu prevents nucleo-cytoplasmic shuttling of annexin II. BMC Biochem
4, 10-10.

Liu, W. J., Gissmann, L., Sun, X. Y., Kanjanahaluethai, A., Müller, M., Doorbar, J., and
Zhou, J. (1997). Sequence close to the N-terminus of L2 protein is displayed on
the surface of bovine papillomavirus type 1 virions. Virology 227, 474-483.

Longworth, M. S., and Laimins, L. A. (2004). The binding of histone deacetylases and
the integrity of zinc finger-like motifs of the E7 protein are essential for the life
cycle of human papillomavirus type 31. J. Virol 78, 3533-3541.

Lowndes, C. (2006). Vaccines for cervical cancer. Epidemiol. Infect. 134, 1-12.

Luo, W., Yan, G., Li, L., Wang, Z., Liu, H., Zhou, S., Liu, S., Tang, M., Yi, W., Dong,
Z., et al. (2008). Epstein-Barr virus latent membrane protein 1 mediates serine 25
phosphorylation and nuclear entry of annexin A2 via PI-PLC-PKCalpha/PKCbeta
pathway. Molecular Carcinogenesis 47, 934-946.
112

Ma, G., Greenwell-Wild, T., Lei, K., Jin, W., Swisher, J., Hardegen, N., Wild, C. T., and
Wahl, S. M. (2004). Secretory leukocyte protease inhibitor binds to annexin II, a
cofactor for macrophage HIV-1 infection. J. Exp. Med 200, 1337-1346.

Malhotra, R., Ward, M., Bright, H., Priest, R., Foster, M. R., Hurle, M., Blair, E., and
Bird, M. (2003). Isolation and characterisation of potential respiratory syncytial
virus receptor(s) on epithelial cells. Microbes Infect 5, 123-133.

Mallon, R. G., Wojciechowicz, D., and Defendi, V. (1987). DNA-binding activity of
papillomavirus proteins. J. Virol 61, 1655-1660.

Marsh, M., and Helenius, A. (2006). Virus entry: open sesame. Cell 124, 729-740.

McMillan, N. A., Payne, E., Frazer, I. H., and Evander, M. (1999). Expression of the
alpha6 integrin confers papillomavirus binding upon receptor-negative B-cells.
Virology 261, 271-279.

McNeely, T. B., Shugars, D. C., Rosendahl, M., Tucker, C., Eisenberg, S. P., and Wahl,
S. M. (1997). Inhibition of human immunodeficiency virus type 1 infectivity by
secretory leukocyte protease inhibitor occurs prior to viral reverse transcription.
Blood 90, 1141-1149.

Medzhitov, R., Preston-Hurlburt, P., and Janeway, C. A. (1997). A human homologue of
the Drosophila Toll protein signals activation of adaptive immunity. Nature 388,
394-397.

Melchior, A., Denys, A., Deligny, A., Mazurier, J., and Allain, F. (2008). Cyclophilin B
induces integrin-mediated cell adhesion by a mechanism involving CD98-
dependent activation of protein kinase C-[delta] and p44/42 mitogen-activated
protein kinases. Experimental Cell Research 314, 616-628.

Merad, M., Ginhoux, F., and Collin, M. (2008). Origin, homeostasis and function of
Langerhans cells and other langerin-expressing dendritic cells. Nat Rev Immunol
8, 935-947.

Mercer, J., Schelhaas, M., and Helenius, A. (2010). Virus Entry by Endocytosis. Annu
Rev Biochem. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20196649
[Accessed March 7, 2010].

Merle, E., Rose, R. C., LeRoux, L., and Moroianu, J. (1999). Nuclear import of HPV11
L1 capsid protein is mediated by karyopherin alpha2beta1 heterodimers. J. Cell.
Biochem 74, 628-637.

113
Mills, K. H. G. (2004). Regulatory T cells: friend or foe in immunity to infection? Nat
Rev Immunol 4, 841-55.

Millward, T. A., Zolnierowicz, S., and Hemmings, B. A. (1999). Regulation of protein
kinase cascades by protein phosphatase 2A. Trends Biochem. Sci 24, 186-191.

Mitra, S. K., and Schlaepfer, D. D. (2006). Integrin-regulated FAK-Src signaling in
normal and cancer cells. Current Opinion in Cell Biology 18, 516-523.

Modis, Y., Trus, B. L., and Harrison, S. C. (2002). Atomic model of the papillomavirus
capsid. EMBO J 21, 4754-4762.

Molling, J., de Gruijl, T., Glim, J., Moreno, M., Rozendaal, L., Meijer, C., van den
Eertwegh, A., Scheper, R., von Blomberg, M., and Bontkes, H. (2007).
CD4+CD25hi regulatory T-cell frequency correlates with persistence of human
papillomavirus type 16 and T helper cell responses in patients with cervical
intraepithelial neoplasia. Int. J. Cancer 121, 1749-1755.

Morel, E., and Gruenberg, J. (2009). Annexin A2 Binding to Endosomes and Functions
in Endosomal Transport Are Regulated by Tyrosine 23 Phosphorylation. Journal
of Biological Chemistry 284, 1604-1611.

Müller, M., Gissmann, L., Cristiano, R. J., Sun, X. Y., Frazer, I. H., Jenson, A. B.,
Alonso, A., Zentgraf, H., and Zhou, J. (1995). Papillomavirus capsid binding and
uptake by cells from different tissues and species. J. Virol 69, 948-954.

Munoz, N., Bosch, F. X., de Sanjose, S., Herrero, R., Castellsague, X., Shah, K. V.,
Snijders, P. J., Meijer, C. J., and the International Agency for Research on Cancer
Multicenter Cervical Cancer Study Group (2003). Epidemiologic Classification of
Human Papillomavirus Types Associated with Cervical Cancer. N Engl J Med
348, 518-527.

Nees, M., Geoghegan, J. M., Hyman, T., Frank, S., Miller, L., and Woodworth, C. D.
(2001). Papillomavirus type 16 oncogenes downregulate expression of interferon-
responsive genes and upregulate proliferation-associated and NF-kappaB-
responsive genes in cervical keratinocytes. J. Virol 75, 4283-4296.

Nelson, L. M., Rose, R. C., LeRoux, L., Lane, C., Bruya, K., and Moroianu, J. (2000).
Nuclear import and DNA binding of human papillomavirus type 45 L1 capsid
protein. J. Cell. Biochem 79, 225-238.

Ohlsson, K., and Tegner, H. (1976). Inhibition of elastase from granulocytes by the low
molecular weight bronchial protease inhibitor. Scand. J. Clin. Lab. Invest 36, 437-
445.

114
Pakula, R., Melchior, A., Denys, A., Vanpouille, C., Mazurier, J., and Allain, F. (2007).
Syndecan-1/CD147 association is essential for cyclophilin B-induced activation
of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and
chemotaxis. Glycobiology 17, 492-503.

Park, J. S., Kim, E. J., Kwon, H. J., Hwang, E. S., Namkoong, S. E., and Um, S. J.
(2000). Inactivation of interferon regulatory factor-1 tumor suppressor protein by
HPV E7 oncoprotein. Implication for the E7-mediated immune evasion
mechanism in cervical carcinogenesis. J. Biol. Chem 275, 6764-6769.

Park, M., Kitchener, H. C., and Macnab, J. C. (1983). Detection of herpes simplex virus
type-2 DNA restriction fragments in human cervical carcinoma tissue. EMBO J 2,
1029-1034.

Parkin, D. M., Almonte, M., Bruni, L., Clifford, G., Curado, M., and Piñeros, M. (2008).
Burden and trends of type-specific human papillomavirus infections and related
diseases in the latin america and Caribbean region. Vaccine 26 Suppl 11, L1-15.

Parkin, D. (2006). The global health burden of infection-associated cancers in the year
2002. Int. J. Cancer 118, 3030-3044.

Parkin, D., Bray, F., Ferlay, J., and Pisani, P. (2005). Global cancer statistics, 2002. CA
Cancer J. Clin. 55, 74-108.

Pena-Alonso, E., Rodrigo, J. P., Parra, I. C., Pedrero, J. M. G., Meana, M. V. G., Nieto,
C. S., Fresno, M. F., Morgan, R. O., and Fernandez, M. P. (2008). Annexin A2
localizes to the basal epithelial layer and is down-regulated in dysplasia and head
and neck squamous cell carcinoma. Cancer Lett 263, 89-98.

Petry, K. U., Scheffel, D., Bode, U., Gabrysiak, T., Köchel, H., Kupsch, E., Glaubitz, M.,
Niesert, S., Kühnle, H., and Schedel, I. (1994). Cellular immunodeficiency
enhances the progression of human papillomavirus-associated cervical lesions.
Int. J. Cancer 57, 836-840.

Piersma, S. J., Welters, M. J. P., van der Hulst, J. M., Kloth, J. N., Kwappenberg, K. M.
C., Trimbos, B. J., Melief, C. J. M., Hellebrekers, B. W., Fleuren, G. J., Kenter,
G. G., et al. (2008). Human papilloma virus specific T cells infiltrating cervical
cancer and draining lymph nodes show remarkably frequent use of HLA-DQ and
-DP as a restriction element. Int. J. Cancer 122, 486-494.

Pillay, K., Coutsoudis, A., Agadzi-Naqvi, A. K., Kuhn, L., Coovadia, H. M., and Janoff,
E. N. (2001). Secretory leukocyte protease inhibitor in vaginal fluids and perinatal
human immunodeficiency virus type 1 transmission. J. Infect. Dis 183, 653-656.

115
Poulin, L. F., Henri, S., de Bovis, B., Devilard, E., Kissenpfennig, A., and Malissen, B.
(2007). The dermis contains langerin+ dendritic cells that develop and function
independently of epidermal Langerhans cells. J. Exp. Med 204, 3119-3131.

Puisieux, A., Ji, J., and Ozturk, M. (1996). Annexin II up-regulates cellular levels of p11
protein by a post-translational mechanisms. Biochem. J 313 ( Pt 1), 51-55.

Pyeon, D., Pearce, S. M., Lank, S. M., Ahlquist, P., and Lambert, P. F. (2009).
Establishment of Human Papillomavirus Infection Requires Cell Cycle
Progression. PLoS Pathog 5.

Rawls, W. E., Tompkins, W. A., Figueroa, M. E., and Melnick, J. L. (1968). Herpesvirus
type 2: association with carcinoma of the cervix. Science 161, 1255-1256.

Raynor, C. M., Wright, J. F., Waisman, D. M., and Pryzdial, E. L. G. (1999). Annexin II
Enhances Cytomegalovirus Binding and Fusion to Phospholipid Membranes†.
Biochemistry 38, 5089-5095.

Rescher, U., and Gerke, V. (2008). S100A10/p11: family, friends and functions. Pflugers
Arch 455, 575-582.

Rescher, U., Ruhe, D., Ludwig, C., Zobiack, N., and Gerke, V. (2004). Annexin 2 is a
phosphatidylinositol (4,5)-bisphosphate binding protein recruited to actin
assembly sites at cellular membranes. J Cell Sci 117, 3473-3480.

Ressing, M. E., van Driel, W. J., Celis, E., Sette, A., Brandt, M. P., Hartman, M.,
Anholts, J. D., Schreuder, G. M., ter Harmsel, W. B., Fleuren, G. J., et al. (1996).
Occasional memory cytotoxic T-cell responses of patients with human
papillomavirus type 16-positive cervical lesions against a human leukocyte
antigen-A *0201-restricted E7-encoded epitope. Cancer Res 56, 582-588.

Richards, R. M., Lowy, D. R., Schiller, J. T., and Day, P. M. (2006). Cleavage of the
papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for
infection. Proc. Natl. Acad. Sci. U.S.A 103, 1522-1527.

Roberts, J. N., Buck, C. B., Thompson, C. D., Kines, R., Bernardo, M., Choyke, P. L.,
Lowy, D. R., and Schiller, J. T. (2007). Genital transmission of HPV in a mouse
model is potentiated by nonoxynol-9 and inhibited by carrageenan. Nat. Med 13,
857-861.

Roden, R. B., Kirnbauer, R., Jenson, A. B., Lowy, D. R., and Schiller, J. T. (1994).
Interaction of papillomaviruses with the cell surface. J. Virol 68, 7260-7266.

116
Romani, N., Reider, D., Heuer, M., Ebner, S., Kämpgen, E., Eibl, B., Niederwieser, D.,
and Schuler, G. (1996). Generation of mature dendritic cells from human blood.
An improved method with special regard to clinical applicability. J. Immunol.
Methods 196, 137-151.

Routes, J. M., Morris, K., Ellison, M. C., and Ryan, S. (2005). Macrophages kill human
papillomavirus type 16 E6-expressing tumor cells by tumor necrosis factor alpha-
and nitric oxide-dependent mechanisms. J. Virol 79, 116-123.

Rudolf, M. P., Fausch, S. C., Da Silva, D. M., and Kast, W. M. (2001). Human dendritic
cells are activated by chimeric human papillomavirus type-16 virus-like particles
and induce epitope-specific human T cell responses in vitro. J Immunol 166,
5917-24.

Rust, R., Kluiver, J., Visser, L., Harms, G., Blokzijl, T., Kamps, W., Poppema, S., and
Berg, A. V. D. (2006). Gene expression analysis of dendritic/Langerhans cells and
Langerhans cell histiocytosis. The Journal of Pathology 209, 474-483.

Ryding, J., French, K. M., Naucler, P., Barnabas, R. V., Garnett, G. P., and Dillner, J.
(2008). Seroepidemiology as basis for design of a human papillomavirus
vaccination program. Vaccine 26, 5263-5268.

Saeki, H., Moore, A. M., Brown, M. J., and Hwang, S. T. (1999). Cutting edge:
secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7
(CCR7) participate in the emigration pathway of mature dendritic cells from the
skin to regional lymph nodes. J. Immunol 162, 2472-2475.

Samsom, J. N., van der Marel, A. P. J., van Berkel, L. A., van Helvoort, J. M. L. M.,
Simons-Oosterhuis, Y., Jansen, W., Greuter, M., Nelissen, R. L. H., Meeuwisse,
C. M. L., Nieuwenhuis, E. E. S., et al. (2007). Secretory leukoprotease inhibitor in
mucosal lymph node dendritic cells regulates the threshold for mucosal tolerance.
J. Immunol 179, 6588-6595.

Sapp, M., and Bienkowska-Haba, M. (2009). Viral entry mechanisms: human
papillomavirus and a long journey from extracellular matrix to the nucleus. FEBS
J 276, 7206-7216.

Sarafian, T., Pradel, L. A., Henry, J. P., Aunis, D., and Bader, M. F. (1991). The
participation of annexin II (calpactin I) in calcium-evoked exocytosis requires
protein kinase C. J. Cell Biol 114, 1135-1147.

Schlaepfer, D. D., Mitra, S. K., and Ilic, D. (2004). Control of motile and invasive cell
phenotypes by focal adhesion kinase. Biochimica et Biophysica Acta (BBA) -
Molecular Cell Research 1692, 77-102.

117
Schlecht, N. F., Kulaga, S., Robitaille, J., Ferreira, S., Santos, M., Miyamura, R. A.,
Duarte-Franco, E., Rohan, T. E., Ferenczy, A., Villa, L. L., et al. (2001).
Persistent human papillomavirus infection as a predictor of cervical intraepithelial
neoplasia. JAMA 286, 3106-3114.

Schneider, A., Papendick, U., Gissmann, L., and De Villiers, E. M. (1987). Interferon
treatment of human genital papillomavirus infection: importance of viral type. Int.
J. Cancer 40, 610-614.

Schön, M. P., and Schön, M. (2008). TLR7 and TLR8 as targets in cancer therapy.
Oncogene 27, 190-199.

Selinka, H., Florin, L., Patel, H. D., Freitag, K., Schmidtke, M., Makarov, V. A., and
Sapp, M. (2007). Inhibition of transfer to secondary receptors by heparan sulfate-
binding drug or antibody induces noninfectious uptake of human papillomavirus.
J. Virol 81, 10970-10980.

Shafti-Keramat, S., Handisurya, A., Kriehuber, E., Meneguzzi, G., Slupetzky, K., and
Kirnbauer, R. (2003). Different heparan sulfate proteoglycans serve as cellular
receptors for human papillomaviruses. J. Virol 77, 13125-13135.

Shimizu, J., Yamazaki, S., and Sakaguchi, S. (1999). Induction of tumor immunity by
removing CD25+CD4+ T cells: a common basis between tumor immunity and
autoimmunity. J. Immunol 163, 5211-5218.

Sibbet, G., Romero-Graillet, C., Meneguzzi, G., and Campo, M. S. (2000). {alpha}6
integrin is not the obligatory cell receptor for bovine papillomavirus type 4. J Gen
Virol 81, 327-334.

Sieczkarski, S. B., and Whittaker, G. R. (2005). Viral entry. Curr. Top. Microbiol.
Immunol 285, 1-23.

Simon, J. W. (1976). The association of Herpes simplex virus and cervical cancer: A
review. Gynecologic Oncology 4, 108-116.

Smith, J. S., Herrero, R., Bosetti, C., Munoz, N., Bosch, F. X., Eluf-Neto, J.,
Castellsague, X., Meijer, C. J. L. M., Van den Brule, A. J. C., Franceschi, S., et al.
(2002). Herpes Simplex Virus-2 as a Human Papillomavirus Cofactor in the
Etiology of Invasive Cervical Cancer. J. Natl. Cancer Inst. 94, 1604-1613.

Smith, J. L., Campos, S. K., Wandinger-Ness, A., and Ozbun, M. A. (2008). Caveolin-1-
dependent infectious entry of human papillomavirus type 31 in human
keratinocytes proceeds to the endosomal pathway for pH-dependent uncoating. J.
Virol 82, 9505-9512.

118
Song, X. Y., Zeng, L., Jin, W., Thompson, J., Mizel, D. E., Lei, K., Billinghurst, R. C.,
Poole, A. R., and Wahl, S. M. (1999). Secretory leukocyte protease inhibitor
suppresses the inflammation and joint damage of bacterial cell wall-induced
arthritis. J. Exp. Med 190, 535-542.

Spoden, G., Freitag, K., Husmann, M., Boller, K., Sapp, M., Lambert, C., and Florin, L.
(2008). Clathrin- and caveolin-independent entry of human papillomavirus type
16--involvement of tetraspanin-enriched microdomains (TEMs). PLoS ONE 3,
e3313.

Stanley, M. A., Pett, M. R., and Coleman, N. (2007). HPV: from infection to cancer.
Biochem. Soc. Trans 35, 1456-1460.

Sterk, L. M. T., Geuijen, C. A. W., van den Berg, J. G., Claessen, N., Weening, J. J., and
Sonnenberg, A. (2002). Association of the tetraspanin CD151 with the laminin-
binding integrins alpha3beta1, alpha6beta1, alpha6beta4 and alpha7beta1 in cells
in culture and in vivo. J. Cell. Sci 115, 1161-1173.

Stern, P. L. (2005). Immune control of human papillomavirus (HPV) associated
anogenital disease and potential for vaccination. J. Clin. Virol 32 Suppl 1, S72-81.

Stoler, M. H. (2000). Human papillomaviruses and cervical neoplasia: a model for
carcinogenesis. Int J Gynecol Pathol 19, 16-28.

Stubenrauch, F., and Laimins, L. A. (1999). Human papillomavirus life cycle: active and
latent phases. Semin. Cancer Biol 9, 379-386.

Sun, X. Y., Frazer, I., Müller, M., Gissmann, L., and Zhou, J. (1995). Sequences required
for the nuclear targeting and accumulation of human papillomavirus type 6B L2
protein. Virology 213, 321-327.

Swairjo, M. A., and Seaton, B. A. (1994). Annexin Structure and Membrane Interactions:
A Molecular Perspective. Annu. Rev. Biophys. Biomol. Struct. 23, 193-213.

Syrjänen, K., Hakama, M., Saarikoski, S., Väyrynen, M., Yliskoski, M., Syrjänen, S.,
Kataja, V., and Castrén, O. (1990). Prevalence, incidence, and estimated life-time
risk of cervical human papillomavirus infections in a nonselected Finnish female
population. Sex Transm Dis 17, 15-19.

Syrovets, T., Jendrach, M., Rohwedder, A., Schule, A., and Simmet, T. (2001). Plasmin-
induced expression of cytokines and tissue factor in human monocytes involves
AP-1 and IKK{beta}-mediated NF-{kappa}B activation. Blood 97, 3941-3950.

119
Syrovets, T., Tippler, B., Rieks, M., and Simmet, T. (1997). Plasmin Is a Potent and
Specific Chemoattractant for Human Peripheral Monocytes Acting Via a Cyclic
Guanosine Monophosphate-Dependent Pathway. Blood 89, 4574-4583.

Tan, P., Anasetti, C., Hansen, J. A., Melrose, J., Brunvand, M., Bradshaw, J., Ledbetter,
J. A., and Linsley, P. S. (1993). Induction of alloantigen-specific
hyporesponsiveness in human T lymphocytes by blocking interaction of CD28
with its natural ligand B7/BB1. J. Exp. Med 177, 165-173.

Todd, R. W., Roberts, S., Mann, C. H., Luesley, D. M., Gallimore, P. H., and Steele, J. C.
(2004). Human papillomavirus (HPV) type 16-specific CD8+ T cell responses in
women with high grade vulvar intraepithelial neoplasia. Int. J. Cancer 108, 857-
862.

Tomee, J. F., Hiemstra, P. S., Heinzel-Wieland, R., and Kauffman, H. F. (1997).
Antileukoprotease: an endogenous protein in the innate mucosal defense against
fungi. J. Infect. Dis 176, 740-747.

Vambutas, A., DeVoti, J., Pinn, W., Steinberg, B. M., and Bonagura, V. R. (2001).
Interaction of human papillomavirus type 11 E7 protein with TAP-1 results in the
reduction of ATP-dependent peptide transport. Clin. Immunol 101, 94-99.

Villadangos, J. A., and Schnorrer, P. (2007). Intrinsic and cooperative antigen-presenting
functions of dendritic-cell subsets in vivo. Nat. Rev. Immunol 7, 543-555.

de Villiers, E. (2001). Taxonomic classification of papillomaviruses. Pap. Report 12, 57-
63.

Visser, J., Nijman, H. W., Hoogenboom, B., Jager, P., van Baarle, D., Schuuring, E.,
Abdulahad, W., Miedema, F., van der Zee, A. G., and Daemen, T. (2007).
Frequencies and role of regulatory T cells in patients with (pre)malignant cervical
neoplasia. Clin. Exp. Immunol 150, 199-209.

de Vos van Steenwijk, P. J., Heusinkveld, M., Ramwadhdoebe, T. H., Löwik, M. J., van
der Hulst, J. M., Goedemans, R., Piersma, S. J., Kenter, G. G., and van der Burg,
S. H. (2010). An Unexpectedly Large Polyclonal Repertoire of HPV-Specific T
Cells Is Poised for Action in Patients with Cervical Cancer. Cancer Res. Available
at: http://www.ncbi.nlm.nih.gov/pubmed/20233872 [Accessed March 28, 2010].

Waisman, D. M. (1995). Annexin II tetramer: structure and function. Mol. Cell. Biochem
149-150, 301-322.

Walboomers, J., Jacobs, M., Manos, M., Bosch, F., Kummer, J., Shah, K., Snijders, P.,
Peto, J., Meijer, C., and Munoz, N. (1999). Human papillomavirus is a necessary
cause of invasive cervical cancer worldwide. J. Pathol. 189, 12-19.
120

Wang, X., Moscicki, A., Tsang, L., Brockman, A., and Nakagawa, M. (2008). Memory T
cells specific for novel human papillomavirus type 16 (HPV16) E6 epitopes in
women whose HPV16 infection has become undetectable. Clin. Vaccine Immunol
15, 937-945.

Weide, I., Tippler, B., Syrovets, T., and Simmet, T. (1996). Plasmin is a specific stimulus
of the 5-lipoxygenase pathway of human peripheral monocytes. 76, 561-8.

Welters, M. J. P., de Jong, A., van den Eeden, S. J. F., van der Hulst, J. M.,
Kwappenberg, K. M. C., Hassane, S., Franken, K. L. M. C., Drijfhout, J. W.,
Fleuren, G. J., Kenter, G., et al. (2003). Frequent display of human papillomavirus
type 16 E6-specific memory t-Helper cells in the healthy population as witness of
previous viral encounter. Cancer Res 63, 636-641.

Weng, X., Luecke, H., Song, I. S., Kang, D. S., Kim, S. H., and Huber, R. (1993). Crystal
structure of human annexin I at 2.5 A resolution. Protein Sci 2, 448-458.

Werness, B. A., Levine, A. J., and Howley, P. M. (1990). Association of human
papillomavirus types 16 and 18 E6 proteins with p53. Science 248, 76-79.

Wiedow, O., Harder, J., Bartels, J., Streit, V., and Christophers, E. (1998).
Antileukoprotease in human skin: an antibiotic peptide constitutively produced by
keratinocytes. Biochem. Biophys. Res. Commun 248, 904-909.

Williams, K. J., and Fuki, I. V. (1997). Cell-surface heparan sulfate proteoglycans:
dynamic molecules mediating ligand catabolism. Curr. Opin. Lipidol 8, 253-262.

Williams, S. E., Brown, T. I., Roghanian, A., and Sallenave, J. (2006). SLPI and elafin:
one glove, many fingers. Clin. Sci 110, 21-35.

Woodman, C. B., Collins, S., Winter, H., Bailey, A., Ellis, J., Prior, P., Yates, M.,
Rollason, T. P., and Young, L. S. (2001). Natural history of cervical human
papillomavirus infection in young women: a longitudinal cohort study. Lancet
357, 1831-6.

Wright, J. F., Kurosky, A., and Wasi, S. (1994). An Endothelial Cell-Surface Form of
Annexin II Binds Human Cytomegalovirus. Biochemical and Biophysical
Research Communications 198, 983-989.

Yan, M., Peng, J., Jabbar, I. A., Liu, X., Filgueira, L., Frazer, I. H., and Thomas, R.
(2004). Despite differences between dendritic cells and Langerhans cells in the
mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime
T cells. Virology 324, 297-310.

121
Yang, R., Day, P. M., Yutzy, W. H., Lin, K., Hung, C., and Roden, R. B. S. (2003). Cell
surface-binding motifs of L2 that facilitate papillomavirus infection. J. Virol 77,
3531-3541.

Yang, R., Yutzy, W. H., Viscidi, R. P., and Roden, R. B. S. (2003). Interaction of L2 with
beta-actin directs intracellular transport of papillomavirus and infection. J. Biol.
Chem 278, 12546-12553.

Yoon, C. S., Kim, K. D., Park, S. N., and Cheong, S. W. (2001). alpha(6) Integrin is the
main receptor of human papillomavirus type 16 VLP. Biochem. Biophys. Res.
Commun 283, 668-673.

Zeitler, J., Hsu, C. P., Dionne, H., and Bilder, D. (2004). Domains controlling cell
polarity and proliferation in the Drosophila tumor suppressor Scribble. J. Cell
Biol 167, 1137-1146.

Zhang, B., Li, P., Wang, E., Brahmi, Z., Dunn, K. W., Blum, J. S., and Roman, A.
(2003). The E5 protein of human papillomavirus type 16 perturbs MHC class II
antigen maturation in human foreskin keratinocytes treated with interferon-
gamma. Virology 310, 100-108.

Zhang, J., Guo, B., Zhang, Y., Cao, J., and Chen, T. (2010). Silencing of the annexin II
gene down-regulates the levels of S100A10, c-Myc, and plasmin and inhibits
breast cancer cell proliferation and invasion. Saudi Med J 31, 374-381.

Zhang, J., and McCrae, K. R. (2005). Annexin A2 mediates endothelial cell activation by
antiphospholipid/anti-{beta}2 glycoprotein I antibodies. Blood 105, 1964-1969.

Zhao, K. N., Sun, X. Y., Frazer, I. H., and Zhou, J. (1998). DNA packaging by L1 and L2
capsid proteins of bovine papillomavirus type 1. Virology 243, 482-491.

Zhou, J., Sun, X. Y., Louis, K., and Frazer, I. H. (1994). Interaction of human
papillomavirus (HPV) type 16 capsid proteins with HPV DNA requires an intact
L2 N-terminal sequence. J. Virol 68, 619-625.

Zhou, J., Sun, X. Y., Stenzel, D. J., and Frazer, I. H. (1991). Expression of vaccinia
recombinant HPV 16 L1 and L2 ORF proteins in epithelial cells is sufficient for
assembly of HPV virion-like particles. Virology 185, 251-257.

Abstract (if available)

Abstract Persistent high-risk human papillomavirus (HPV) infection is causally associated with the generation of several cancers, including cervical cancer. While most women infected with HPV clear their lesions, the average time to clearance is one year, indicating that HPV has evolved specific mechanisms that allow it to circumvent the human immune system and establish persistent infections. Currently, there is no treatment for persistent HPV infections, and therefore our long-term goal is to elucidate how HPV escapes immune mediated clearance in order to develop novel ways to treat HPV infections and HPV-induced lesions. During its natural life cycle, HPV infects the basal cells of the epithelium and interacts with Langerhans cells (LC), the resident antigen presenting cells of the epithelium. Due to their location, LC are responsible for initiating immune responses against pathogens entering the epithelium. However, HPV does not activate LC, preventing the induction of an HPV specific adaptive immune response and implicating an HPV immune escape mechanism that targets human LC. The HPV protein responsible for inducing this immune escape has not been determined.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

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

PDF

Human papillomavirus type 16 entry via the annexin A2 heterotetramer leads to infection and immune evasion

PDF

Investigating immune escape mechanisms between high & low risk mucosal human papillomavirus genotypes and cutaneous human papillomavirus genotypes

PDF

The annexin A2 heterotetramer as a host cell cofactor in human papillomavirus infection

PDF

Theta defensins inhibit high-risk human papillomavirus infection through charge-driven capsid clustering

PDF

Infection by herpes simplex virus type 1 increases human papillomavirus type 16 uptake and infection through the annexin A2/S100A10 heterotetramer

PDF

Contribution of herpes simplex virus type 2 in the aquisition of human papillomavirus infection

PDF

Human myeloid-derived suppressor cells in cancer: Induction, functional characterization, and therapy

PDF

Immune escape in head and neck squamous cell carcinoma

PDF

Rationalized immunotherapy by immune signature characterization

PDF

Generation and characterization of fully human anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies

PDF

Immune responses by glia during neurotropic coronavirus induced encephalomyelitis

PDF

Lym-1 epitope targeted chimeric antigen receptor (CAR) T cells for the immunotherapy of cancer

PDF

Studying the relationship of annexin A2, langerin, and Birbeck granules

PDF

Generation and characterization of humanized anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies

PDF

The effects of hepatitis C virus infection on host immune response and signaling pathways

PDF

Tri-specific T cell engager immunotherapy targeting tumor initiating cells

PDF

Developing peptide and antibody-mimetic ligands for the cell surface receptors β2AR and DC-SIGN

PDF

Investigation into the use of repurposed influenza vaccines for immunotherapy of HPV-induced tumors

PDF

Characterization of invariant natural killer T cells in a novel humanized HBV-transgenic model

Asset Metadata

Creator Raff, Adam Benjamin (author)

Core Title The silenced sentinel: a human papillomavirus type 16 immune evasion mechanism targeting Langerhans cells

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Systems Biology

Publication Date 07/30/2011

Defense Date 05/11/2010

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

Tag annexin A2,antigen presenting cells,ANXA2,HPV,human papillomavirus,immune escape,immune evasion,L2,Langerhans cells,minor capsid protein,OAI-PMH Harvest,p11,receptor,S100A10

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Kast, W. Martin (committee chair), Chen, Si-Yi (committee member), Kaslow, Harvey R. (committee member), Woodley, David (committee member)

Creator Email adam.raff@gmail.com,araff@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3237

Unique identifier UC155432

Identifier etd-Raff-3836 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-355460 (legacy record id),usctheses-m3237 (legacy record id)

Legacy Identifier etd-Raff-3836.pdf

Dmrecord 355460

Document Type Dissertation

Rights Raff, Adam Benjamin

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

Repository Email cisadmin@lib.usc.edu