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

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INVESTIGATING IMMUNE ESCAPE MECHANISMS BETWEEN
HIGH & LOW RISK MUCOSAL HUMAN PAPILLOMAVIRUS GENOTYPES AND
CUTANEOUS HUMAN PAPILLOMAVIRUS GENOTYPES

by

Carly Anne Movius

___________________________________________________________

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

May 2011

Copyright 2011 Carly Anne Movius

ii

Acknowledgements

First and foremost, I would like to thank my parents and sister for their continual
support and love. Thank you for encouraging me to always to reach for and pursue my
dreams. Thanks to all of my family members for guidance and encouragement!!!
Darren, my love and best friend, thanks for always being there for me day in and
day out. I appreciate your support and love!
I would like to acknowledge my mentors/PI Dr. W. Martin Kast and Dr. Diane Da
Silva. Thanks for allowing me to be part of the Kast lab to work on this MS thesis
project. Thanks for the training and guidance through the duration of my time with the
lab. I feel as though I have grown both as an individual and as a scientist during my time
in the lab and feel prepared to start a PhD program after my training in your lab.

iii

Table of Contents

Acknowledgements iii

List of Figures vi

Abstract v

Introduction 1 Human Papillomavirus 4
Pathology of Papillomavirus Infection 1
Table 1: HPV Genotypes Associated with Benign and
Malignant Lesions 2
Human Papillomavirus Type 16 and Langerhans Cells 4
L2 Capsid Protein is Responsible for Immune Escape Mechanism 10
Clinical Treatment of Human Papilloma Viral Infection 15
Toll-Like Receptor Agonists Reverse Immune Suppression 17
My Work 20

Chapter One: Materials and Methods 23

Chapter Two: Results 29
Surface Activation Markers in LCs are Not Up-Regulated
after Treatment with High and Low Risk HPV Genotypes and
Cutaneous HPV5 29
The ability for LCs to Migrate is Hindered after HPV Treatment 32
HPV Genotypes Prevent Cytokines and Chemokines Secretion
in LCs 34
Region in L2 Responsible for HPV16 Immune Evasion on LCs
is Conserved Among HPV Genotypes 37
TLR Agonists Reverse Induce Up-Regulation of Cell Surface
Activation Markers In LCs After HPV Treatment 40
TLR Agonists R848 and PolyI:C Stimulate Migration in LCs
Suppressed by HPV 44

Conclusions 48

References 53

iv

List of Figures

Figure 1: HPV entry into basal keratinocytes 5

Figure 2: Immune evasion of Human Papillomavirus Type 16 in 9
Langerhans Cells

Figure 3: Neutralizing L2 minor capsid sequences involved 13
in Human Papillomavirus uptake

Figure 4: L2 minor capsid prevents LC maturation 14

Figure 5: Reversal of Human Papillomavirus Type 16 Immune 19
Suppression on Langerhans Cells by TLR Agonists

Figure 6: Activation Markers in LCs are Similar after Treatment with 31
Cutaneous HPV and with High and Low Risk Mucosal HPV
Genotypes

Figure 7: Migration Response of LC in Response to Treatment with 33
Cutaneous HPV Genotypes and High & Low Risk Mucosal
HPV Genotypes

Figure 8: Cytokine and Chemokine Levels in LCs are Similar After 36
Treatment with High and Low Risk HPV Genotypes and
Cutaneous HPV Genotypes

Figure 9: Alignment and Conserved Consensus Sequence of HPV L2 39
Proteins to HPV16 L2 (aa 108-126)

Figure 10: TLR Agonists Reverse the Immune Suppression in 42
LCs Caused by HPV Genotypes and Surface Activation
Markers are Up-Regulated

Figure 11: TLR Agonists R848 and PolyIC Reverse the immune 46
Suppression in LCs caused by HPV genotypes and
Allow for Migration

v

Abstract

Cervical cancer is associated with high risk HPV genotypes 93 % of the time.
HPV infections can take months and even up to a year to clear, and 30 % of HPV
infections will develop into cervical cancer. A novel immune suppression mechanism
used by HPV16 has been characterized in Langerhans Cells (LCs), such that there is an
activation of PI3K pathway and a down regulation of AKT pathways, mediated in part by
PP2A. HPV16 suppresses phenotypic activation and immune function in LCs. This
study reveals that other high risk mucosal HPV genotypes (HPV18, HPV31 and HPV45)
and a low risk mucosal HPV genotype (HPV11) and a cutaneous HPV genotype (HPV5)
share this particular immune escape mechanism based on a conserved sequence in L2
minor capsid protein (aa 108-126). However, a cutaneous HPV genotype (HPV1) does
not share this immune escape mechanism in LCs, as there is little homology to the
HPV16 L2 sequence (aa 108-126). In addition this study shows that this particular
immune suppression mechanism can be reversed with intracellular toll-like receptor
(TLR) agonists 3 and 8, but only marginally with a TLR3 agonist. This demonstrates the
possibility for therapeutic compounds such as these which could be developed to treat the
10 % of the world’s population currently estimated to be infected with HPV, as an
alternative treatment to ablative surgical procedures.
1

Introduction

Human Papillomavirus

Human Papillomavirus (HPV) has been linked to cervical, vulvar, anal, penile and
head and neck cancers. Epidemiological studies have found that 93% of cervical cancers
lesions test positive for HPV DNA when screened by PCR (Bosch, Manos et al. 1995;
Allameh, Moghim et al. 2011). Approximately 75% of all sexually active women
between the ages of 18 and 45 will acquire and HPV infection at least once (Kanodia,
Fahey et al. 2007; Allen, Lewis et al. 2010). The Centers for Disease Control (CDC)
estimates that 9-13% of the world’s population is currently infected with HPV and
expects 500,000 new HPV infections emerging in the global population each year. The
greatest burden of cervical cancer is in developing countries, whereas approximately 83%
of total cervical cancer cases occur versus developed countries. Yet, the mortality
associated with this type of cancer is 50% (Parkin and Bray 2006; Kyrgiou, Valasoulis et
al. 2010).
HPV belongs to the Papillomaviridae (PV) family and have a tropism for
cutaneous or mucosal epithelia, causing warts and precancerous lesions localized at
specific sites on the body. A summary of the diseases associated with the various types
of mucosal and cutaneous HPVs are provided in Table 1. PVs have been found to infect
mammals and birds, yet the class of PV that infects the epithelia of one species will rarely
infect the epithelia of other species. This particular family of viruses is encapsulated,
non-enveloped containing double-stranded circular DNA with a genome size of 8 kB (de
Villiers, Fauquet et al. 2004; Mistry, Simonsson et al. 2007; Allen, Lewis et al. 2010)
2

Table I: HPV Genotypes Associated with Benign and Malignant Lesions; Source: (Bharti, Shukla et al.
2009)
Disease HPV type(s) associated
Benign and pre-cancerous Lesions
Plantar warts 1, 2, 4, 63
Common warts 2, 1, 7, 4, 26, 27, 29, 41, 57, 65, 77, 1, 3, 4, 10, 28
Flat warts 3, 10, 26, 27, 28, 38, 41, 49, 75, 76
Other cutaneous lesions (e.g., epidermoid cysts) 6, 11, 16, 30, 33, 36, 37, 38, 41, 48, 60, 72, 73
Epidermodysplasia verruciformis 2, 3, 10, 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23,
24, 25, 36, 37, 38, 47, 50
Recurrent respiratory papillomatosis 6, 11
Laryngeal papillomatosis 16,11,6,10
Focal epithelial hyperplasia of Heck 13, 32
Conjunctival papillomas 6, 11, 16
Condyloma Acuminate (genital warts) 6, 11, 30, 42, 43, 45, 51, 54, 55, 70
Cancerous lesions
Cervical carcinoma 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
Oral carcinoma 16, 18
Esophageal carcinoma 16, 18
Laryngeal carcinoma 6, 11, 16, 18
Conjunctival carcinoma 6, 11, 16, 18
Anal carcinoma 16, 18, 31, 33
Vaginal carcinoma 16, 18, 31, 33
Vulvar carcinoma 16, 18, 31, 33
Lung carcinoma 18
Bladder carcinoma 16
Penile carcinoma 16, 18, 31, 33
Non-melanoma skin cancer 5, 8
Table I: HPV Genotypes Associated with Benign and Malignant Lesions; Source:
(Bharti, Shukla et al. 2009)

3

Over 200 genotypes of HPV have been sequenced and are clustered into families
based on the major L1 capsid, the type of infection caused (whether mucosal or
cutaneous) and their ability to either induce cancer or persist as benign warts (Bharti,
Shukla et al. 2009). From the alpha-paillomavirus genus, there are approximately 18
HPV genotypes considered high-risk mucosal that are known to lead to the progression of
cervical cancer; such types include HPV16, HPV18, HPV31, HPV33, HPV34, HPV35,
HPV35, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV67, HPV68,
HPV69, HPV70, HPV73 and HPV85. The types most frequently associated with cervical
cancer are HPV16 and 18 (de Villiers, Fauquet et al. 2004; Kanodia, Da Silva et al.
2008). Low risk mucosal HPV genotypes from alpha-papillomavirus group are
associated with benign condylomas and the highest occurrence of genital warts is
associated with HPV6 and HPV11 (de Villiers, Fauquet et al. 2004; Kyrgiou, Valasoulis
et al. 2010). Certain HPV genotypes are also known to have a tropism for the cutaneous
epithelium. Like the mucosal HPV genotypes, there are cutaneous types that are also
known to cause malignant lesions in immune compromised individuals, such as HPV5
and HPV8 (Forslund 2007). Additionally, there are cutaneous HPV genotypes which
only cause benign warts, such as HPV1 which is associated with foot warts (Carter,
Hagensee et al. 1993).

4

Pathology of Human Papillomavirus Infections

HPV is transferred to the anogenital tract on a new host as a sexually transmitted
disease. The virus particles end up infecting the basal keratinocytes after initially binding
to the basement membrane (BM) resulting from micro tears in the epithelium.
Attachment is to an abundant heparin sulfate proteoglycan (HSPG) receptor of the BM
via the major L1 capsid of HPV, and then the virus is transferred to the primary HSPG
receptor of basal cells (Horvath, Boulet et al. 2010). Following binding to the initial
receptor the virus undergoes a conformational change which exposes the N-terminus of
the minor L2 capsid protein, and a conserved sequence among all HPV genotypes is
subjected to furin cleavage (Day and Schiller 2009). The result of this furin cleavage is
such that there is an exposed site on the L2 capsid that is vital to binding a secondary
receptor which is responsible for HPV uptake (Fig. 1) (Day and Schiller 2009; Horvath,
Boulet et al. 2010). Neutralization studies have shown that aa 108-120 on the L2 minor
capsid becomes exposed after furin cleavage at an adjacent site has a role in binding to
the secondary receptor (Kawana, Kawana et al. 2001; Yang, Day et al. 2003; Sapp and
Bienkowska-Haba 2009). Our lab has recently identified a potential receptor responsible
for viral uptake.

5

Figure 1: Depiction of HPV entry into the basal keratinocytes. Initial binding takes
place by the virus binding to a HPSG receptor by the major L1 capsid protein on the
ECM. The virus then is passed to a HPSG on the surface of the cell in which furin
cleavage takes places. This critical event allows for an exposed site in the minor capsid,
L2 of aa 108 – 120 to bind to a secondary receptor to allow for viral uptake. Source: (Day
and Schiller 2009)

6

Because HPV infects cells above the basement membrane, the professional
Antigen Presenting Cells (APCs) HPV interacts with are Langerhans Cells (LCs). APCs
initially recognize pathogens and are responsible for mounting an appropriate immune
response against the pathogen for the successful clearance of an infection. LCs are the
resident APC that reside above the basement membrane in the epidermis, while Dendritic
Cells (DCs) occupy the dermis, and thus are below the basement membrane
(Cunningham, Abendroth et al. 2010). Although HPV has been demonstrated to interact
with both LCs and DCs (Da Silva, Velders et al. 2001; Bousarghin, Hubert et al. 2005) it
is critical to understand the interaction between the LC and HPV, as this is the only APC
that HPV encounters during an infection. Moreover, the pathway leading to HPV16
uptake by LCs as shown by recent work done in our laboratory has shown that the
mechanism is the same as in the basal cells of the epithelium. The difference in HPV
uptake in LCs is that uptake is associated with langerin, a surface molecule constitutively
expressed on LCs involved in antigen uptake and processing (Merad, Ginhoux et al.
2008; Schiller, Day et al. 2010).

Human Papillomavirus Type 16 and Langerhans Cells

HPV interacts with LCs during infection. LCs are APCs responsible for capturing
and processing antigens in the epidermal epithelial tissue layers and presenting them in
restricted MHC class molecules to antigen-specific T-cells which mount a specific
immune response against a pathogen. Prior to LCs encountering pathogens, the APCs are
in an inactivated or immature resting state characterized by low expression levels of
7

surface activation markers such as MHC class II, CD80 and CD86, a low expression level
of CCR7 which causes LCs to be non-migratory and unable to respond to signals secreted
by the lymph node, and cytokines and chemokines are not secreted from LCs. However,
when LCs encounter pathogens they undergo maturation and are in an active state
capable of mounting an immune response against a pathogen. Immune activated LC are
characterized by an up-regulation of surface activation markers such as MHC class II,
CD80 and CD86, as well as an up-regulation of CCR7 allowing for chemokine directed
migration to the draining lymph node, and cytokines and chemokines which orchestrate
the immune system are released in the tissues. The activation of LCs interacting with
specific T-cells results in a T-cell response against the pathogen (Banchereau and
Steinman 1998; Bousarghin, Hubert et al. 2005; Fausch, Fahey et al. 2005).
Fausch and others from our laboratory have discovered a novel immune escape
mechanism used by HPV16 on LC in which the mechanism of uptake causes an
activation of the PI3K pathway, while the AKT, MAPK, ERK and NF-κB pathways are
down regulated; and this disruption of signaling is mediated in part PP2A (Fig 2C).
Interestingly, signaling disruption occurs just 15 minutes after HPV has interacted with
LC (Fausch, Fahey et al. 2005). Fausch et al. demonstrated that indeed this specific
signaling modulation in LCs by the uptake of HPV16 prevents human-derived LC from
maturing into active APCs capable of stimulating a CD8
+
T-Cell response. Thus, the
expression of surface activation markers is not up regulated (Fig 2A), in vitro migration
of LC does not take place (Fig 2B, and specific cytokines and chemokines such as IL-12
are not secreted which is needed for a Th1 response (Fausch, Da Silva et al. 2002).
8

Stimulating a Th1 response induces the secretion of IFNγ, IL-2 and TGFβ from T-cells
critical for mediating the clearance of viral pathogens such as HPV (Pinto and Arredondo
et. al., 2005).

9

Figure 2A-2C: Immune evasion of Human Papillomavirus Type 16 in Langerhans
Cells A. Expression of surface activation markers: MHC II-DR, DP, DQ, MHC I-
A,B,C, CD80, CD86 and CCR7 on LCs in response to i. no treatment ii. CD40L (a
known activator of LCs), iii. HPV16 VLP and iv. VLP in addition of CD40L. B. In Vitro
Migration of LC as a response to treatment with PBS, TNFα, LPS and HPV16 VLP,
where by TNFα and LPS are known to stimulate migration in APCs. C. HPV16 Actives
PI3K pathway and downregulates AKT, ERK, MAPK and NF-κB pathways and is
mediated by PP2A, and is the mechanism which defines the immune evasion mechanism.
Sources: Fig 2Aand2B modified from (Fausch, Da Silva et al. 2002), Copyright 2009.
The American Association of Immunologists, INC. Fig 2C modified from (Fausch, Fahey
et al. 2005) Copyright 2005. The American Association of Immunologists, INC.

10

L2 Minor Capsid Protein Mediates Immune Escape Mechanism

The specific uptake of HPV into LC has been debated and remains controversial.
There is experimental evidence for both a cavoelaei-dependant pathway of entrance as
well as a clathrin-independent, caveolai-independent, actin-independent mode of uptake.
The difference between the mode of uptake is in the structure of the virus-like particles
(VLPs) used in the experiments. Live HPV cannot be generated in the lab, as the virus
life cycle is linked to the differentiation the epithelial cells. Researchers can either
produce a VLP, expressing the major L1 capsid in addition to the minor L2 capsid protein
or a particle consisting only of L1 can be generated. The difference in producing either
HPV VLP L1 or HPV VLP L1L2 and then testing the mode of uptake results in two
different results. The VLPs generated in the laboratory that most reflect the virus found
in nature is HPV VLP L1L2, since HPV found in nature contains L1 and L2. Thus, the
mechanism of uptake is by a clathrin-independent, caveolaei-independent and actin-
independent pathway (Fahey, Raff et al. 2009; Pereira, Hitzeroth et al. 2009; Sapp and
Bienkowska-Haba 2009; Horvath, Boulet et al. 2010; Schiller, Day et al. 2010).
Elucidating the mechanisms of how HPV is taken up in LCs, in combination of
with neutralization and binding assays (Kawana, Yoshikawa et al. 1999; Kawana,
Kawana et al. 2001; Gambhira, Karanam et al. 2007) reveals stretches of highly
conserved L2 sequences among HPV genotypes (aa 108-126 and aa 17-36) (Fig 3) which
bind to epithelial cells and are responsible HPV uptake. Fahey and others in our
laboratory discovered that the presence of the L2 protein in the virus capsid is responsible
for the immune evasion in LCs (Fahey, Raff et al. 2009). LCs were treated with either
11

HPV16 L1 VLP or HPV16 L1L2 VLP and immune function was determining by
evaluating the expression levels of surface activation markers, testing for in vitro
migration, detecting the presence of cytokines and chemokines and testing the ability for
these capsids to induce antigen-specific CD8
+
T-Cell responses. LCs treated with HPV16
L1 VLP became mature, resulting in the loss of the immune evasion (Fig 4). The
opposite was true for LCs treated with HPV16 L1L2 resulting in LCs that remained
immature (Fig. 4). The presence of L2 in the virus capsid prevented expression of
surface activation markers such as MHC class II, CD80 and CD86 (Fig 4A), prevented in
vitro CCL21 migration (Fig. 4B), prevented the secretion of cytokines and chemokines
such as the TNFα, IL-12p70, IL-6, IL-8, IP-10, MCP-1 and RANTES (Fig. 4C).
Furthermore, HPV16 capsids with the L2 protein prevented a CD8
+
T-cell response (Fig.
4D) (Fahey, Raff et al. 2009). LCs in an inactivated state presenting HPV16 antigens to
T-cells have potentially a tolerizing effect on T-cells and thus, an immune response
against HPV does not occur, leading to a persistent infection.
In addition to testing LC immune function, Fahey et. al. investigated the
disruption of the PI3K/AKT signaling cascade. Capsids comprised of L1 were
insufficient in causing a disruption of the PI3K/AKT signaling cascade; however the
L1L2 capsid was effective in causing activation of PI3K and down regulating the AKT
pathway. The results indicated that the presence of L2 is needed to disrupt the
PI3K/AKT signaling cascade leading to immune suppression in LCs (Fausch, Fahey et al.
2005; Fahey, Raff et al. 2009). There are highly conserved L2 sequences between HPV
genotypes. These conserved regions are involved in HPV uptake in LCs leading to
12

immune escape; and therefore, it is plausible that the immune escape mechanism
mediated by the minor L2 capsid protein may be conserved among other high risk and
low risk mucosal HPV genotypes (Fahey, Raff et al. 2009).

13

Figure 3A-3B: Neutralizing L2 Minor Capsid Sequences Involved In HPV Uptake.
A. Mabs neutralize a highly conserved aa 108-120 on L2 capsid protein among HPV
genotypes. Experiments involving this stretch of amino acids revealed that this region is
involved in HPV uptake: Source B. Mab were found to neutralize aa 17-36 on L2 minor
capsid protein highly conserved among HPV genotypes. Sources: Fig 3A modified from
(Kawana, Yoshikawa et al. 1999) with permission from J. of Virology (#
2639611339149) Fig 3B modified from (Gambhira, Karanam et al. 2007) with
permission from J. of Virology; (# 2639610984100.

14

Figure 4A-4D: L2 Minor Capsid Prevents LC Maturation A. LCs when treated with
HPV16 L1 allowed for the expression of surface activation molecules - MHC II, CD80
and CD86. HPV16 L1L2 prevented the up regulation the of surface activation markers.
B. HPV16 VLP L1 caused CCL21 directed migration of LC in contrast to HPV16 VLP
L1L2 as migration did not occur. C. LCs were treated with 1. No treatment 2. LPS 3.
HPV16 L1 and 4. HPV16 L1L2. LCs secreted cytokines and chemokines treated with
HPV16 L1. L1L2 prevented secretion of chemokines and cytokines into the milieu. 4.
The ability for LCs to elicit a T-cell as a result of L1 vs L1L2 capsids of the HPV16 VLP
was determined. L1 only capsids caused a robust T-cell response whereas addition of L2
prevented this T-Cell response. Source: Figure Adapted from (Fahey, Raff et. al. 2009)
Copy Right 2009. The American Association of Immunolgoists, Inc.

15

Clinical Treatment of Human Papilloma Viral Infections

The immune suppression HPV16 induces on LC partially explains why HPV
infections may take up to several months to a year to clear clinically (Fahey, Raff et al.
2009). HPV infections that are diagnosed in abnormal Pap smears are categorized as
either low-grade or high-grade squamous intraepithelial lesions or categorized by the
severity of the cervical intraepithelial neoplastic (CIN) lesions. The majority of patients
diagnosed with CIN lesions are women in their child bearing years. Therapeutic
treatments for lesions focus on cancer prevention rather than fighting viral infection. If
the lesion is low grade patients are monitored closely over two years with more frequent
Pap smears in hopes that lesions spontaneously regress. High grade CIN2 and CIN3
lesions are removed by Loop electrosurgical excision procedure (LEEP) in which
surgeons carve out tissue appearing neoplastic. Surgical ablative procedures on CIN2
lesions reappear in 10% of the cases while excised CIN3 lesions are associated with
100% recurrence (Bharti, Shukla et al. 2009).
HPV may be present in normal looking tissue at the time of surgery and may even
still be infecting the basal epithelial cells. This tissue is not cut away in order to preserve
as much healthy tissue as a means to prevent sterility. However, the most common side
effects in women who undergo removal of CIN lesions is regrowth of the lesion and
complications during pregnancy. Although CIN lesions are precancerous 30% will lead to
cervical cancer demonstrating the need for biomarkers indicating progression toward
cancer. This would enable surgeons to cut away only lesions in danger of progressing
towards cervical cancer. (Bharti, Shukla et al. 2009; Kyrgiou, Valasoulis et al. 2010).
16

Screening programs are effective in developed countries which make-up only 17% of
total cases of cervical cancer. Developing countries lacking proper screening methods
display a disproportionate 83% of total cervical cancer cases, demonstrating the
effectiveness of screening programs (Karanam, Jagu et al. 2009). Setting up the proper
amount of screening programs needed to reduce cervical cancer cases in developing
countries including ablative surgeries and proper follow up care after CIN lesion removal
is very expensive rendering this an impractical idea. Taken together, this demonstrates
the need for therapeutics specifically designed at hindering HPV infections and
stimulating the immune system in clearing an HPV infection.
GARDASIL (Merck, Whitehouse Station, NJ) and CERVARIX (GSK,
Middlesex, UK) are approved preventive vaccines on the market, but remain only
effective at preventing an HPV infection if the person has not been exposed to HPV
before. The vaccines are made against the L1 capsid protein only and show limited cross
neutralization against HPV genotypes for which they are not designed. In the case of
GARDASIL, it only will be effective against HPV16, HPV18, HPV6 and HPV11, while
CERVARIX only has the potential to protect against HPV16 and HPV18 (Karanam, Jagu
et al. 2009; Kyrgiou, Valasoulis et al. 2010). There is still a need for effective therapies
designed against the remaining 13 high risk oncogenic HPV genotypes as well as
treatments for benign genital warts. In addition, this vaccine is marketed as an anti-
cancer vaccine, but the target age of vaccination for effective protection is in both boys
and girls before they are sexually active since the spread of HPV is through sexual
17

activity. Convincing parents to vaccinate young women at this age is a dilemma and
convincing parents to vaccine boys is also a challenging.
Arguably, CERVARIX and GARDASIL have the potential to reduce cervical
cancer cases in developing countries; however, the cost for a full round of the vaccine is
$350 rendering it too expensive for governments to afford without a significant price
reductions. Furthermore, the stability of the vaccine requires proper storage conditions in
areas where clinics are limited in refrigeration units (Pereira, Hitzeroth et al. 2009). Also,
a total of three immunizations are required for immunity against HPV. In remote areas of
developing countries many clinics are designed to offer care to several villages, and the
clinic in relation to a village may be a long distance thus, it may be impractical to expect
patient compliance.

Toll-Like Receptor Agonists Reverse Immune Suppression

Currently on the market, approved for the use against benign anogenital warts is
ALDARA (3M Healthcare, St. Paul, MN) containing 5 % Imiquimod, an intracellular
toll-like receptor (TLR) 7 agonist designed to stimulate the immune system to clear HPV
infections. While Imiquimod (IMQ) is effective at clearing warts, HPV infections
usually reoccur (Kyrgiou, Valasoulis et al. 2010). In addition there have been serious
complications with this immune modulating product including urinary retention,
ulceration and severe eczema at secondary sites other than where the cream is placed
(McQuillan and Higgins 2004; Taylor, Maslen et al. 2006). Researchers from our
laboratory have discovered that intracellular TLR 8 agonists are much more effective at
18

stimulating an immune response in LCs actively being repressed by HPV16 than the TLR
7 agonist IMQ. The TLR 8 agonists found to reverse immune escape in LCs were
Resiquimod (R848) and 3M002 (Fig 5) (Fahey, Raff et al. 2009). The compounds, R848
and 3M002 at minimal recommended concentrations were more effective than IMQ was
at recommended concentrations in stimulating an immune response in LCs treated with
HPV16. Immune function in LCs with TLR 8 agonists showed significantly increased
activation markers (Fig 5a), significantly increased secretion of inflammatory cytokines
and chemokines (Fig 5b) was effective in stimulating migration (Fig 5c) and lastly,
caused a robust CD8
+
T-cell response (Fig 5c). These results are in direct contrast to
those found when IMQ was administered to HPV16 infected LCs, which was only
slightly effective in reversing the immune suppression (Fahey, Raff et al. 2009). Given
the plausibility of HPV16 sharing the same immune escape mechanism as other HPV
genotypes, it begs the question of the effectiveness of the TLR agonists to reverse the
suppression in other high and low risk HPV genotypes.

19

Figure 5A-5D: Reversal of Human Papillomavirus Type 16 Immune Suppression
on Langerhans Cells by TLR Agonists. Source: modified figures from (Fahey, Raff et
al. 2009). Copy Right 2009. The American Association of Immunologists A. TLR 8
agonists R848 and 3M002 had a robust increase in MHC I, MHC II, CD80 and CD86
activation markers when treated with LC that had been treated with HPV16. The
phenotypic activation caused by IMQ was not as effective as TLR 8 the TLR 8 agonists
were. B. R848 and 3M002 is able to reverse the inhibition of inflammatory cytokines
and chemokines such as IL-12p70, involved in viral clearance. IMQ and other TLR 7
agonists do not reverse the suppression of cytokine and chemokine secretion by HPV16
in LCs. C. IMQ does not up-regulate CCR7 to allow for migration to chemokine
CCL21, whereas R848 and 3M002 allows for up-regulation of CCR7 restoring the ability
of LCs to migrate towards CCL21. D. R848 and 3M002 cause a specific T-Cell
response in suppressed LC and IMQ does not cause a specific T-Cells

20

My Work

The L2 capsid protein is a highly conserved among HPV genotypes, and our
laboratory has recently discovered a stretch of amino acids (108-126 ) on the L2 protein
binds to a receptor on LC which is involved in HPV uptake. Subsequently, this uptake
pathway leads to PP2A mediated immune suppression through up-regulation of PI3K
pathways and down regulation of AKT pathways. The L2 protein is responsible for this
immune suppression mechanism in LCs, which are the resident APC at the site of HPV
infections. This active suppression of LCs via HPV16 prevents phenotypic activation of
surface markers, prevents migration and prevents cytokine and chemokine secretion.
This ultimately results in a tolerizing effect on T-Cells preventing an immune response
against HPV (Fahey, Raff et al. 2009).
Since the L2 capsid protein is highly conserved among HPV genotypes, I
investigated if other high and low risk mucosal and cutaneous HPV genotypes share the
same immune escape mechanism as HPV16. The high risk HPV mucosal genotypes
linked to cervical cancer selected were HPV16, HPV18, HPV31 and HPV45. HPV11
was selected to represent low risk HPV mucosal genotypes and is associated with benign
genital warts. The cutaneous HPV genotypes selected were HPV1, associated with
Plantar Warts and HPV5 which arise as cancerous lesions in immune suppressed patients.
The HPV genotypes were made as virus-like particles (VLPs) and pseudovirions (PsVs)
which are the capsid proteins of HPV that self-assemble into VLPs when the proteins are
overexpressed in producer cell lines. These capsids are structurally identical to virions on
the outside, however they lack viral DNA. They also are capable of having producing the
21

same immune suppression in LCs as the HPV virus in nature. To test the hypothesis that
other high and low risk mucosal HPV genotypes, as well as high and low risk cutaneous
HPV genotypes evade the immune system similar to HPV16, immune function in LCs
was assessed. Immune function was tested by analyzing the cell surface expression
levels of MHC class II, CD80 and CD86 by FACS analysis, investigating whether HPV
genotypes prevented the chemokine directed migration like that of HPV16 and by testing
the chemokine and cytokine secretion levels after treatment with different HPV
genotypes. From these investigations, we found that other high and low risk mucosal
HPV genotypes as well as the cutaneous HPV5 genotype show similar immune
suppression in LCs as HPV16. However, LCs exposed to HPV1 become activated,
suggesting that HPV1 infection uses a distinct mechanism of immune escape compared to
the other genotypes tested.
Because HPV1 likely does not share the same immune escape mechanism as
HPV16 with regards to LC interactions, I hypothesized that the region on the HPV16 L2
protein that binds to the receptor on LCs is not homologous to the region on HPV1. To
test this hypothesis and to investigate the homology between HPV genotypes that share
the immune escape mechanism with HPV16, the L2 protein sequences of several
genotypes were compared to the HPV16 L2 protein. Each L2 sequence was aligned
against the L2 protein of HPV16 and the percentage of the conserved amino acids was
calculated in the region that we believe binds to the secondary receptor involved in HPV
uptake. The result confirmed our hypothesis in that there is low homology between
HPV1 and HPV16 in this region due to a proline rich insertion of amino acids that could
22

potentially interrupt the binding of HPV1 L2 to the receptor mediating uptake into the
pathway that leads to LC suppression.
The immune suppression caused by HPV16 on LCs can be reversed rather
robustly by the TLR 8 agonists R848 and 3M002 compared to the TLR 7 agonist, IMQ,
which weakly reverses the immunosuppression (Fahey, Raff et al. 2009). A 5 %
imiquimod cream has been approved for the use in the treatment of benign condylomas,
but there have been reports of serious complications involving urinary retention,
ulceration and cases of eczema on secondary sites other than where the 5 % IMQ cream
has been applied (McQuillan and Higgins 2004; Taylor, Maslen et al. 2006). To test the
hypothesis that the immune suppression caused by other HPV genotypes can be reversed
by intracellular TLR 8 and 3 agonists, immune function was tested by analyzing
phenotypic activation and functional activation. The TLR 8 agonist tested for immune
suppression reversal was R848, while the TLR 3 agonist tested was PolyI:C (InvivoGen,
San Diego, Ca). The results reveal that TLR agonists reverse other HPV genotypes
immune suppression on LCs and demonstrate the potential for either a TLR3 or a TLR8
to be developed and used as therapeutic agent in HPV infections linked to cervical
cancer.

23

Chapter 1: Materials and Methods

Production of Virus-Like-Particles (VLPs) and Pseudovirions (PsVs)

VLPs were produced in the laboratory as for HPV1 L1L2, HPV11 L1L2, HPV16 L1L2
and HPV18 L1L2. SF9 insect cells were seeded in a 175 cm
2
cell culture in TNM-FH
Graces Insect Media containing 10 % FBS, 1X antibiotic/antimycotic and 4 mM
Glutamine. When the cells reached 50 % confluence, they were infected with a seed
stock of bacculovirus stock. Amplified baculovirus was harvested after 7 days and used
to infect Trichoplusia ni (Hi5) insect cells for HPV capsid protein expression. Hi-5 cells
were grown as non-adherent cells in X-Cell 405 serum free media with 1X
antibiotic/antimycotic in spinner flasks and brought to a final concentration of 5-8 x 10
6

cells/ml. Clarified supernatant containing recombinant baculovirus from the Sf9 cells at
an MOI of 10. Hi5 cells were gently rocked in a minimal volume of media in 75 cm
2
tissue culture flasks for three hours. After the initial incubation period, infected Hi-5
cells were transferred to Erlenmeyer flasks with 200 ml of X-Cell Media and were
incubated 72 hours 27° C at 85 rpm. Following incubation cells were centrifuged 10
minutes at 650 x g and were snap frozen until VLP purification. VLPs were first
extracted from insect cells in extraction buffer composed of 5 mM MgCl
2
, 5 mM CaCl
2
,
150 mM NaCl, 0.01% Triton X-100, 20 mM Hepes at a pH of 7.5 with 10 µg/ml E-64
protease inhibitor and 2X Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford,
Il). Sonication was performed followed by a 15 minute centrifugation at 10,000 rpm at
4°C. The supernatant was saved for further purification. Purification of VLPs was
performed sequential ultracentrifugation through a secrose cusion and cesium chloride
24

equilibrium gradient. Supernatants were layered onto 40 % sucrose then centrifuged for
2.5 hours at 25,000 rpm at 4°C. The pellet that remained was re-suspended in 27% CsCl,
sonicated for 10 seconds, placed in 13.9 ml Quickseal tubes (Beckman Coulter, Brea, Ca)
and was centrifuged overnight at 50,000 rpm at 20°C. The VLP band was drawn out of
the Quickseal tube with a 22 gauge needle and re-suspended in 27% CsCl and the
ultracentrifugation was repeated. Finally, the VLPs were dialyzed against 0.5 M
NaCl/PBS solution for 72 hours.
PsVs were generated for HPV5 L1L2, HPV16 L1L2, HPV31 L1L2 and HPV45
L1L2 in 293TT cells. 293TT cells were grown in media containing IMEM, 10% FBS,
with 400 µg/ml hygromycin B (Roche). PsV16 were made as a self-packaging vector
stock as described in Current Protocols (Buck and Thompson 2007). HPV1, HPV5,
HPV18, HPV31 and HPV45 sheLLs plasmids (A gift from Chris Buck, NCl) were
transfected into 293TT cells. The protocol was described in Current Protocols (Buck and
Thompson 2007). A standard salt extraction was performed with additives Benzonase
and Plasmid-Safe and PsVs were maturated overnight (Buck and Thompson 2007). An
optiprep gradient was prepared to purify the PsVs by ultra-centrifugation as described in
standard production protocol of papillomavirus from the Laboratory of Cellular Oncology
(http://home.ccr.cancer.gov/Lco/pseudovirusproduction.htm).
Western blot analysis confirmed the presence of specific L1 and L2 proteins; while an
ELISA with genotype specific neutralizing antibodies confirmed the presence of intact
HPV particles.

25

Generation of Langerhans Cells from Peripheral Blood Monocytes

Langerhans Cells (LCs) were derived from Peripheral Blood Mononuclear Cells
(PBMCs) for the purpose of testing immune function after incubation with Human
Papilloma Virus (HPV) genotypes. PBMCs were obtained through leukapheresis of
healthy anonymous donors and cryopreserved until needed for the immune function
assays. To generate LC, adherent monocytes were cultured for seven days in complete
RPMI-1640 media containing 10% FBS (Omega Scientific, Tarzana, Ca), 2 mM
Glutamax (Life Technologies, Carlsbad, Ca), 10 mM non-essential Amino Acids, 10 mM
sodium pyruvate (Life Technologies) and 50 mM 2-betamercaptaethanol (Sigma-
Alderich, St. Louis, Mo). Cryopreserved cells were thawed, washed twice with media,
plated into a 175 cm
2
tissue culture flask and incubated at 37° C for two hours to allow
monocytes to adhere. Following incubation, non-adherent cells were washed and 40 mLs
of fresh media was added to the tissue culture flask, supplemented with a cytokine
cocktail composed of 1400 U/ml rGM-CSF, 750 U/ml rIL-4 and 10 ng/ml TFGb
(BioSource International, Camarillo, Ca). Cytokines were replenished on day 1, day 3
and day 5 during the seven day culture.

Activation of Langerhans Cells by HPV genotypes

After the LCs had been produced via a seven day culture as described above, the
non-adherent LCs were harvested from the T-175 tissue culture flasks and centrifuged at
300 x g, 5 minutes and washed with Hanks Balanced Salt Solution (HBSS) (Sigma-
Alderich). The LCs were then counted and divided equally in 1 ml of HBSS/2 million
26

cells. Five microgram of desired HPV genotype was used to activate 1 million cells. The
HPV genotypes selected to activate LCs were as follows HPV1 L1L2, HPV5 L1L2,
HPV11 L1L2, HPV16 L1L2, HPV18 L1L2, HPV31 L1L2 and HPV 45 L1L2.
Laboratory generated HPV was used in the assays. Virus-like-particles (VLPs) were
used as for HPV1 L1L2, HPV11 L1L2, HPV16 L1L2 and HPV18 L1L2, while
Pseudovirions (PsV) were used for HPV5 L1L2, HPV31 L1L2 and HPV45 L1L2. LPS
(7µg/ml, Sigma-Alderich) was used as a control. LCs were then incubated with the
specific HPV genotype, LPS or LC only for an hour at 37°C. The cells were then re-
plated in fresh complete media at a concentration of 1 x 10
5
cells/mL with 500 U/ml
rGM-CSF and incubated at 37°C for 48 hours.

Toll-Like Receptor Agonist Activation Assay

Four hours following treatment of LC with the various HPV genotypes or LCs
only, Toll-Like Receptor (TLR) 3 and 8 agonists or 7 µg/ml LPS was added to the HPV
treatments groups and incubated for 48 hours at 37°C. TLR 7/8 agonist was 5µg/ml
imidazoquinoline (R848) (cat# tlrl-r848-5); TLR 7 agonist was 10 µg/ml Imiquimod
(IMQ) (cat# tlrl-imq); and the TLR3 agonist uwas 10 µg/ml poly(I:C) (cat# tlrl-pic-5)
(InvivoGen, San Diego, Ca) Treated cells were subsequently incubated at 37°C for
48hrs assessed for immune function as previously described. (Fahey, Raff et al. 2009).

Flow Cytometric Analysis of Immune Surface Markers on Langerhans Cells

Flow cytometry was used to detect the presence of immune cell surface markers such
as MHC class II, MCH class I, CD80, CD83 and CD86 on Langerhans Cells (LCs).
27

After the seven day culture that generated LCs and following 48 hours of LC activation,
non-adherent cells were harvested from the tissue culture flask and centrifuged 5 minutes
300 x g. LCs were rinsed twice with FACS buffer containing 1X PBS, 2% FBS (Omega
Scientific) and 0.01 % NaN
3
. LCs were stained in FACS buffer on ice, in the dark for 30
minutes with the following antibodies: HLA-DP, DQ, DR-FITC, HLA-A, B, C-FITC,
CD80-FITC, CD83-PE and CD-86-FITC (BD Biosciences). The primary antibodies used
for isotype controls were a rabbit IgG2a and an anti-human CCR7, the secondary
antibody used was goat anti-rabbit IgG-FITC (BD Biosciences). Between the primary
and secondary antibody staining cells were washed twice with FACS buffer. Following
antibody staining, LC were fixed with 2 % paraformaldehyde, protected from light to
prevent quenching of FITC and PE fluorophores and kept at 4°C until the samples were
analyzed by flow cytometry. The samples were run on a Cytomics FC500 Flow
Cytometer Analyzer (Beckman Coulter). The compensation was set using HLA-DP, DQ,
DR-FITC and HLA-A, B, C-PE antibodies (BD Biosciences). Data analysis was
performed by CXP2.2 software provided by Beckman Coulter in which the fold change
in mean fluorescence for the surface activation markers was calculated.

Determining the ability of Langerhans Cells to Migrate via In Vitro Migration
Assay

Migration experiments were performed to detect the ability of a LC to migrate in
vitro mimicking LC migration in vivo. LC treatment groups were harvested 48 hours
post activation, washed with media and brought to a concentration of 1.5 x 10
6
cells/ml.
Migration assays were performed in a Costar 24-well plate containing a semipermeable
28

insert with a pore size of 5.0 µm and a diameter of 6.5 mm. The membrane was pre-
coated with 600 µl RPMI-1640 media overnight and replaced with either fresh media or
media containing 250 ng/ml CCL21 (R&D Systems #366-6C). To the top of the chamber
100 µl of each treatment group was placed in the insert containing 1.5 x 10
5
cells. The
transwell plate was left to incubate for 4 hours at 37 °C. Following incubation cells in
the lower chamber were counted. Cells that migrated to the wells containing the
chemokine CCL21 are cells which have up-regulated CCR7 and will chemotax to the
chemokine, while those cells which have migrated to the chamber lacking CCL21 are
cells that have spontaneously migrated. Migration index is calculated by the number of
cells migrating to CCL21 over the number cells migrating spontaneously. A higher
migration index is indicative of a more migratory LC.

Analysis of Chemokine and Cytokine Secretion from Langerhans Cells by Bioplex
Assay

Supernatant was collected from the treated LCs 48 hours post activation and
immediately frozen. A panel of twelve immune chemokines and cytokines were
analyzed in a 96-well plate assay via a Human Cytokine/Chemokine Milliplex Map kit
(Millipore, Cat# MPXHCYT-60K). The chemokines chosen for analysis were IL-8, IP-
10, MCP-1, MIP-1α, MIP-1β and RANTES. The inflammatory cytokines IL-1β, IL-6 and
TNFα and a suppressive cytokine, IL-10 were analyzed. Two cytokines known to be
involved in an anti-viral mediated response, IL-12p70 and IFNα were analyzed. This
protocol was performed according to the manufactures guidelines. The samples were
then run on a Luminex 100
TM
v. 1.7 with Bio-Plex software (BioRad).
29

Chapter 2: Results
Surface Activation Markers in LCs are Down Regulated by Treatment with High
and Low Risk HPV Genotypes and Cutaneous HPV5

A novel active immune escape mechanism on LCs when treated with HPV16 has
been characterized (Fausch, Da Silva et al. 2002). In addition, it has been concluded that
the L2 minor capsid protein of HPV16 is responsible for the immune evasion and is tied
to the uptake mechanism (Fahey, Raff et al. 2009). Since the L2 capsid protein is highly
conserved among HPV genotypes, I hypothesized that other high and low risk mucosal
HPV genotypes and cutaneous HPV genotypes share the immune escape mechanism.
Therefore, if LCs are exposed to other HPV genotypes then the activation makers should
be minimally up-regulated just as is observed for HPV16 (Fig 2A). Other HPV high risk
genotypes selected to be used in the assays were HPV18, HPV31 and HPV45. HPV11
was selected to represent low risk HPV genotypes. HPV1 and HPV5 were used in the
assay and represent cutaneous HPV genotypes.
In addition to peptide presentation within the context of MHC molecules,
constimulatroy molecules provide an important signal to prime T-Cells to effectively
clear an infection. The activation phenotype in LCs after 48 hours of treatment with the
HPV genotypes was tested by flow cytometry. The fold change in surface activation
markers expression was determined to evaluate the up-regulation of MHC class II, CD80
and CD86 markers critical in priming a T-cell. Flow cytometric analysis revealed that
like HPV16, the other high risk mucosal HPV genotypes and the low risk mucosal HPV
genotype do not up-regulate surface activation markers such as MHC class II, CD80 and
30

CD86 (Fig. 6). In regards to the cutaneous HPV genotypes, HPV5 had similar surface
expression compared to HPV16; however, HPV1 showed significant up-regulation of
MHC class II, CD80 and CD86 as compared to HPV16 (Fig 6). These findings suggest
that HPV1 does not share the same immune escape mechanism as HPV16, while the
other high and low risk HPV genotypes and cutaneous genotypes that emerge in
immunosuppressed patients share the same immune escape mechanism. The implications
of not up-regulating surface activation markers, but expressing a basal level of MHC
class molecules results in tolerizing T-cells in that they do not get activated and will not
clear the HPV infection.

31

Figure 6: High and Low Risk Mucosal HPV Genotypes and Cutaneous HPV
Genotypes All Suppress Phenotypic Activation of LC, except for HPV1.
Flow cytometric analysis reveals that surface activation marker expression in LCs
between high and low risk mucosal HPV genotypes and a cutaneous HPV genotype are
similar to LCs treated with HPV16. HPV1 showed significant up-regulation of surface
activation markers (MHC class II, CD80 and C86) as compared to HPV16. The
treatment groups are on the x axis, whereby (-) is LC only. LPS was used as a control.
High risk mucosal HPV genotypes treated on LC include HPV16, HPV18, HPV31 and
HPV45. HPV11 was a low risk HPV genotype. HPV5 and HPV1 are cutaneous HPV
genotypes. Cells were stained for surface activation markers and then ran on FACS.
Fold change of surface marker expression was calculated based on mean fluorescence
intensity normalized against the LC untreated. Data is a representation of mean ± SD of
which represents an average of 6 donors, the assay was repeated 8 times (*, p < 0.5 and
**, p < 0.01).

32

Langerhans Cell Migration is Inhibited by Low and High-Risk HPV Genotypes

When APCs are activated by pathogens in peripheral tissues, C-C Chemokine
Receptor 7 (CCR7) is up-regulated on the cell surface which enables LCs to respond to
chemicals such as CCL21 secreted by cells in the draining lymph node (LN). Activated
LCs will migrate to the LN, activating and stimulating T-cell proliferation. It has been
shown previously that LCs treated with HPV16 for 48 hours in in vitro migration assays
are non-migratory, as a result of treatment with HPV16 (Fig 2B) (Fausch, Da Silva et al.
2002). Since, the HPV genotypes may share the same immune evasion mechanism, LCs
were tested for migration capacity with other high risk mucosal HPV genotypes (HPV18,
HPV31, and HPV45) and a low risk mucosal HPV genotype (HPV11) and other
cutaneous HPV genotypes (HPV1 and HPV5).
In vitro migration assays were performed. LCs were incubated with HPV
genotypes for 48 hours, and were placed into the upper chamber of transwell
semipermeable membrane. Cells that are chemotactic towards CCL21 will migrate to the
bottom chamber containing CCL21. Spontaneous migration is controlled for by plating
treated LCs in the top chamber without any chemokine present in the bottom chamber.
The results of the in vitro migration assay reveal that the other high and low risk
mucosal HPV genotypes and both of the cutaneous HPV genotypes suppress the ability
of LCs to migrate in response to CCL21 (Fig 7). These findings are similar to the
inhibition of migratory ability of LCs treated with HPV16 (Fig 7), indicating that the LCs
do not migrate in response to HPV genotypes due to a conserved immune evasion
mechanism demonstrated by several classes of HPV genotypes.
33

Figure 7: Migration of LC is Inhibited in Response to Treatment with Cutaneous
HPV Genotypes and High and Low Risk Mucosal HPV Genotypes.
In vitro migration assay show that LCs after treatment with HPV genotypes have similar
ability to respond to chemokine signals directing migration. Human-derived LCs were
treated with HPV genotypes for 48 hours and then were incubated for 4 hours in
transwell plates on a semi-permeable membrane with a chemokine, CCL21 in the bottom
chamber. Cells that had migrated to the bottom chamber were counted. This figure
represents the MI (# of activated migrated cells/# of spontaneously migrated cells) of LCs
after treatment with LCs only (-), LPS as a control which is known to activate LCs
causing them to migrate, HPV16, 18, 31 and 45 (high risk HPV genotypes), HPV 11 (a
low risk HPV genotype), HPV5 and HPV11 (cutaneous HPV genotypes). The data
represent the mean ± SD of 6 donors.

34

HPV Genotypes Prevent Cytokines and Chemokines Secretion in LCs

When APCs are activated by viral pathogens specific cytokines and chemokines are
secreted which are important signals critical to directing the immune system for
intracellular pathogens via a Th1 response (Berger 2000). A cytokine that is secreted by
APCs which is critical to inducing the Th1 response is IL-12. Previous data showed that
after LCs are treated with HPV16, immune suppression prevents cytokines and
chemokines from being secreted and it was shown specifically that IL-12 was not
secreted by LCs (Fausch, Da Silva et al. 2002). Thus, the lack of cytokine and
chemokine signals provided to T-Cells prevents an immune response needed to clear a
HPV infection.
If the immune suppression mechanism is conserved between HPV genotypes then
LCs exposed to other high and low risk mucosal HPV genotypes as well as cutaneous
HPV genotypes will not secrete cytokines and chemokines needed for an immune
response against the viral pathogen. To test this, LCs were treated with the various HPV
genotypes or LPS for 48 hours and supernatant was collected. A Bioplex cytokine assay
was performed to screen for a panel 12 cytokines and chemokines. The analytes included
6 chemokines (MCP-1, MIP-1α, MIP-1β, RANTES, IL-8 and IP-10), two cytokines
needed for a Th1 response (IL-12p70 and IFNγ), 3 inflammatory cytokines (IL-1b, IL-
6,and TNFα), and one immune suppressive cytokine (IL-10),
Analysis showed that high risk HPV genotypes (HPV18, HPV31 and HPV45) and the
low risk HPV genotype HPV11 and the cutaneous HPV5 genotype all prevent LCs from
secreting cytokines and chemokines, and was similar to the effect HPV16 had on LCs
35

(Fig. 8). HPV1 treated LCs showed elevated levels of cytokine and chemokine secretion
for IL-6, MIP-1b, RANTES and IP-10 (Fig. 8). Since the levels of cytokine and
chemokine secretion in LCs after treatment with other high and low risk mucosal HPV
genotypes and at least one other cutaneous genotype are similar to the cytokine and
chemokines secretion levels in LCs after HPV16, it seems likely that he immune escape
mechanism is likely conserved among HPV genotypes.

36

Figure 8: Cytokine and Chemokine Levels in LCs are Similar After Treatment with
High and Low Risk HPV Genotypes and Cutaneous HPV Genotypes
The cytokine and chemokine secretions in LCs after treatment with HPV genotypes
showed similar levels of secretion as what was observed in LCs after treated with HPV16
when analyzed for a panel of 12 analytes. Supernatant from LCs were collected after 48
hours of treatment with LC only, LPS, high risk HPV genotypes (HPV16, HPV18,
HPV31 and HPV45), HPV11 (a low risk HPV genotype) or cutaneous genotype (HPV5
and HPV1). Bioplex assays performed in triplicate were used to detect the levels of a
panel of 12 cytokines. The data depict four analytes selected from the panel of twelve.
This assay was performed a total of three times, each with similar results. Graphs are of
one assay selected to represent the data. The data is a mean ± SD of analyte
concentration assay in triplicate.

37

Region in L2 Responsible for HPV16 Immune Evasion on LCs is Conserved Among
HPV Genotypes

Immune function in LCs after treatment with HPV18, HPV31, HPV45, HPV11 and
HPV5 is similar to immune function in LCs after treatment with HPV16. However,
immune function in LCs differs after treatment with HPV1 when compared with LCs
treated with HPV16. These findings suggest that the immune evasion mechanism is
conserved between HPV genotypes, however, HPV1 does not use this conserved
mechanism. Fahey et. Al. reported that the presence of the L2 minor capsid protein is
responsible for the immune evasion mechanism of HPV16 in LCs (Fahey, Raff et al.
2009). Recently, a region on the L2 protein (aa 108-126) has been identified to bind to a
secondary receptor involved in uptake in LCs (Kast lab, unpublished data). Remarkably,
this region of L2 (aa 108-126) from HPV16 is conserved among the L2 regions of the
other HPV genotypes analyzed in this study, with the exception of HPV1.
The L2 protein sequence for HPV18, HPV31, HPV45, HPV5 and HPV1 was blasted
against HPV16 L2. Each of the L2 proteins was aligned against HPV16 L2. The region
108-126 was analyzed for conserved sequences and for amino acids that could function in
the same manner. Analysis of the alignment revealed that this region on L2 was
conserved between HPV genotypes (Fig. 9). For HPV genotypes that appear to share the
same immune evasion mechanism the range in conserved consensus sequence was 30 –
53 % (Fig. 9). For HPV1, which does not share the immune evasion mechanism the
consensus sequence is 21% and differs marginally from HPV5 and HPV18, which appear
to be evading the immune system the same way. What is apparent and different between
38

HPV1 and HPV16 when the sequences are aligned is an insertion of 5 amino acids.
Furthermore, this stretch of amino acids in this region of L2 on HPV1 is rich in prolines.
Prolines in amino acids generally restrict protein flexibility.
From the analysis of alignment sequences it can be hypothesized that the addition of
the 5 amino acids and the properties from the proline rich amino acid sequence 105-128
prevent HPV1 from suppressing immune function on LCs, however this hypothesis
remains to be tested.

39

Figure 9: Alignment and Conserved Consensus Sequence of other HPV L2 Proteins
to HPV16 L2 (aa 108-126) The sequence of amino acids (108-126) on HPV 16 L2 was
shown to be conserved between HPV genotypes that share the same immune evasion
mechanism. However, the sequence alignment between HPV1 and HPV16 revealed that
in HPV1, this stretch of AA’s reveals an insertion of 5 aa’s and also reveals the region
contains many prolines. The L2 protein of HPV18, HPV31, HPV45, HPV5 and HPV1
was blasted against HPV16. Sequences were individually aligned against HPV16 by
Cobalt:Red. The data show the alignment sequence to HPV16 (top line). The dashes are
empty space providing for any insertions of aa that did not line up with HPV16. The aa
in bold are conserved sequences and the shaded grey are aa that function in the same
manner. The table to the left is aligned with the sequences. Column 1 states the
genotype, column 2 is the % of conserved aa in the aligned region of the genotype and
the third column is the stretch of amino acids that aligned with HPV16 L2 (aa 108-126).

40

TLR Agonists Reverse Induce Up-Regulation of Cell Surface Activation Markers In
LCs After Exposure to HPV Genotypes

Intracellular TLRs (TLR3, TLR7 and TLR8) in APCs recognize and bind pattern-
associated molecular patterns on viruses. TLR7 and TLR8 recognize viral ssRNA, while
TLR3 recognize both ssRNA and dsRNA. The recognition of ssRNA or dsRNA on these
intracellular TLRs leads to signaling cascades which activate transcription factors,
namely NF-κB and IRF7. The activation of these transcription factors in turn activate
APC by up-regulating surface activation molecules, inducing the migration to the
draining lymph and causing the secretion of inflammatory cytokines and several
interferons which induce a Th1 immune response, needed to clear intracellular pathogens
such as viruses (Kumar, Kawai et al. 2009). Currently approved on the market for the
treatment of benign genital warts is a 5 % Imiquimod cream, a TLR 7 agonist. However
the product has been linked to side effects and is associated with recurrent HPV infection
(McQuillan and Higgins 2004; Taylor, Maslen et al. 2006; Bharti, Shukla et al. 2009).
Previous studies demonstrated that TLR7 agonist IMQ was ineffective at stimulating
an immune response capable of activating LCs, while a TLR8 agonist R848 does have
the potential to overcome the immune suppression in LCs caused by HPV16 allowing for
the up-regulation of surface activation markers MHC class II, CD80 and CD86 (Fig. 5A)
(Fahey, Raff et al. 2009). Because the immune suppression appears to be conserved
between genotypes and TLR 8 agonists were effective and reversing the immune
suppression in LCs by HPV16, the TLR8 agonist R848 and TLR 9 agonist PolyI:C were
tested for the ability to reverse the immune suppression of other high and low risk
41

mucosal HPV genotypes and the cutaneous HPV genotypes that are thought to be
actively preventing the activation of LCs.
Human LCs were treated with HPV genotypes HPV16, HPV18, HPV31, HPV45,
HPV11, HPV5 and HPV1, and then subsequently treated with TLR agonists IMQ, R848
or PolyI:C. Flow cytometry was performed and the surface expression of MHC class II,
CD80 and CD86 levels were determined. Both R848 and especially PolyI:C robustly
induce surface marker expression, reversing the immune suppressing phenotype caused
by HPV genotypes (Fig. 10). Treatment of HPV-exposed LC with IMQ did induce LC
activation with most of the HPV genotypes, however the expression levels for CD86, a
critical surface activation marker needed to activate T-Cells, was significantly higher
with PolyI:C in all genotypes than was seen for IMQ. Therefore, compared to IMQ, a
TLR3 agonist such as PolyI:C is a much stronger candidate in activating LCs, and
demonstrates potential to allow for a better overall clearance of HPV in HPV-associated
lesions.

42

Figure 10A-10B: TLR Agonists R848 and PolyIC Reverse the Immune Suppression
in LCs Caused by HPV Genotypes and Surface Activation Markers are Up-
Regulated. LC were treated with HPV genotypes and each graph is a representation of
the HPV genotypes tested and include: HPV31, HPV45, HPV18, HPV16, HPV11, HPV5
and HPV1. The treatments groups of LCs were 1. LCs only 2. LCs-HPV 3. LCs-HPV-
LPS 4. LCs-HPV-IMQ 5. LCs-HPV-R848 6. LCs-HPV-PolyI:C. 48 hours following LCs
with HPV + TLR agonist, LCs were stained for MHC class II, CD80 and CD86, and the
expressions levels were detected by FACS. The data is fold change in surface markers
expression which represents mean fluorescence increase. This figure shows HPV
genotypes performed in one assay represented by two different donors A and B. Two of
the HPV genotypes were performed in both donors and show similar results. Data
represented is the data point of one assay representative of one donor.

43

Figure 10A-10B, Continued

44

TLR Agonists R848 and PolyI:C Stimulate Migration in LCs Suppressed by HPV

In addition to testing if TLR agonists can induce the expression of surface
activation markers in LCs immune suppressed by HPV genotypes, LCs were additionally
tested for the ability to migrate by performing in vitro migration assays. Previously, it
was shown that migration could be induced in HPV16 treated LC by TLR agonists R848
and 3M002, whereas IMQ did not stimulate migration in LCs (Fig 5C) (Fahey, Raff et al.
2009). I tested the hypothesis that immune suppressed LCs by HPV genotypes would
regain the ability to migrate after treatment with TLR agonists.
To test for a restoration of migration function, human-derived LCs were first
treated with each of the genotypes: HPV16, HPV18, HPV31 and HPV45 representing
high risk mucosal HPV genotypes; HPV11, a low risk mucosal HPV genotype, and
cutaneous HPV genotypes, HPV5 and HPV1. LPS is known to activate LCs inducing
migration was used as a control and untreated LC were used as a negative control to
assess basal levels of migration. After an initial 4 hour incubation of LCs with HPV
genotype, control or no treatment, TLR agonists were subsequently added and cells were
incubated for 48 hrs. Following incubation with TLR agonists on HPV treated LCs in
vitro migration assays were performed in transwell plates for 4 hours. Cells in the bottom
chamber were counted and MI was calculated.
Migration assays showed that IMQ was not effective in reversing the inability of
LCs to migrate as a result of HPV immunosuppression (Fig 11), which was similar to
previous findings (Fig 5C). However, R848 and PolyI:C, TLR8 and TLR3 agonists,
respectively, were both effective in stimulating migration of LCs suppressed by HPV
45

genotypes (Fig 11). Further, the ability for PolyI:C to induce migration in HPV treated
LCs was rather robust in HPV18, HPV31, HPV45, HPV11, HPV1 and HPV5 in
comparison to R848 which has seems to have intermediate effects.
These data suggest that R848 and especially PolyI:C have the ability to induce
migration in LCs after being suppressed by HPV genotypes and compounds that
recognize TLR3 and TLR8 will have the ability to stimulate a more profound and
effective immune response against HPV.

46

Figure 11A-11B: TLR Agonists R848 and PolyIC Reverse the Immune Suppression
in LCs Caused by HPV Genotypes and Allow for LC Migration.
LCs were treated initially for 4 hours with the appropriate HPV treatments, and then
subsequently were treated with TLR agonists, LPS as a control or null treatment. Cells
were plated for 4 hours in a migration assay. Each graph is a representation of one HPV
genotype from two different donors A and B. Two of the HPV genotypes were performed
in each of the donors and similar results were observed. The data points represent the
fold change in MI controlled to the untreated LC treatment group.

47

Figure 11A-11B, Continued

48

Conclusions

HPV is linked to 93% of all cervical cancer cases worldwide and is associated
with anogenital cancers and head and neck cancers. Cervical cancer is the second leading
cause of cancer in women world, with a disproportionate 83 % of the cases occurring
developing countries. Yet, the mortality rate for women with cervical cancer is 50 %.
There are two preventative vaccines on the market, GARDASIL and CERVARIX.
However, GARDASL is only effective in preventing HPV16, HPV18, HPV11 and HPV6
infections, while CERVARIX is only effective in preventing HPV16 and HPV18 (Bharti,
Shukla et al. 2009; Kyrgiou, Valasoulis et al. 2010). Thus, these will not be effective in
preventing cervical cancer linked to 16 other genotypes. In addition these vaccines are
too costly to provide protection to developing countries. While ALDARA, a 5 %
imiquimod cream is approved for the use of genital warts, additional therapies are needed
targeting persistant HPV infection or in stimulating the immune system to clear an
infection (Kyrgiou, Valasoulis et al. 2010).
HPV infections may take several months to a year to clear, suggesting that HPV
has acquired the ability to suppress the immune system. Previous work has shown that
when HPV16 comes in contact with LCs, surface activation markers are not up-regulated,
migration to the draining LN does not occur and cytokine and chemokine secretion is not
induced (Fausch, Fahey et al. 2005; Fahey, Raff et al. 2009). It is known that the L2
capsid protein between HPV genotypes have highly conserved sequences. Members in
our lab determined that amino acids 108-126 on the L2 protein of HPV16 bind to the
49

receptor on LCs involving uptake. Furthermore, it is the L2 protein on HPV16 that is
responsible for the immune evasion in LCs.
Because the L2 protein is highly conserved between HPV genotypes, it seemed
likely that the immune suppression mechanism was conserved among HPV genotypes.
The data that assess LC immune function demonstrate this mechanism of immune
evasion is conserved among high and low risk mucosal HPV genotypes and cutaneous
HPV genotypes; however, there may HPV genotypes that do not use this mechanism,
such as HPV1. LC immune function was tested by determining levels of surface
activation markers, determining the ability for LCs to migrate and measuring the levels of
cytokine and chemokine secretion. One of the cutaneous HPV genotypes showed
differences in LC function when compared to HPV16. HPV1 was the only genotype that
showed significant differences in the levels of surface activation markers MHC class II,
CD80 and CD86. Furthermore, this was the only genotype that appeared to have higher
levels of inflammatory cytokines, such as MIP-1β, RANTES, IP-10 and IL-6. The other
high and low risk mucosal HPV genotypes, as well as the other cutaneous HPV
genotypes used in the assays prevented LCs from up-regulating surface activation
markers, prevented chemokine-site directed migration, and did not induce secretion of
cytokines and chemokines. These results were similar to previous experiments
performed on LCs that had been exposed to HPV16 (Fahey, Raff et al. 2009).
HPV genotypes that share the immune evasion mechanism will cause LCs to
present MHC molecules along with low or absent levels of CD80 and CD86 to antigen-
specific T-cells and will have a tolerizing effect on T-Cells preventing an immune
50

response. To test the ability of LCs to stimulate proliferation of T-Cells after activation
with the other HPV genotypes, mixed lymphocyte reaction assays should be performed.
To further confirm, that the immune evasion mechanism is conserved, the activation of
PI3K and inactivation of AKT pathways need to be confirmed by western analysis.
Because the immune evasion mechanisms of high and low risk mucosal HPV
genotypes and at least one other cutaneous HPV genotype are suppressing the immune
activation of LC and because HPV1 is not suppressing LC activation, I investigated the
amino acid sequence 108-126 on L2 on HPV16 aligned to HPV18, HPV31, HPV45,
HPV11, HPV5 and HPV1 to see there was conserved homology in this stretch of amino
acids between these HPV genotypes and HPV16. The alignment sequences provide an
explanation as to why HPV1 causes LCs to behave differently (Fig. 9). HPV1 has an
insertion of 5 amino acids and has many prolines in the sequence that aligned to HPV16
L2 (108-126). This most likely prevents L2 from binding properly to the receptor of LCs
in the same manner that HPV16 does and explains why HPV1 does not share the immune
escape mechanism. Results of the alignment sequences for the remaining HPV
genotypes to HPV16 for amino acids 108-126 show that this sequence to be conserved
and suggest that L2 is responsible for the immune evasion mechanism shared in other
HPV genotypes. To further confirm this statement LC immune function should be tested
with the L1L2 capsids versus the L1 only capsids of other HPV genotypes, similar to
what was performed with HPV16 (Fahey, Raff et al. 2009).
Previous data supports that HPV16 immune suppression induced in LCs can be
overcome by TLR 8 agonist R848 (Fig. 5) (Fahey, Raff et al. 2009). The shared immune
51

evasion between high risk and low risk mucosal HPV genotypes and the other cutaneous
HPV genotype, caused us to investigate whether the shared immune suppression
mechanism in LCs by other HPV genotypes was able to be reversed by intracellular
TLR8 and TLR3 agonists, R848 and PolyI:C.
The results confirmed that both of the TLR agonists, R848 and PolyI:C are able to
reverse the immune suppression in LCs induced by other HPV genotypes. R848 and
PolyI:C, when compared to IMQ showed a much more activated phenotype than IMQ.
PolyI:C was able to induce high surface activation levels for CD86 and induced the
strongest migration response in LCs. R848 stimulated an intermediate up-regulation of
CD86 and migratory ability in LCs. IMQ showed the least amount of CD86 up-
regulation and was not able to stimulate migration in LCs treated with other high and low
risk mucosal HPV genotypes and when treated with cutaneous HPV genotypes.
There are several reasons as to why the TLR3 agonist may be more effective at
activating LCs. While the signaling cascade in LCs involving TLRs reversing the
immune suppression by HPV genotypes is not clear, it is possible to speculate why TLR3
and TLR8 agonists are effective at reversing the immune suppression, while IMQ is not.
IMQ binds TLR3 and activates signaling cascade TRIF which activates transcription
factor IRF3 and then IFNβ is transcribed, furthermore TRIF activates the IRAK/TRAF
pathway which activates transcription factor NF-κB and then immunomodulatory genes
are transcribed. TLR7 and 8 initially activate MyD88 which activates IRAK/TRAF
which in turn activates transcription factor NF- κB leading to the transcription of
immunomodulatory genes (Miller and Modlin, 2007). The signaling cascade provides an
52

explanation as to why a TLR3 agonist can overcome HPV immune suppression in LCs.
By activating TLR3 an alternative signaling cascade activates IRAK/TRAF which
mediated by TRIF and that TRIF also activates another pathway that TLR7 and 8
agonists do not. This may provide an explanation for as to why immune suppressed LCs
by HPV genotypes are then able to be activated following treatment with PolyI:C. In the
case of TLR7 and 8 agonists differing in there capacity to activate LCs, perhaps there is
more TLR8 present in LCs, leading to stronger signaling transduction in order to
transcribe genes required for LC maturation. Taken together, these data suggest that a
TLR3 agonist compound may be more effective at stimulating an productive immune
response able to clear HPV infections.
Developing an affordable stable compound would be effective in treating the
approximate 10% of the global population currently infected with HPV. A product such
as this would provide an alternative to LEEP procedure preventing complications during
pregnancy. Finally, this would also be effective at reducing the burden of HPV infections
and cervical cancer cases in developing countries.

53

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Asset Metadata

Creator Movius, Carly Anne (author)

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

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 04/26/2012

Defense Date 03/29/2011

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

Tag HPV,immune evasion mechanisms,Langerhans cells,OAI-PMH Harvest

Language English

Advisor Kast, W. Martin (committee chair), Machida, Keigo (committee member), Yuan, Weiming (committee member)

Creator Email c.a.movius@gmail.com,cmovius@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3776

Unique identifier UC1205562

Identifier etd-Movius-4573 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-464808 (legacy record id),usctheses-m3776 (legacy record id)

Legacy Identifier etd-Movius-4573.pdf

Dmrecord 464808

Document Type Thesis

Rights Movius, Carly Anne

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 uscdl@usc.edu

Abstract (if available)

Abstract Cervical cancer is associated with high risk HPV genotypes 93 % of the time. HPV infections can take months and even up to a year to clear, and 30 % of HPV infections will develop into cervical cancer. A novel immune suppression mechanism used by HPV16 has been characterized in Langerhans Cells (LCs), such that there is an activation of PI3K pathway and a down regulation of AKT pathways, mediated in part by PP2A. HPV16 suppresses phenotypic activation and immune function in LCs. This study reveals that other high risk mucosal HPV genotypes (HPV18, HPV31 and HPV45) and a low risk mucosal HPV genotype (HPV11) and a cutaneous HPV genotype (HPV5) share this particular immune escape mechanism based on a conserved sequence in L2 minor capsid protein (aa 108-126). However, a cutaneous HPV genotype (HPV1) does not share this immune escape mechanism in LCs, as there is little homology to the HPV16 L2 sequence (aa 108-126). In addition this study shows that this particular immune suppression mechanism can be reversed with intracellular toll-like receptor (TLR) agonists 3 and 8, but only marginally with a TLR3 agonist. This demonstrates the possibility for therapeutic compounds such as these which could be developed to treat the 10 % of the world’s population currently estimated to be infected with HPV, as an alternative treatment to ablative surgical procedures.