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

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

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Content CONTRIBUTION OF HERPES SIMPLEX VIRUS TYPE 2 IN THE
ACQUISITION OF HUMAN PAPILLOMAVIRUS INFECTION
by
Tania B. Porras
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulllment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
May 2015
Copyright 2015 Tania B. Porras
Dedication
To the memory of my father-in-law, Jos e Miguel.
ii
Acknowledgements
First of all, I would like to thank my husband Miguel for his personal support and pa-
tience at all times; and to my parents-in-law, Elsa and Jose Miguel for their unconditional
help, strength and encouragement. I also want to thank my parents, and my siblings.
I would like to express my deepest gratitude and appreciation to my advisor, Dr. Marin
Kast, and to my supervisor Dr. Diane Da Silva for their constant and excellent research
guidance which made possible the completion of my research work. This thesis would not
have been possible without their help, support and patience. I'm also thankful to Heike
Brand, Joseph Skeate, Andrew Woodham, and other members of the Kast Lab, for their
help, suggestions, support, friendship, and for making this time a memorable experience.
iii
Table of Contents
Dedication ii
Acknowledgements iii
List of Figures vi
Abstract vii
Chapter 1 Introduction 1
Chapter 2 Background 5
2.1 Human herpes simplex viruses . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2 Papillomaviruses and human cancers . . . . . . . . . . . . . . . . . . . . . . 13
2.3 Secretory Leukocyte Protease Inhibitor (SLPI) . . . . . . . . . . . . . . . . 15
2.4 HSV, HPV, and cervical cancer . . . . . . . . . . . . . . . . . . . . . . . . . 19
Chapter 3 Materials and Methods 21
3.1 Cell lines, antibodies and recombinant proteins . . . . . . . . . . . . . . . . 21
3.2 Propagation and titration of HSV-2 . . . . . . . . . . . . . . . . . . . . . . 22
3.3 HPV-16 Pseudovirus (PsV) and Virus-Like Particles (VLP) . . . . . . . . . 23
3.4 Infection of HaCaT cells with HSV-2(G) . . . . . . . . . . . . . . . . . . . . 24
3.5 Quantication of HSV-2 infection by
ow cytometry . . . . . . . . . . . . . 24
3.6 Assessment of slpi gene expression in HSV-2(G) infected HaCaT cells . . . 25
3.7 Quantication of secreted SLPI by HSV-2 infected HaCaT cells . . . . . . . 26
3.8 Uptake of HPV-16 L1 and L1L2 pHrodo
TM
-labeled VLPs after HSV-2(G)
infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.9 Infection of HaCaT cells with HPV-16 L1L2 PsVs post HSV-2(G) infection 27
Chapter 4 Results 29
4.1 HSV-2(G) infection causes down-modulation of SLPI after 24h . . . . . . . 29
4.2 Down-modulation of SLPI after 24h of HSV-2(G) infection causes minimal
eect on HPV uptake and infection . . . . . . . . . . . . . . . . . . . . . . . 30
4.3 HSV-2(G)-induced down-regulation of SLPI increases and it is maintained
after 48h post infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4.4 HSV-2(G) infection of HaCaT cells and cell viability . . . . . . . . . . . . . 37
4.5 Increased HPV-16 uptake and infection after HSV-2 infection . . . . . . . . 38
iv
4.6 Restored SLPI secreted protein levels reduces HPV-16 uptake . . . . . . . . 42
4.7 HPV L2 capsid protein is required for Enhanced VLP uptake . . . . . . . . 43
Chapter 5 Discussion and Conclusions 46
Bibliography 53
v
List of Figures
2.1 Herpes simplex virus life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 Human Papillomavirus life cycle . . . . . . . . . . . . . . . . . . . . . . . . 16
4.1 HSV-2(G) infection downmodulates SLPI in HaCaT cells after 24 of infection 31
4.2 pHrodo
TM
-red labeled HPV-16 L1L2 VLP concentration optimization. . . . 33
4.3 HPV-16 uptake and infection is not altered after 24 hours of HSV-2(G)
infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
4.4 Sustained down-regulation of slpi gene expression on HaCaT cells infected
with HSV-2(G) for 48h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.5 HSV-2(G) infection for 48h reduces secretion of SLPI protein by HaCaT cells. 36
4.6 Percentage of HSV-2(G) infected HaCaT cells . . . . . . . . . . . . . . . . . 37
4.7 Acyclovir treatment prevents in vitro replication of HSV-2(G) in infected
HaCaT cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.8 HPV-16 L1L2 pHrodo-VLP uptake increases after HSV-2(G) infection . . . 40
4.9 HPV-16 L1L2 GFP PsV infection increases after HSV-2(G) infection . . . . 41
4.10 VLP uptake by HSV-2(G) infected cells after normalizing SLPI levels . . . 43
4.11 HPV-VLP uptake by HSV-2(G) infected HaCaT cells is inversely propor-
tional to SLPI concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.12 L2 capsid protein is necessary for A2t mediated endocytosis of HPV-16 VLPs 45
5.1 Model of HPV:A2t interaction . . . . . . . . . . . . . . . . . . . . . . . . . 50
vi
Abstract
Despite the fact that Human Papillomavirus (HPV) is responsible for almost all cases
of cervical cancer around the world, the epidemiological link between Herpes Simplex
Virus (HSV) and cervical cancer has persisted for decades. Although both HSV-2 and
HSV-1 cause genital herpetic ulcerative disease, HSV-2 remains the main causative agent
of ano-genital infections. Host immune evasion strategies are crucial to facilitate pathogen
infection and persistence, and HSV-mediated down-regulation of the Secretory Leukocyte
Protease Inhibitor (SLPI) constitutes one of those mechanisms. SLPI, ubiquitously found
in body
uids, is known for its an anti-microbial, anti-in
ammatory and anti-protease
properties. SLPI also constitutes the natural cellular ligand for annexin A2/S100A10 or
A2t, which is the preferred cellular receptor for the internalization and infection of HPV.
Therefore, we hypothesized that reduced SLPI protein levels after an HSV-2 infection
would facilitate HPV interaction with A2t, resulting in an enhanced internalization and
infection of HPV in a human keratinocyte cell line. We showed a signicant increase in
HPV internalization in epithelial cells previously infected with HSV-2 compared to HSV
negative cultures. Restoring SLPI concentrations by adding human recombinant SLPI
(rhSLPI) to HSV-2-infected cultures, HPV infection decreased conrming the protective
role of SLPI against HPV infection. Additionally, we provided evidence that proper HPV
binding and internalization through the A2t receptor requires interaction with the HPV
vii
L2 capsid protein. We concluded that HSV-2 intermittent reactivations and reinfections
would create the perfect SLPI-depleted microenvironment for HPV to infect and per-
sist. Our ndings not only contribute to understanding the contribution of HSV-2 in
the increased susceptibility to HPV infection, but they also provide the basis to explore
SLPI-based anti-viral therapeutic alternatives to use against HPV as a strategy to prevent
cervical cancer.
viii
Chapter 1
Introduction
Human papillomavirus (HPV) is one of the most common sexually transmitted infec-
tions worldwide. High-risk HPV (hr-HPV) or oncogenic papillomaviruses are the etiologic
agent of almost all the approximately 530,000 new cases of cervical cancer annually diag-
nosed worldwide. Of the dierent hr-HPV genotypes, HPV-16 and HPV-18 are the most
prevalent and are found in over 70% of those cases [10]. HPV-16 and HPV-18 are also
responsible for other genital malignancies such as anal, penile and vaginal cancers, as well
as for head and neck squamous cell carcinomas (HNSCC) that aect both men and women
[4].
Although the majority of HPV infections are naturally cleared within the rst two
years, persistent HPV infections, representing approximately 10% of the total number of
cases, increase the risk for developing HPV associated cervical cancer. It has been shown
that immune evasion mechanisms contribute to the persistence of HPV infections [30].
Since the HPV life cycle depends on the dierentiation and maturation of epithelial cells,
viruses initially infect basal cells, and new virions are only assembled and released in
the upper layers of the epithelium, thus avoiding recognition by immune cells. HPV also
1
has the ability to block T cell activation by suppressing Langerhans cell (LC) activation.
These LCs are a subset of antigen presenting cells specically found in epithelial layers.
Additionally, by maintaining DNA as an episome (extrachromosomal circular DNA) in
basal epithelial cells, HPV not only hides from immune surveillance, but also serves as a
reservoir of latent or persistent HPV infection [3].
Herpes simplex virus (HSV) was originally identied as the causal agent of cervical
cancer, and despite the discovery of HPV as the etiologic agent, the association between
HSV and cervical cancer has persisted. Although both HSV-2 and HSV-1 are recognized
as causal agents of genital herpetic ulcerative disease, HSV-2 is more commonly associ-
ated to ano-genital infections and has a recurrence rate 8 to 10 times greater than that of
HSV-1. It has been suggested that although the HSV-2 genome does not contain onco-
genes, HSV-2 has sucient oncogenic properties to induce the malignant transformation
of HPV infected cells [28]. Two DNA regions within the HSV-2 genome known as the
minimal transformation fragments II and III (mtrII and mtrIII)have shown to induce neo-
plastic transformation in human keratinocytes immortalized with HPV [11, 12]. However,
inconsistent ndings of HSV-2 DNA in cervical cancer biopsies contradict that idea [19].
Unquestionable however, is the fact that similarly to HPV, HSV-1 and HSV-2 have also
developed strategies to evade host immune defenses. Upon HSV infection, the virion host
shuto (VHS) protein interferes with cellular protein synthesis by degrading host mRNAs
[20]. Similarly, the immediate early (IE) product, the infected cell protein 27 (ICP27), also
contributes to the reduction in cellular protein synthesis by inhibiting pre-mRNA splicing.
Other immediate early proteins, ICP0 and ICP4 have been also linked to reduced synthesis
of cell immune mediators such as pro-in
ammatory cytokines and IFN-/ by interfering
2
with Toll-Like Receptor-2 (TLR-2) and NF-B signaling [53]. Additionally, ICP47 pre-
vents MHC class I antigen presentation, and inhibits activation of IFN signaling pathways
[40, 50, 60], and in vitro infection of a cervical carcinoma cell line with HSV-1 and HSV-2
results in the modulation of the host cell response as evidenced by reduced expression of
Secretory Leukocyte Protease Inhibitor (SLPI) [18]. Interestingly, there is evidence that
SLPI, an anti-in
ammatory and antimicrobial serine protease inhibitor commonly found
in mucosal surfaces, constitutes the cellular ligand for the annexin A2/S100A10 or A2t
heterotetrameric receptor. A2t has not only been identied as the primary internalization
receptor for HPV, but also as a key component in viral intracellular tracking [16, 58].
Furthermore, treating human keratinocytes with SLPI blocks infection with HPV-16 in
vitro [58]. Additionally, an inverse correlation between SLPI protein expression and HPV
positivity in biopsies from patients with head and neck squamous cell carcinomas has
been observed [23]. Those ndings not only provide evidence for the protective role of
SLPI against HPV infection, but they also add signicance to the down-modulation of
this protein as part of an immune escape mechanism employed by HSV.
To date, there has not been any direct association between HSV-2 down-modulation
of SLPI expression and hr-HPV internalization and infection, and therefore is the prin-
cipal objective of the present work. In this study, we hypothesized that in vitro HSV-2
infection of human keratinocytes results in a signicant down-regulation of both slpi gene
transcription and SLPI protein secretion, facilitating HPV-16 infection by increasing the
availability of the primary uptake receptor, At2. Since HSV-2 is a lifelong infection that
intermittently reactivates from latency in the neurons and re-infects epithelial cells of the
cervix, permanent shedding of viruses occurs from the epithelium. Thus, we believe that
3
HSV-2 infection constantly alters the cervical local microenvironment by suppressing SLPI
secretion and promoting hr-HPV infections. Findings from this study will contribute to
the understanding of a synergistic mechanism between HSV-2 infection and the increased
susceptibility to HPV infection and persistence. We also aim to provide insights that
support research on anti-viral therapeutic alternatives used against HPV cervical cancer,
and strengthen current HPV-prophylactic-vaccine strategies.
4
Chapter 2
Background
2.1 Human herpes simplex viruses
Human herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) belong to the alpha-
herpesviruses of the Herpesviridae family. Both have the ability to infect epithelial and
neuronal cells. Alphaherpesviruses are spherical viruses of approximately 200nm composed
of genome, capsid, tegument and envelope. The HSV genome is a single copy of linear
double stranded DNA (dsDNA) of approximately 130-250kbp packed into an icosahedral
capsid shell. Surrounding the capsid is a proteinaceous layer called tegument, and the
outermost layer or envelope is a proteic-lipid-bilayer made out of both viral and cellular
components [2].
The icosahedral herpes virus capsid is composed of a total of 162 capsomers made out
of ve highly conserved viral proteins: the major capsid protein (MCP) or viral protein
VP5, VP19C, VP23, the smallest capsid protein (SCP) or VP26, and the portal protein
(PORT) [2].
The tegument matrix is comprised of more than 30 dierent proteins. Although the
activity of each has not been well characterized, many play active roles in viral replication,
5
assembly, and viral egress. In HSV-1, for example, the tegument protein products UL7,
UL17, UL25, and UL37 are essential for viral replication. The -trans-inducing factor
(-TIF) or VP16 acts as a transcription activator of immediate-early and early genes, and
also participates in viral assembly. The VHS protein's principal role is to halt cellular
protein synthesis by degrading mRNAs [50, 60]. Additionally, VP1-2 protein binds to
HSV DNA facilitating both its release into the nucleus as well as guiding encapsidation
during viral assembly [2].
Finally, the HSV envelope contains a total of 10 specic viral glycoproteins embedded
in a lipid layer derived from the cell membrane. Those glycoproteins are important for
viral adsorption or initial viral attachment to cell surface receptors, and for viral-cell
membrane fusion [2, 52].
2.1.1 HSV-1 and HSV-2 life cycle
Herpes simplex viruses have the ability to undergo both lytic or productive infections,
and latent or non-lytic infections. The replicative cycle takes place in the skin or mucosal
epithelial cells while latent infections require HSV to reach and remain dormant in sensory
neurons. Reactivation out of the dormant state is considered a spontaneous event, and
dormant virions may repeatedly reactivate and lead to secondary infections [24] (Figure
2.1.
Viral adsorption to target cells occurs through interaction between glycoprotein B
(gB) and gC with cellular proteoglycans such as heparan sulfate proteoglycans (HSPGs).
This interaction increases the proximity between viruses and target cell facilitating inter-
action of viral gD with cellular entry receptors. Dierent receptors for HSV that have
6
Epi t hel i al
cel l s
HSV Pr i mar y i nf ect i on
Sensor y neur ons
Recur r ent
i nf ect i on
Vi r al
sheddi ng
Figure 2.1: Representation of Lytic and Non-lytic cycle of HSV. Lytic cycle of HSV
which includes viral adsorption, viral uncoating, viral transcription and viral genome
replication, viral assembly and viral egress occur in epithelial cells. Some of those new
released viruses enter the sensory neuron terminals and establish latency in the nuclei
of the neurons. During reactivation of a latent HSV infection, new viral particles are
assembled and released form the neuron. Those can re-infect epithelial cells near the site
of the primary infection resulting in a recurrent infections. Figure modied from Expert
Reviews in Molecular Medicine. Volume 5; 5 December 2003. Cambridge University Press.
7
been described include the herpesvirus entry mediator (HVEM), a member of the TNF
receptor family; nectin-1 and nectin-2, members of the immunoglobulin superfamily; and
the modied 3-O-sulfated heparan sulfate (3-OS HS). Entry receptor preference by HSV-1
and HSV-2 viruses has been well characterized, and while both can bind to HVEM and
nectin-1, only HSV-2 utilizes nectin-2 as entry receptor. Binding of gD to one of those
entry receptors triggers fusion of viral envelope and cell membrane, a process in which
viral gB and gD/gH heterodimers are also involved [52]. This membrane fusion results
in the internalization and cytosolic release of the HSV nucleocapsid and tegument. Once
in the cytoplasm, HSV is transported along microtubules towards the nucleus where, in
a not well-understood mechanism, the HSV capsid uncoats and viral genome is delivered
into the nucleus through the nuclear pores. The capsid and the majority of the tegument
proteins are left in the cytoplasm [2, 47].
During a lytic cycle, HSV replicates and bursts the host epithelial cell to release new
virions. Free virions can then go on to cause secondary epithelial infection or infect sensory
neurons and invade neuronal nuclei in trigeminal or sacral ganglion where latent infection
is established [2, 21, 24].
2.1.1.1 Lytic or productive cycle
In epithelial cells, after delivery of viral DNA to the nucleus, viral transcription begins
followed by viral protein synthesis. Viral gene expression occurs in three phases: (i)
Immediate-early (IE), (ii) Early (E) and (iii) Late phases. During the immediate-early
phase, VP16 interacts with cellular transcription factors to initiate expression of ve IE-
genes: ICP4, ICP0, ICP27, ICP22, and ICP47. The rst three encode for proteins that are
8
essential to activate early and late gene expression, and to stimulate export and translation
of viral mRNAs. Interestingly, the ICP0 protein also induces targeted ubiquitination and
proteasome degradation of cellular proteins that may block viral gene expression. Finally,
the ICP22 protein is involved in viral replication while ICP47 acts in conjunction with the
VHS protein to interfere with MHC class I antigen presentation through inhibition of the
TAP antigen transporter [17].
Once early gene transcription phase advances, proteins involved in viral DNA repli-
cation are synthesized. Expression of late genes begins as soon as viral DNA replication
starts. All structural components required for capsid assembly and envelope formation
are synthesized during this phase. Those components are transported to dierent cellular
compartments; for example, some migrate back to the nucleus, while others are incorpo-
rated in the endoplasmic reticulum. In the nucleus, HSV capsomers form mature capsids,
and DNA is encapsidated. After this process, DNA-containing capsids bud through the
nuclear membrane and are released into the cytoplasm where capsids associate to the
membranes of the endoplasmic reticulum. Tegument proteins and envelope containing
viral glycoproteins surround these capsids to form mature virions. Complete virions can
either infect other cells via virus-induced cell fusion, or be released from the cell [2].
2.1.1.2 Latent or non-lytic cycle
After primary infections with either HSV-1 or HSV-2, latent infections are established
in sensory neurons for the life of the host. From there, viruses spontaneously reactivate
causing secondary infections, resulting in reinfection or transmission of HSV to other hosts.
9
During productive initial infection, released HSV virions enter neuronal axons. In the
neurons, HSV travels along microtubules towards the nuclei where viral DNA remains
as an extra-chromosomal element. In this state, DNA replication is repressed and the
majority of viral genes are transcriptionally inactive. However, genes implicated in latency
maintenance are transcribed, those are known as latency-associated transcripts (LATs).
Although the function of LATs is not completely known, it is generally accepted that LATs
anti-apoptotic properties ensures establishment of latency, and when absent, reactivation
will proceed.
Intermittent recurrences generally result from reactivation of latent HSV infections.
When a reactivation event is triggered, viruses travel from sensory ganglia back to the
site of primary infection and infect epithelial cells [8]. Recurrences in the genital tract by
HSV-2 are more frequent than HSV-1. Within the rst year after a symptomatic genital
infection, approximately 20-50% of the cases caused by HSV-1 will reactive, while 70-90%
of the cases by HSV-2 will do the same. HSV shedding during reactivation events is usually
asymptomatic, making it dicult to treat and control unintentional sexual transmission
of the disease. Additionally, HSV spreading within the same host via viral-mediated cell-
cell fusion constitutes an elegant way to escape host immune defenses contributing to the
persistence and recurrence of HSV infections [21].
2.1.2 Genital herpes
Herpes simplex virus type 1 (HSV-1) and Type 2 (HSV-2) have been recognized as
the etiologic agents of genital herpes. Although genital infections with HSV-1 are more
10
common in certain populations, the large majority of cases are caused by HSV-2. Esti-
mates indicate that only in the United States approximately a half million people become
infected with HSV-2 every year, and approximately 20% and 60% of the population is
seropositive for HSV-2 and HSV-1, respectively [31, 35].
Traditionally, due to specic tropism, HSV-1 infection occurs primarily in the oro-
labial area while HSV-2 infections are primarily associated to genital herpetic episodes.
In spite of this dynamic, within the last two decades the number of cases of genital herpes
infections caused by HSV-1 has increased, specially among young males. This shift is
believed to be caused by changes in sexual behavior and an increase in the frequency
of oral sex. While genital-genital contact accounts for the majority of HSV-2 infections,
oral-genital contact constitutes the main route of HSV-1 transmission [21].
Clinical signs of genital herpes may vary from having no symptoms to very painful
ulcers. Although in many cases HSV-2 primary infections and reactivations are asymp-
tomatic, viral replication and shedding is almost always present. Therefore many people
remain unaware of having an infection, and unintentionally spread the virus among the
population. On the other hand, symptomatic episodes of genital herpes are initially char-
acterized by erythema followed by the formation of vesicles and ulcers. Current available
antiviral therapies utilize nucleotide analogs such as acyclovir, valacyclovir, and fanciclovir
to prevent viral replication and to limit duration of the symptoms; however, these neither
eliminate the virus nor prevent reactivation of latent infections [21, 35].
11
2.1.3 Alteration of mucosal immune response during genital herpes
Herpesviruses, like other human viruses, utilize immune evasion mechanisms to infect
and persist in a host. The three major components of antiviral responses: innate, acquired
and intrinsic immunity play important roles in controlling pathogen invasion. However, in
the particular case of HSV, some viral proteins interfere with those responses creating an
immuno-depleted microenvironment that allows not only HSV survival but also increase
the risk of acquiring other viral pathogens including human immunodeciency virus (HIV)
and human papillomavirus [8, 9].
One of the major lines of innate immunity against viral pathogens is the production of
cytokines, particular those of the interferon family (IFNs). In general, the activity of IFNs
consist of inducing cellular signaling pathways that lead into transcription of ISGs (IFN-
stimulated genes). These genes encode for anti-microbial proteins, molecules involved in
antigen presentation, and other cytokines. In fact, activation of IFN pathways during
HSV infection has been shown to be sucient in inhibit viral infection [45]. Unfortu-
nately, certain HSV proteins interfere with these processes. ICP0, for example, due to its
E3-ubiquitin ligase activity, mediates degradation of transcription factors that activate ex-
pression of INF- and IFN- genes. ICP0 also induces proteolysis of TLR-2, consequently
blocking the TLR activation pathway and dramatically altering NF-B activation [60].
In addition to ICP0, the VHS protein can directly degrade host mRNAs or block mRNA
translation by binding to the initiation factor eIF4H [17, 20, 50]. Reduction of mRNA
translation negatively in
uences pro-in
ammatory cytokine production, MHC class I and
II peptide loading and presentation, and antimicrobial peptide secretion [18, 40, 60]. In
fact, reduced expression of antimicrobial proteins such as beta-defensin 1 and SLPI is
12
observed during HSV infections and may indirectly facilitate infection and colonization of
other pathogens.
2.2 Papillomaviruses and human cancers
HPV have been associated with several conditions in humans. Broadly, according
to their oncogenic potential, non-oncogenic or low-risk HPV (lr-HPV) cause clinically
benign lesions whereas carcinogenic or high-risk (hr-HPV) papillomaviruses are strongly
associated with ano-genital and head and neck squamous cell carcinomas [4, 22].
Infections with hr-HPV in the ano-genital area are usually cleared by host immune
mechanisms. However, in cases in which hr-HPV infections persist for several years, the
risk of developing low-grade and high-grade lesions that eventually progress into invasive
cervical carcinomas increases. Globally, cervical cancer is one of the most common cancers
in women. Approximately 530,000 new cases are annually diagnosed, with an alarming
mortality rate of 50%. Invasive squamous cell carcinomas and adenocarcinomas of the
cervix, as well as their respective precursor lesions are almost always positive for any hr-
HPV, with HPV-16 and HPV-18 being the most prevalent genotypes [22]. In men, more
than 50% of penile cancers cases worldwide diagnosed are associated to HPV, and 40%
and 60% of oropharyngeal cases in Europe and in North America, respectively are also
causally associated to HPV [39]. Additionally, more than 90% of the anal cancer cases
among men who have sex with men (MSM) and immuno-compromised individuals are also
positive for HPV [4].
13
2.2.1 HPV infectious cycle
Human papillomaviruses are dsDNA non-enveloped viruses surrounded by a proteinaceuos
icosahedral capsid of approximately 50-60 nm in diameter [6]. HPV genome encodes for
eight dierent proteins. Early proteins E1, E2, E4 and E5 regulate transcription and viral
DNA replication. The viral E6 and E7 proteins contribute to cell transformation and im-
mortalization. Finally, the structural proteins L1 and L2 assemble around the HPV-DNA
to form the virion's capsid [3, 13].
Human Papillomaviruses have a unique life cycle that depends exclusively on infecting
undierentiated and actively dividing basal epithelial keratinocytes. The replication cycle
is completed in the mid- and upper-cell strata where cells dierentiate and eventually
slough o from the epithelium. Because of the strict tropism for basal keratinocytes for
initial infection, HPV requires exposure of basal cells located near the basement membrane
through physical or chemical disruption of the epithelium [43]. Visible lesions, abrasions
and cuts, and even non-apparent micro-traumas or micro-abrasions have been hypothe-
sized to serve as entry points for HPV. This rened tropism and dependence of HPV for
basal cells ensures viral replication, and constitutes an important determinant for viral
persistence and transmission (Figure 2.2.
Binding and entry of HPV to basal cells is a multistep process that not only requires
interaction between viral and cell proteins but also activation of cellular signaling cascades.
Initial interaction occurs either between HPV L1 capsid protein and heparan sulfate pro-
teoglycans (HSPGs) expressed on the cell surface and basement membrane, or between
HPV L1 and non-HSPG molecules such as laminin-332 on the extracellular matrix. Alpha-
6 beta-4 integrin and tetraspanin may also participate in the binding process of HPV to
14
target cells. Subsequent cleavage of HPV L2 capsid proteins by cellular pro-protein con-
vertases such as furin, results in capsid conformational changes that reduce molecular
anity between L1 and HSPGs, facilitating HPV binding to its internalization receptor,
the heterotetrameric protein complex of annexin A2 and S100A10 (A2t) [7, 16, 25, 44, 58].
Then, in a non-clathrin, non-caveolin, non-dynamin, but actin-dependent endocytic pro-
cess, HPV is internalized. Acidication of HPV-endocytic vesicles facilitates viral uncoat-
ing and release of L2-HPV DNA complex. L2 protein chaperons HPV-DNA to the Golgi
compartment, and from there, in a retrograde fashion while bound to dynein, L2-HPV-
DNA is transported to the nucleus along the cellular microtubule network. Once inside
the nucleus HPV DNA is replicated and transcribed by the host cell machinery [3].
Expression of early viral proteins E1 and E2 maintain viral DNA as an episome in
basal keratinocytes and facilitate correct segregation of viral episomes into progeny cells.
As a consequence, one daughter cell remains in the basal layer, creating a reservoir for
HPV DNA (HPV latent infection), while the other daughter cell migrates to epithelial
upper layers and undergoes terminal dierentiation. During the dierentiation process,
host mRNA proles shift and dictate which HPV genes are transcribed, eventually leading
to the completion of new HPV virions. Within infected cells, low expression of the viral
oncoproteins E6 and E7 prevent cells from exiting cell cycle and retard normal terminal
dierentiation of the keratinocytes, favoring virus proliferation [29].
2.3 Secretory Leukocyte Protease Inhibitor (SLPI)
The surface epithelium is an important physical barrier that separates and protects us
from external pathogens. Epithelial cells that form the mucosal epithelia such as the one
15
Figure 2.2: Representation of a normal stratied epithelial layer, and epithelium infected
with HPV.
lining ano-genital, respiratory and oral cavities not only form that barrier, but they also
secrete a variety of antimicrobial compounds, proteases and enzymes that degrade mi-
crobial cells walls and facilitate immune cell migration by degrading extracellular matrix
components among many other functions [31]. In fact, this constitutes both the primary
non-specic line of defense against pathogens, and the activator of adaptive immunity.
However, without any control, in
ammatory reactions activated in response to pathogen
invasion or as a consequence of tissue injury may lead into self-tissue deterioration and
contribute to the development of dierent diseases. An important player in maintain-
ing this balance is the secretory leukocyte protease inhibitor (SLPI), which acts as both
antimicrobial and anti-in
ammatory mediator. [54]
16
Some examples of secreted compounds of the innate immune system such as mucins,
proteases (granzymes, tryptase, elastase, proteinase 3, etc), and antimicrobial peptides
like defensins, elans, and secretory leukocyte protease inhibitor act as potent molecular
defenses that prevent adherence and invasion of pathogens. SLPI is a 11.7-kDa serine
protease inhibitor secreted by immune and epithelial cells, and belongs to the WFDC-
motif-containing [WAP(whey acidic protein) four-disulde core] protein family. SLPI is
constitutively found in dierent human
uids including saliva, nasal, bronchial and in-
testinal secretions, as well as in cervical mucus, and semen. Initially described as a pro-
tease inhibitor of neutrophil elastase and cathepsin-G, SLPI is currently known for its
anti-in
ammatory, anti-protease, anti-fungal and anti-microbial biological functions that
protect local tissues against harmful consequences of in
ammation. [48, 55, 56]
As an in
ammatory mediator, it has been demonstrated that SLPI inhibits pro-
in
ammatory response both in vivo and in vitro by intracellular and extracellular mech-
anisms. Intracellularly, SLPI suppresses TNF and nitric oxide production by blocking
LPS-induced activation of NF-B in human monocytes in vitro. Specically, SLPI sta-
bilizes and prevents degradation of IB and IB, and IRAK (IL-1-receptor-associated
kinase) as well as inhibits NF-B-DNA interaction by binding to the p65 subunit of NF-
B, decreasing IL-1, IL-8 and TNF- gene expression [34, 54, 55]. In SLPI knockout mice
(SLPI-/-) undetectable levels of IL-1, TNF- and NO-2 were observed after LPS admin-
istration by comparing to SLPI wild type (SLPI +/+) control animals. [41] In addition to
this, extracellularly SLPI can also indirectly modulate immune response against certain
pathogens by binding to pathogen-associated molecular patterns such as LPS of Gram
negative bacteria and lipoarabinomannan of Mycobacteria prior to TLR activation. [48]
17
During in
ammation the delicate balance between induction of innate immune re-
sponse and tissue repair is predominantly controlled by neutrophil elastase and its inhibitor
SLPI. In fact, increased in
ammation and TGF- production, and human neutrophil elas-
tase activity correlate with impaired wound healing in SLPI knockout mice. [41] A plau-
sible explanation for this phenomenon is that epithelial cell growth is suppressed by the
massive recruitment of neutrophils in the presence of epithelin, the cleavage product of
pro-epithelin. Under normal physiological conditions, by degrading neutrophil elastase,
SLPI prevents cleavage of pro-epithelin to epithelin, aiding normal wound healing and
tissue repair. [41, 55]
Finally, another important function of SLPI is its anti-microbial capacity through the
targeting of Gram positive and Gram negative bacteria, some fungi and certain viruses. In
the upper respiratory tract, SLPI is one of the most abundant and eective anti-microbial
compounds against Pseudomonas aeruginosa, Staphylococcus aureus, S. epidermidis, Es-
cherichia coli and Candida albicans and Aspergillus fumigatus [54]. Another well char-
acterized antiviral activity of SLPI is its anti-HIV capacity. [14, 26, 36] SLPI has been
described as the most potent anti-HIV-1 molecule in human saliva, explaining why oral
HIV transmission is uncommon. Specically, in an in vitro study performed on primary
human monocytes, SLPI showed signicant inhibition of HIV infection. [38] Importantly,
SLPI has been identied as a natural endogenous ligand for a membrane-bound receptor
called annexin A2, which turned out to be an initial binding receptor for phophatidylserine
located on the outer envelope of HIV-1. Therefore, the interaction of SLPI with annexin
A2 on the cell surface of macrophages prevents initial binding of the virus, reducing subse-
quent recognition of specic cellular receptors. [36] In addition to these nding, it has also
18
been shown that annexin A2 expressed in human epithelial cells facilitates HPV infection
through interaction of L2 HPV capsid protein with the S100A10 minor subunit of the
heterotetrameric complex. [58]
The ability of SLPI to eectively block HPV infection has been observed in both ep-
ithelial and Langerhans cells (LC), the local antigen-presenting cell of the epithelium [58].
Epidemiological data has also shown that in vivo, an inverse correlation exists between
the level of SLPI and the risk of having an HPV-positive head and neck squamous cell
carcinoma (HNSCC) [23]
2.4 HSV, HPV, and cervical cancer
Until the late 1980's it was believed that the primary etiologic agent in both oral and
cervical cancers was HSV [15, 51]. Scientists went as far as suggesting that HSV-infection
lead to oncogenic transformations through hypomethylation of host cellular DNA [37].
However, even after HPV DNA was found in the overwhelming majority of cervical can-
cer tissues the epidemiologic link between HSV and cervical cancer persisted. HSV-2
has been implicated as a cofactor in the development of cervical carcinoma; specically
a two fold increase in the risk of developing cervical adenocarcinoma has been observed
in patients seropositive for both hr-HPV and HSV-2 versus patients seropositive for HPV
alone [61]. In addition to the possible direct role that HSV-2 may have in HPV-cervical
cancer development, HSV-2 can indirectly facilitate HPV infection. Undoubtedly, ulcer-
ative lesions during active HSV-2 infection facilitate HPV access to basal keratinocytes
and basal membrane. Furthermore, suppression of host immune response against HSV
infections can also support HPV acquisition and persistence. In particular, blockage of
19
viral peptide-presentation via MHC class I by HSV-2 ICP47 protein, as mentioned earlier,
suppresses recruitment and activation of CD8+ cytotoxic T lymphocytes which indirectly
facilitates HPV infection and persistence in the epithelium [43, 61].
In addition to these immune evasion strategies, it has been demonstrated that HSV-1
and HSV-2 infections alter the expression of mucosal immune mediators such as human
beta defensin-1 and SLPI. Down-regulation of these components is not only a possible
mechanism of host immune evasion, but also a direct insult to the immune mechanism
in place to prevent infection by other pathogens such as HIV and HPV [18, 46]. The
consequence of HSV-2 mediated-alteration of innate mucosal immunity in the acquisition
and persistence of hr-HPV infections remain unknown; however, it is the main objective
of the present study.
20
Chapter 3
Materials and Methods
3.1 Cell lines, antibodies and recombinant proteins
In vitro spontaneously transformed human skin keratinocytes or HaCaT cells (Cell
Lines Service, Eppelheim, Germany) were cultured in Dened Keratinocyte Serum-Free
Media (K-SFM) (Gibco
R
) complemented with the provided nutrient supplement (insulin,
EGF, FGF), and grown at 37

C with 5% of CO
2
. Green monkey kidney epithelial cells
or Vero cells, purchased form the ATCC, were maintained in complete media (Iscove's
Modied Dulbecco's Medium, IMDM, 10% FBS and 1X PenStrep) (Lonza) at 37

C with
5% of CO
2
.
The antibodies used were anti HSV-1/2 gB (10B7) mouse monoclonal antibody (Santa
Cruz Biotechnology), PE/Cy7 Goat anti-mouse IgG (Biolegend) antibody, H16.E70 mouse
anti-HPV-16 L1 (gift form Neil Christensen, Penn State). Recombinant human SLPI
(rhSLPI) was purchased from R & D Systems (Minneapolis, MN)
21
3.2 Propagation and titration of HSV-2
Wild type Human Herpesvirus 2 strain G [HSV-2(G)] (VR-734
TM
) was purchased from
ATCC. Amplication and titration of HSV-2(G) were done in Vero cells grown at 37

C
with 5% of CO
2
. Brie
y, a monolayer of Vero cells grown in a 150mm tissue culture
dish was infected with HSV-2(G) at a multiplicity of infection (MOI) of 0.01 pfu/cell.
After 2 hours of virus absorption (incubation and rocking every 15 minutes), additional
IMDM media was added to the cells. Infection was carried out until 100% of cells showed
cytopathic eect (CPE) by visualizing them under the microscope. Rounded up and
detached cells from the bottom of the plate were the common CPE observed in HSV-
2(G) infected Vero cells. After virus propagation, media was carefully collected from the
plates and cleared via centrifugation for 5 minutes at 1200 rpm at room temperature.
Then, concentration of the viruses present in the suspension was performed by ultra-
centrifugation for 1 hour at 28000 rpm at 4

C. HSV was then resuspended in IMDM
media, aliquoted and stored at80

C.
Since we wanted to study the eect of HSV-2(G) infection on the expression of SLPI in
HaCaT cells, we used that cell line to quantitate the amount of HSV-2(G) viral particles
contained in the HSV-2(G) stock suspension. Virus titer was determined by a plaque assay
through the assessment of plaque-forming units per mL (pfu/mL). In this case, monolayers
of HaCaT cells were infected with serial dilutions of the virus stock, incubated for 2 hours
followed by removal of the infectious media and overlaid with 0.4% carboxymethylcellulose
(CMC) containing K-SFM. Three days later, plaques were visualized previous xation and
staining with crystal violet. Plaques were manually counted and viral titre was calculated.
22
3.3 HPV-16 Pseudovirus (PsV) and Virus-Like Particles
(VLP)
Amplication and purication of HPV-16 L1L2 GFP expressing pseudovirions (PsVs)
were done following a previously published protocol [5, 32]. In general, 293TT cells were co-
transfected with a codon-optimized expression vector containing HPV-16 L1 and L2 genes,
and a GFP expressing-reporter plasmid DNA (pCIneoGFP). After 48h, cells were lysated,
and PsVs were puried by iodixanol (OptiPrep
TM
) density gradient centrifugation. The
infectious titer of PsV preparations (in infectious units/mL, IU/mL) was determined by

ow cytometric analysis of 293TT cells treated with serially diluted doses of the PsV stock.
Neutralization of PsVs was validated in infection assays with H16.E70 prior to cellular
exposure, and minimal infection rates of less than 1% were observed on cell cultures
treated with those neutralized PsVs.
HPV-16 L1L2 Virus-Like Particles (VLPs) and HPV-16 L1 VLPs were obtained by
using the baculovirus expression vector system (BEVS) in Sf9 cells. Once puried and
titrated, VLPs were labeled with pHrodo
TM
Red, SE labeling kit (Life Technologies).
Following the manufacturer's instructions, pHrodo
TM
-labeled VLPs were puried by gel-
column ltration using 2% of agarose beads. Dierent fractions collected during the
ltration process were compared to BSA standard solutions (0.5, 0.25, 0.1, 0.05mg/mL)
in a protein gel stained with Coomassie Brilliant Blue after SDS-PAGE electrophoresis.
pHrodo-VLP concentration was expressed in mg/mL.
23
3.4 Infection of HaCaT cells with HSV-2(G)
HaCaT cells were grown to 80-90% con
uency in a 175cm
2
tissue culture
ask (BD
Falcon); following treatments with 10mL of 1X trypsin-EDTA in PBS (Life Technologies)
for 10 minutes at 37

C, and with 10mL of dened trypsin inhibitor (DTI) (Life Technolo-
gies), cell were collected and span down for 5 minutes at 1500 rpm at room temperature.
Cell pellet was re-suspended in K-SFM, then cell viability and total cell number were
calculated using The Countes
R
(Life technologies) after staining cells with 0.4% Trypan
blue. A total of 5x10
5
cells per well were plated in 24-well cell culture plates followed
by incubation for 24 hours at 37

C with 5% CO
2
. After the incubation period, media
was removed and cells were infected, in duplicate, with HSV-2(G) at multiplicity of in-
fection (MOI) of 0.7, 1, 2.5 and 5. Uninfected cells, incubated with complete K-SFM,
were used as mock infected control. HSV-2(G) viruses were adsorbed for 3 hours; after
that, infectious media was discarded, cells were rinse twice with 1X sterile PBS to remove
unadsorbed virions, and fresh complete K-SFM media containing 0.8mM of Acycloguano-
sine (Acyclovir) (Sigma-Aldrich) was added to every well including the uninfected cells.
Plates were incubated at 37

C with 5% CO
2
. Acyclovir was added to the cells to prevent
completion of the HSV lytic cycle. This allows the maintenance of intact cells prior to
HPV infection.
3.5 Quantication of HSV-2 infection by
ow cytometry
Percentage of HSV-2 infected HaCaT cells was measured by
ow cytometry after spe-
cic staining for viral glycoproteins expressed on the cell surface. At 24 and 48 hours
24
after HSV-2(G) infection, HaCaT cells were trypsinized and collected from the wells, and
transferred to 5mL polystyrene round-bottom tubes. Then, the cell suspension was spun
down for 5 minutes 1500 rpm at 4

C. After decanting the supernatant, cell pellet was
rinsed once with 2mL of FACS buer (80% PBS, 20% FBS and 0.01% NaN
3
), and nally
resuspended in 200L of FACS buer. Cells were incubated, on ice, for 45 minutes with
0.5g per million cells of anti HSV-1/2 gB (10B7) mouse monoclonal antibody (Santa
Cruz Biotechnology). Unbound antibodies were removed by washing cells twice with 2mL
of FACS buer and afterward cells were resuspended in 200L of FACS buer. PE/Cy7
Goat anti-mouse IgG (Biolegend) antibody was used as secondary antibody at a concen-
tration of 0.25g per million cells. Cells were incubated on ice and protected from direct
light for 30 minutes. Next, cells were washed twice with FACS buer and resuspended in
500L of FACS buer. Immediately,
uorescent signals were measured by
ow cytometry,
and percentage of HSV-2(G) infected cells was calculated and compared to uninfected
control cell population.
3.6 Assessment of slpi gene expression in HSV-2(G) in-
fected HaCaT cells
To access whether HSV-2(G) infection modulates slpi gene expression, HaCaT cells
were infected with HSV-2(G) for 24 and 48h, as described above. Then slpi gene expression
was quantitated by (quantitative reverse transcription PCR) RT-qPCR. After infection,
media from the HSV-2(G) infected and uninfected HaCaT cells was poured o from the
25
wells, cells were lysed and RNA was extracted using RNeasy
R
Plus Mini Kit (QIAGEN)
following recommended directions from the manufacturer.
Concentration and purity of the total RNA isolated was then determined by spectrom-
etry using Nanodrop
R
2000 (Thermo Scientic). Expression analysis was performed by
initially reverse transcribing RNA to cDNA using the iScript
TM
cDNA Synthesis Kit (Bio-
Rad), followed by quantitative PCR utilizing a SensiFAST
TM
SYBR No-ROX kit with
primers: SLPI-F: 5'-CTGTGGAAGGCTCTGGAAA, SLPI-R: 5'-GTCAACAGGATCCA
GGCATT, 18S-F: 5'-AAACGGCTACCACATCCAAG, and 18S-R: 5'-CCTCAATGGATCCTCGTTA.
PCR reaction was monitored using a CFX Real-Time Detection System (Bio-Rad). The
basic reaction conditions were 95

C for 5 minutes followed by 40 cycles of 94

C for 15 sec-
onds, 59

C for 15 seconds, and 72

C for 20 seconds. Lastly, the results were normalized,
for each RNA preparation, to 18S mRNA.
3.7 Quantication of secreted SLPI by HSV-2 infected Ha-
CaT cells
To access whether HSV-2(G) infection modulates SLPI protein translation, HaCaT
cells infected with HSV-2(G) for 24 and 48h, as indicated before. Then SLPI secreted
protein levels were measured via ELISA. Following manufacturer's protocol, Quantikine
R
ELISA was performed on the supernatant of HSV-2(G) infected HaCaT cells. In general,
the assay consist in a quantitative sandwich ELISA, in which cell culture supernatants,
diluted 1:20 were added to microplate wells previously coated with anti-human SLPI mon-
oclonal antibodies. Then wells were incubated for 2 hours, and unbound SLPI was removed
26
by decanting supernatants and washing wells with provided wash buer. An enzyme-linked
polyclonal antibodies specic for SLPI were added to the wells and incubated for 2 hours.
Following washes to remove unbound antibodies, substrate solution was added. SLPI con-
centration was calculated based on optical density readings compared to standard curve
obtained at the same time of the experiments. SLPI secreted protein concentration was
expressed in picograms per mL (pg/mL). The SLPI concentration detection limit of this
kit was 62.5 pg/mL.
3.8 Uptake of HPV-16 L1 and L1L2 pHrodo
TM
-labeled VLPs
after HSV-2(G) infection
HaCaT cells infected with HSV-2(G) at MOI 2.5 were incubated with HPV16 L1L2
pHrodo
TM
Red-labeled VLPs at a concentration of 2g per 10
6
cells (approximately,
7 particles/cell) for 16 hours. Then cells were trypsinized and collected. Fluorescence
emission of both pHrodo
TM
Red and from antibody staining for HSV-2-cell-surface antigens
were measured by FACS. Positive signals were compared and normalized to uninfected
controls.
3.9 Infection of HaCaT cells with HPV-16 L1L2 PsVs post
HSV-2(G) infection
To access changes in HPV16 L1L2 PsV uptake after HSV-2(G) infection, dual infection
with HSV-2(G) and HPV16 PsVs was performed. Initially, HaCaT cells were infected for
24 and 48h with HSV-2(G) at MOI of 0.7. Then, HPV16 L1L2 PsVs at a MOI of 200 were
27
added. Cell were incubated for 4 hours to facilitate virus adsorption. Then, fresh media
was added to the cells, and plates were incubated overnight. Next day, infectious media
was removed, cells were washed twice with PBS, and cells were incubated for 24 hours in
fresh K-SFM. Forty eight hours after addition of PsVs, cells were trypsinized, collected and
analyzed via
ow cytometry. Specic antibody-
uorescence signal from HSV-2(G) positive
cells, along to GFP
uorescence from HPV16 infected cells were measured. Positive signals
were compared and normalized to uninfected controls.
28
Chapter 4
Results
Given the protective role of SLPI against viral infections including HIV and HPV
[23, 36, 58], and its down-regulation after HSV infections in vitro [18], the present study
shows results that indicate a possible association between reduced expression of SLPI
during HSV-2(G) infection, and enhanced susceptibility of human keratinocytes to HPV
infection in vitro.
4.1 HSV-2(G) infection causes down-modulation of SLPI
after 24h
It has been demonstrated that infection with HSV-1 and HSV-2 causes sustained down-
regulation of SLPI in CaSki cells, an HPV-16-transformed cervical cancer cell line [18];
therefore we hypothesized that similar eect would be observed after infecting HaCaT cells
with HSV-2(G). HaCaT cells are immortalized epithelial cells not transformed by HPV.
The eect of HSV-2(G)infection on HaCaT cells in regard to slpi gene transcription and
protein secretion was accessed by gene expression analysis by RT-qPCR, and quantitation
of secreted SLPI protein by ELISA.
29
HaCaT cells were infected for 24 hours with HSV-2(G) at MOI of 1, 2.5 and 5 in the
presence of 0.8mM of acyclovir. Twenty four hours after HSV-2(G) infection, signicant
reduction of slpi gene expression of 48.3%, 56.3% and 64.7% was observed on cells infected
at MOI of 1, 2.5 and 5, respectively compared to the uninfected cells as shown in gure
4.1-A. Additionally, at the same time point, secreted SLPI protein concentration was
measured in cell supernatants. In comparison to SLPI protein concentration secreted
by uninfected cells (157.3ng/mL), HaCaT cells infected for 24h with HSV-2(G) secreted
signicantly less amount of SLPI, which represented a reduction of 20.9% (MOI of 1),
34.2% (MOI of 2.5) and 39.1% (MOI of 5) (Figure 4.1-B).
4.2 Down-modulation of SLPI after 24h of HSV-2(G) infec-
tion causes minimal eect on HPV uptake and infection
Previous studies have shown that SLPI is the natural cellular ligand for the HPV inter-
nalization receptor A2t in human keratinocytes. Additionally, introduction of exogenous
recombinant human SLPI (rhSLPI) blocks HPV infection of HaCaT cells in vitro [58].
Since a signicant reduction in SLPI protein secretion after HSV-2(G) infection of
HaCaT cells was observed, we wanted to test the hypothesis that diminished extracellu-
lar SLPI concentration, as a consequence of HSV infection, facilitates HPV infection by
unblocking the A2t receptor. To address that, HSV-2(G) infected and uninfected HaCaT
cells were infected with HPV-16. However, since the life cycle of HPV requires dierentia-
tion of human epithelial cells making it dicult for in vitro studies, we used an alternative
model of infection using either HPV-16 L1L2 pHrodo
TM
red-labeled virus-like particles
30
Normalized SLPI gene
expression
0 1 2.5 5
0.0
0.2
0.4
0.6
0.8
1.0
1.2
HSV-2(G) multiplicity of infection
**
**
**
A
Secreted SLPI protein (ng/mL)
0 1 2.5 5
0
25
50
75
100
125
150
175
*
**
**
HSV-2(G) multiplicity of infection
B
Figure 4.1: A: slpi gene expression normalized to 18S mRNA. B: SLPI secreted protein
concentration measured by ELISA. Black bars indicate uninfected HaCaT cells, while
gray bars correspond to HSV-2(G) infected cells. Single asterisk indicates statistically
signicant dierence compared to uninfected control cells with a P value <0.05, while
double asterisks indicate statistically signicant dierence with a P value <0.01.
(VLPs) or HPV-16 L1L2 GFP expressing pseudo-virions (PsVs). Both types of particles
are morphologically similar because both are formed by L1L2 capsids, however, VLPs
are empty capsids labeled with the pH-sensitive
uorescent dye, pHrodo
TM
red and PsVs
contain a (Green Fluorescence Protein) GFP reported DNA plasmid. While VLPs are
utilized to monitor the amount of particles internalized by HaCaT cells though endocytic
31
pathway, PsVs allow to quantitate the amount of HaCaT cells infected through reporter
gene transduction and expression of GFP. Those particles, either VLPs or PsVs were
added to HaCaT cells previously infected with HSV-2(G) for 24h.
Since
uorescence of pHrodo
TM
-red label increases in acidic environments, and the
infectious pathway of HPV involves endocytic vesicle formation and pH-dependent matu-
ration into early and late endosomes, VLP pHrodo
TM
red
uorescence was considered a
sign for HPV-16 VLP internalization. Therefore, after 16h pHrodo
TM
-VLP
uorescence
was measured by FACS. Additionally, 48h after addition of PSVs, GFP expressing cells
were quantitated also by
ow cytometry.
Initially, optimization of HPV-16 L1L2 pHrodo
TM
-labeled VLP concentration was per-
formed on normal or uninfected HaCaT cells. A range from 2g to 10g of VLPs per 10
6
cells was tested. PHrodo
TM
-red
uorescence was detectable in all groups of cells treated
with dierent concentrations of VLPs, as showed in gure 4.2. Since the lowest VLP con-
centration of 2g per million cells was sucient to obtain a signicant amount of HPV-VLP
infected cells (18%) compared to untreated cells, this concentration was selected as the
optimal to use for further experiments.
As can be seen in the gure 4.3, both HPV-VLP and HPV-PsV uptake and infection
did not signicantly changed between uninfected and HSV-2(G) infected cell groups. By
comparing the percentage of pHrodo
TM
-
uorescent cells on the HSV-2(G) infected pop-
ulations with the percentage on the uninfected HaCaT cells, there was not a signicant
change in terms of HPV-16 VLP uptake (Figure 4.3 A). Similarly, no observable changes
in HPV-16 PsV infection on HSV-2(G) infected HaCaT cell groups versus infection on
HSV negative control cells were measured (Figure 4.3, B).
32
Percentage of HPV-16 L1L2 VLP uptake
(pHrodo-red positive cells)
0 2 2.5 4 5 7.5 10
0
10
20
30
40
50
60
mg of VLPs per 10
6
cells
Figure 4.2: Percentage of pHrodo
TM
-red HPV-16 VLP infected cells was calculated based
on the pHrodo
TM

uorescence accessed by FACS.
These results indicate that after 24h of HSV-2(G) infection, susceptibility of HaCaT
cells to HPV-16 infection remains stable, and does not dier from the observed levels of
HPV-16 infection on HSV negative cells. In fact, although the SLPI protein concentration
secreted by HSV-2(G) infected cells was statistically signicantly lower when compared
to that secreted by uninfected cells (Figure 4.1), biologically it seems to be irrelevant in
the context of HPV infection. Indeed, SLPI concentrations, after 24h of HSV-2(G), were
not suciently diminished to signicantly reduce the amount of SLPI bound to A2t cell
receptor.
4.3 HSV-2(G)-induced down-regulation of SLPI increases
and it is maintained after 48h post infection
As previously observed, reduction of about 50% on slpi gene transcription, and between
20-30% decrease in SLPI protein secretion, 24h after HSV-2 (G) infection, had no eect on
HPV infection on HaCaT cells. Therefore, we increased the HSV-2(G) infection period to
33
Percentage of HPV-16 L1L2 VLP uptake
(pHrodo-red positive cells)
0 1 2.5 2.5
H16.E70
0
5
10
15
20
25
30
35
**
HSV-2(G) multiplicity of infection
A
Percentage of HPV-16 L1L2 PsV uptake
(GFP positive cells)
0 1 2.5 2.5
H16.E70
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
**
HSV-2(G) multiplicity of infection
B
Figure 4.3: HPV-16 uptake and infection is not altered after 24 hours of HSV-2(G) in-
fection. A: percentage of pHrodo
TM
-
uorescent cells which indicates HPV-16 L1L2 VLP
endocytosed by HaCaT cells previously infected with HSV-2(G) at MOI indicated in the
x axes. B: percentage of HPV-16 PsV infected cells as indicated by the percentage of GFP
expressing HaCaT. Double asterisks indicates statistically signicant dierence with a P
value <0.01. H16.E70 refers to neutralizing monoclonal antibody against HPV L1 capsid
protein used to treated either VLPs or PsVs that was included as internal control.
34
Normalized slpi gene
expression
0 0.7 1 2.5 0 0.7 1 2.5
0.0
0.2
0.4
0.6
0.8
1.0
1.2
24h 48h
**
** *
**
HSV-2(G) multiplicity of infection
*
Figure 4.4: HSV-2(G) infection induces downregulation of slpi gene expression on HaCaT
cells. slpi gene expression of both uninfected (MOI of 0) and HSV-2(G) infected cells
was normalized 18S reference gene. Results show mean and standard deviation of three
independent experiments. Single asterisk indicate statistically signicant dierence of slpi
gene expression comparing HSV-2 infected cells to the uninfected control cell at specic
infection time with a P value <0.05, while double asterisks with a P value <0.01.
48h to test if a prolonged HSV infection is required to increase HaCaT cell susceptibility
for HPV infection in vitro. We believed that both SLPI mRNA degradation and reduced
protein secretion increase as HSV-2(G) infection progresses in time. Additionally, we
tested if mimicking an in vivo HSV-2(G) infection by infecting the cells with a lower
MOI would also result in a signicant down-modulation of SLPI. Thus, HaCaT cells were
infected with HSV-2(G) at MOI of 0.7, 1 and 2.5 for 48h.
As the data shows in gure 4.4, transcriptional down-regulation of the slpi gene was
observed after 24h of HSV-2(G) infection, conrming our previous results. However,
greater reduction of slpi mRNA transcription was observed at 48 hours, indicating that
this down-modulation in HaCaT cells is maintained after a long infection period. A
dramatic decline on SLPI transcripts up to 76.9%, 72% and 77% was detected on cells
infected at MOI of 0.7, 1 and 2.5, respectively as shown in gure 4.4).
35
Secreted SLPI protein (ng/mL)
0 0.7 1 2.5 0 0.7 1 2.5
0
25
50
75
100
125
150
175
200
225
24h 48h
HSV-2(G) multiplicity of infection
**
*
*
**
**
Figure 4.5: HSV-2(G) infection reduces the secretion of SLPI protein by HaCaT cells. The
amount of secreted SLPI was quantitated by ELISA. Results show mean concentration and
standard deviation of tree independent experiments. Single asterisk indicate statistically
signicant dierence compared to uninfected control cell at specic infection time with a
P value <0.05, while double asterisks with a P value <0.01.
Similarly, reduction of SLPI secreted protein measured on supernatant of cells after
HSV-2(G) infection was quantied. While on average, uninfected cells secreted 128ng/mL
of SLPI, those infected for 24 hours with HSV-2(G) secreted about 20-30% less SLPI. Cells
infected with MOI of 0.7 of HSV-2 secreted 93ng/mL; MOI of 1, 101ng/mL, and MOI of
2.5, 91ng/mL. More signicantly, after 48 hours of infection, the SLPI protein level on
supernatant of cells infected with an MOI of 0.7 was reduced by 66.4% (43ng/mL) when
compared to the amount secreted by uninfected HaCaT cells (168ng/mL). Likewise, more
than 57% reduction was noticed on cells infected with MOI of 1 and 2.5 at the same time
point (Figure 4.5).
These results clearly indicate that in HaCaT cells, down-modulation of both slpi gene
expression and SLPI protein secretion after in vitro infection with HSV-2(G) persists and
increases over time under acyclovir treatment.
36
0. 7 1 2. 5 0. 7 1 2. 5
Figure 4.6: Percentage of HSV-2(G) infection on HaCaT cells
4.4 HSV-2(G) infection of HaCaT cells and cell viability
To conrm HSV-2 infection of HaCaT cells, HSV-2(G) infection was measured by

ow cytometry after antibody staining for HSV-gB antigens expressed on cell surface of
infected cells. As indicated in the gure 4.6, percentage of infected cells was proportional
to MOI used, and between 15% and 34% of the cells were infected.
Additionally, we wanted to conrm that acylovir treatment of HSV-2 infected cells
would prevent cell death due to the viral active lytic cycle during the longer infection
period of 48h. Therefore, the number of viable cells was determined by Trypan-blue dye
exclusion test at dierent time points. For this particular assay, HSV-2(G) infected HaCaT
cells with MOI of 2.5 were maintained in culture without and with 0.8mM of acyclovir,
and every 12h, cells were collected and stained with Trypan blue. When compared the two
groups of HSV infected cells treated with and without acyclovir, we observed signicant
dierence in the percentage of viable cells as the infection time advanced, see gure 4.7.
As expected, after 72 hours of lytic cycle or uncontrolled HSV-2 infection only half of the
37
Time after HSV-2(G) infection (moi 2.5)
Cell viability
(percentage of alive cells)
12h 24h 36h 48h 60h 72h
0
10
20
30
40
50
60
70
80
90
100
HSV-2(G) ACV
HSV-2(G) No ACV
Figure 4.7: Viability of HaCaT cell infected with HSV-2(G) at an MOI of 2.5. Percentage
of cells was determined after by Trypan blue exclusion test.
cells (48%) remained alive, compared to 90% when viral replication was inhibited through
addition of acyclovir.
Together, these results clearly indicate that HSV-2(G) infection inhibits slpi gene
transcription and reduces SLPI protein secretion of HaCaT cells. This eect was observed
in cells infected for 24 hours, and it became more signicant after 48 hours of infection
indicating that even in the absence of viral replication, HSV-2(G) consistently decreases
transcription and secretion of SLPI.
4.5 Increased HPV-16 uptake and infection after HSV-2 in-
fection
Since the major eect on SLPI down-modulation was observed after 48 hours of HSV-
2(G) infection, we decided to carry out HSV-2 infections on HaCaT cells after that period
of time before pHrodo
TM
-red HPV-16 VLP and HPV-16 GFP PsV exposure. After HSV
infection, either pHrodo
TM
-labeled VLPs or HPV-16 GFP PsVs were added to the cells.
38
Additionally, to prevent changes in the SLPI protein concentration, cell growth medium
was not replaced before adding VLPs and PsVs.
By comparing the percentage of HPV-16 VLP uptake between uninfected HaCaT cells
and HSV-2(G) infected cells, a dramatic dierence in the amount of endocytosed VLPs
was observed as indicated in gure 4.8. Specically, when compared uptake of uninfected
(non-HSV) cells to uptake of HSV infected cells, we observed a shift from 13.5% VLP-
positive to 33.6%, which represents a 2.5 fold-increase in VLP uptake. This indicates, that
HSV-2(G) infection promotes HPV-16 L1L2 VLP binding and internalization on HaCaT
cells. In fact, increased HPV-16 VLP uptake can be inversely correlated to the observed
down-regulation of SLPI after HSV-2(G) infection.
Similarly, HPV-16 infection of HSV-2(G) infected cells increased as seen in gure 4.9.
While about 9% of normal (non-HSV-2) HaCaT cells were infected with HPV-16 GPF
PsVs (gure 4.9B), this number almost doubled when cells were previously infected with
HSV-2(G) (gure 4.9 C). Those numbers represents an 1.8 fold increase in HPV-16 PsV
infection after HSV-2(G) infection.
Results have shown that after HSV-2(G) infection, secretion of SLPI by HaCaT cells
substantially decreases; therefore we assume that the ratio SLPI:HPV-internalization re-
ceptor (A2t) reduces as well making the latter accessible to interact with HPV-16 VLPs.
This is supported by the observed increase of HPV-16-VLP internalization and HPV-16-
PsV infection. Additionally, as seen in both gure 4.8 D (quadrant of double positive
cells) and gure 4.9 C, no dual infection HPV-16 and HSV-2(G) was observed.
39
85. 7% 0. 3%
0. 3% 13. 7%
43. 3% 21. 8%
0. 9% 34. 0%
99. 1% 0. 3%
0. 1% 0. 5%
72. 7% 23. 3%
0. 5% 3. 5%
HSV- 2( G) i nf ect i on
pHr odo- HPV- 16 VLP
A B
C D
Percentage of HPV-16 L1L2 VLP uptake
(pHrodo-red positive cells)
0 0.7 0.7
H16.E70
0
5
10
15
20
25
30
35
**
**
HSV-2(G) multiplicity of infection
Figure 4.8: HPV-16 L1L2 pHrodo-VLP uptake increases after HSV-2(G) infection. Figures
on top show FACS results showing HSV-2(G) infected cells in the x-axis and pHroro
TM
-
VLP
uorescent cells in the y-axis. A: Control of normal HaCaT cells, without HSV or
VLP; B: non-HSV infected cells with VLP added; C: HSV-2(G) infected cells and neu-
tralized VLPs with anti-HPV-16 L1 monoclonal antibody (H16.E70), and D: cells infected
with both HSV-2(G) and pHrodo
TM
-VLPs. The bar-graph shows the mean percentage of
pHrodo-red HPV-16 VLP infected cells calculated based on the pHrodo
TM

uorescence
accessed by FACS form two independent experiments. Double asterisks indicate signicant
dierence (P value of <0.01) in VLP uptake compared to HSV-uninfected cells.
40
0 0.7 0.7
H16.E70
0
5
10
15
20
Percentage of HPV-16 L1L2 PsV uptake
(GFP positive cells)
**
**
HSV-2(G) multiplicity of infection
Figure 4.9: HPV-16 L1L2 GFP PsV infection increases after HSV-2(G) infection. Fig-
ures on top show FACS results showing HSV-2(G) infected cells in the x-axis and GFP
expressing cells (HPV-16 infected cells) in the y-axis. A: Control of normal HaCaT cells,
without HSV or PsVs; B: non-HSV infected cells with PsVs added; C: HSV-2(G) infected
cells and neutralized PsVs with anti-HPV-16 L1 monoclonal antibody (H16.E70), and D:
cells infected with both HSV-2(G) and with HPV-16 L1L2 GFP expressing PsVs. The
bar-graph shows the mean percentage of HPV-16 GFP PsV infected cells calculated based
on the GFP
uorescence accessed by FACS. Double asterisks indicate signicant dierence
(P value of <0.01) in VLP uptake compared to HSV-uninfected cells.
41
4.6 Restored SLPI secreted protein levels reduces HPV-16
uptake
Since in vitro HSV-2(G) infection of HaCaT cells results in increased HPV-16 VLP
uptake due to reduction in SLPI secretion, it was hypothesized that restoring the SLPI
extracellular concentration by adding rhSLPI (recombinant human SLPI) to the HSV-
infected cell cultures would result in reduction of HPV-VLP uptake.
Initially HaCaT cells were infected with HSV-2(G) at MOI of 2.5 for 48h, then me-
dia was removed, and dierent concentrations of rhSLPI were added to the cells prior
addition of VLPs. Since a reduction of about 125ng/mL of SLPI was measured by com-
paring amount secreted by uninfected cells (168ng/mL) to HSV-2(G) infected HaCaT cells
(43ng/mL) (gure 4.5), similar concentrations of rhSLPI were used during this experiment.
As observed in the gure 4.10, no signicant dierence in the L1L2 VLP uptake was ob-
served between rhSLPI-untreated cells and cells incubated with dierent concentrations
of rhSLPI.
Although SLPI concentration added back was equilibrated to a similar level of secreted
by normal cells (HSV uninfected), no eect was observed in regard to HPV VLP uptake.
According to this, apparently concentrations as high as 160ng/mL are not sucient to
block HPV uptake. Hence, we increased the amount of rhSLPI and measured VLP uptake
on HSV-2 infected cells.
Data shown in gure 4.11 indicates, after increasing the amount of rhSLPI added to
HSV-2(G) infected cells, HPV-VLP uptake was signicantly reduced. Signicant reduction
on HPV-VLP uptake was observed as cells were treated with >1000ng/mL of rhSLPI. In
42
Percentage of HPV16 L1L2 VLP uptake
(pHrodo-red positive cells)
0 40 80 120 160
0.0
2.5
5.0
7.5
10.0
12.5
15.0
rhSLPI added (ng/mL)
Figure 4.10: Dierent concentration of rhSLPI were added after HSV-2(G) infection pro-
ceeded for 48h, then 2g per million cells of L1L2 pHrodo-VLPs were added to the cells.
Graph shows percentage of VLP uptake measured by amount of pHrodo-
uorescent cells,
all cell groups were infected with HSV-2(G).
facts, more signicant results were obtained as the rhSLPI concentration was higher than
2500ng/mL, and complete blockage of HPV uptake was reached at 50000ng/mL.
These results clearly suggest that SLPI protein blocks and prevents HPV uptake, and
that reduction of this protein during HSV-2(G) increases HaCaT cells ability to internalize
HPV-16 as seen for the increment on HPV-16 L1L2 VLP uptake. In fact, studies indicate
that SLPI binds to the Annexin A2t heterotetramer receptor by interacting with annexin
A2. This interaction prevents HPV L2-mediated recognition and binding to the receptor
[58].
4.7 HPV L2 capsid protein is required for Enhanced VLP
uptake
It has been shown that the natural cellular ligand for A2t receptor is SLPI, while
during HPV infection, the viral ligand for A2t is the HPV-L2 capsid proten. To evaluate
43
Percentage of HPV16 L1L2 VLP uptake
(pHrodo-red positive cells)
0
1000
1250
1500
2500
10000
25000
50000
0.0
2.5
5.0
7.5
10.0
12.5
15.0
rhSLPI added (ng/mL)
*
**
*
**
**
**
*
Figure 4.11: Dierent concentration of rhSLPI were added after HSV-2(G) infection pro-
ceeded for 48h, then 2g per million cells of L1L2 pHrodo-VLPs were added to the cells.
Graph shows percentage of VLP uptake measured by amount of pHrodo-
uorescent cells,
all cell groups were infected with HSV-2(G). Double asterisks indicate signicant dier-
ence (P value of<0.01) in VLP uptake between untreated HaCaT cells (no rhSLPI) versus
SLPI treated cells.
the eect of reduced SLPI on HPV L2-mediated endocytosis, we treated normal and HSV-
infected HaCaT cells with either L1L2 HPV-16 VLP-pHrodo or L1 HPV-16 VLP-pHrodo
labeled particles and measured HPV-VLP uptake.
Initially, by comparing L1 VLP and L1L2 VLP uptake by uninfected HaCaT cells, we
observed a signicant dierence in the amount of HPV infected cells. While 5% of cells
were infected with L1 VLP, almost 9% were with L1L2 VLP (Figure 4.12). On the other
hand in a reduced SLPI micro-environment, after HSV-2(G) infection, 6% versus 28% of
the cells were infected with L1 and L1L2 VLP particles, respectively. This means that
L2-containing particles are more eciently endocytosed by the cells than L1 VLP particles
even in the absence of SLPI indicating that the enhanced uptake for the HPV-16 VLPs is
through the A2t infectious pathway.
44
0 0 0.7 0.7
0
5
10
15
20
25
30
35
Percentage of HPV-16 VLP uptake
(pHrodo-red positive cells)
HSV-2(G) multiplicity of infection
*
**
L1L2 VLP
L1 VLP
Figure 4.12: HPV-VLP uptake of L1L2 and L2 particles by HSV-2(G) infected and unin-
fected HaCaT cells. After 48 oh HSV-2(G) infection, 2g per million cells of VLP-pHrodo
labeled particles were added, and pHrodo
uorescence measured 16h later. Single and
double astesiks indicate signicant dierence with P values of < 0:05 and <0.01, respec-
tively.
Together, the data indicate that under both normal and reduced SLPI levels, L1 VLPs
are less eciently internalized by HaCaT cells compared to L1L2 VLPs, which suggests
that the HPV L1L2 capsid is required for enhanced VLP uptake in an SLPI decient
micro-environment.
45
Chapter 5
Discussion and Conclusions
Clinical studies performed before the advent of PCR strongly suggested that HSV-2
was the cause of cervical cancer [51]. Even after HPV was identied the real primary
etiologic agent of cervical cancer, researchers still suggest that concomitant HSV-2 and
HPV genital infections increase the risk of HPV-associated invasive cervical carcinoma
[28]. Understanding how molecular mechanisms used by HSV to infect and persist in a
host may increase susceptibility to infection and persistence of HPV oncoviruses is crucial
to understand the interplay between those commonly sexually transmitted viruses that
may lead into cancer progression.
In the present work, we studied the role of HSV-2 infection in the increased suscep-
tibility of human keratinocytes to HPV-16 infection. Exposure of epithelial basal cells in
herpetic lesions and HSV alteration of innate immune response are some of the proposed
mechanisms that may facilitate HPV infection and persistence [18, 20, 40, 50, 60]. We
provide evidence that transcriptional down-regulation of SLPI and reduced SLPI protein
secretion during HSV-2 in vitro infection contribute to the increased risk of HPV-16 ac-
quisition and infection. Interestingly, down-modulation of SLPI has been recognized as
46
an important viral immune evasion mechanism during HSV infections. Signicantly lower
concentrations of SLPI have been measured in cervicovaginal lavage from women infected
with HSV compared to that found in HSV negative women [31]. Since HSV-2 infections
are never completely cleared and become latent for the life of the host exhibiting periodic
reactivations, constant shedding of HSV-2 viruses in the cervical mucosa may permanently
create a perfect immuno-suppressed environment for HPV infections to occur and persist.
Although this is not the rst study that shows that an in vitro infection of a human
cell lines with HSV-2 causes down-modulation of SLPI transcription and secretion, we
extended these studies and linked this phenomenon to an increased susceptibility to HPV-
16 infection. Fakioglu et al. in 2008, demonstrated that HSV-2 and HSV-1 infection of a
human cervical epithelial cells line (CaSki) down-regulated SLPI expression orchestrated
by the immediate-early proteins ICP0 and ICP4, but independent of viral replication and
VHS protein activity [18]. They reported that HSV-2(G) infected cells released approxi-
mately 70% less SLPI than uninfected control cells. Although we observed similar results
only after 48 of HSV-2(G) infection, dierences in the cell lines used may account for
this observation. Since CaSki cells are already HPV-16 infected cells, genomic instabil-
ity resulting from the HPV-DNA integration process alters expression of several genes.
Although chromosomal location 20q12, where the slpi gene is localized, has not been rec-
ognized as an HPV DNA preferential insertional site, it has been found that virtually
all chromosomes of HPV-cervical cancer cells contain integrated HPV genomes at various
locations [59]. This allows for the hypothesis that slpi gene expression in CaSki cells may
be already altered, and its down-regulation becomes more dramatic after HSV-2 infection.
In fact, it has been observed that viral oncoprotein E6 has the ability to down-regulate
47
transcription of anti-serine protease elan in human keratinocytes [57]. Since HaCaT are
non-HPV immortalized cells, we considered using them a better model to study of the
eect of HSV-2 down-regulation of SLPI on HPV infection.
Our lab previously showed that HSV-1 infected HaCaT cell cultures secreted 50 %
less SLPI than uninfected cells 24 post infection [49]. However, using the same cell line,
multiplicity and duration of infection, we observed that HSV-2 reduces SLPI protein secre-
tion by 30%. Only after 48h of infection, was the reduction comparable to that observed
with HSV-1. We believe that variations in the patterns of viral gene expression between
HSV-1 and HSV-2 may explain those dierences. Although HSV-1 and HSV-2 have a
high genomic identity (>80%), it has been proposed that the specic cell tropism and
the dierent pathologies that those viruses cause, are due to dierences in preferences in
cellular receptor usage, in viral transcription and replication as well as in viral protein ki-
netics [1]. In fact, a comparative study between the HSV-1 and the HSV-2 transcriptomes
by microarray analysis suggested that HSV-2 transcript accumulation occurs at a lower
rate and level than in HSV-1, even though the overall gene expression of those viruses
was similar over time [1]. This in part may explain the longer HSV-2 infection period
required to observe dramatic changes in SLPI secretion. Since it has been suggested that
degradation of SLPI mRNA is mediated by the IE proteins ICP0 and ICP4 [18], perhaps
only after 48h of HSV infection, concentrations of those proteins become high enough
to signicantly reduce SLPI expression. However, it would be appropriate to evaluate
the stability of those viral proteins during such a longer infection period under acyclovir
treatment. Additionally, determining the half-life and stability of secreted SLPI during
48
HSV-2 infection would clarify whether lower SLPI concentration in supernatants are due
to HSV-2 mediated down-regulation or to protein instability.
Our results demonstrate that 2 and 2.5 fold increase in HPV-16 uptake and HPV-16
infection, respectively of HaCaT cells result from the overall reduction of mRNA and se-
creted protein levels of SLPI observed during HSV-2(G) infections. In fact, by adding
back 50g of rhSLPI to HSV-2 infected HaCaT cells we were able to prevent HPV infec-
tion. Since SLPI has been described as the natural cellular ligand for A2t receptor, our
observations indicate that SLPI and HPV competitively bind with A2t on the cell surface.
These ndings are supported by previous studies in our laboratory that indicate that: (i)
HPV-16 binding to its receptor involves interaction between the L2 capsid protein and
the S100A10 subunit of the A2t heterotetrameric receptor, and (ii) SLPI protein binds
to the Annexin A2 subunit of the same receptor [58]. Although, HPV and SLPI bind to
dierent parts of the A2t receptor, it has been proposed that SLPI binding to Annexin
A2 may cause either steric repulsion between L2 and S100A10, or it may induce molecular
conformational changes on the receptor reducing anity of L2 for S100A10 [49]. Our
observations that HPV-16 L2 capsid protein is required for proper binding and internal-
ization through the A2t receptor even in the absence of SLPI (post HSV-2(G) infection),
strengthen the current model of HPV adsorption and uptake which proposes that after
initial binding to HSPGs, conformational changes in the HPV capsid structure that result
in the exposure of the N-terminus of L2 protein increase anity of L2 for the A2t uptake
receptor (Figure 5.1.
SLPI protein is ubiquitous in human secretions including saliva, seminal plasma, cer-
vical mucus, and bronchial secretions. Due to its anti-serine protease, anti-in
ammatory
49
Figure 5.1: Model of HPV and A2t interaction. A: SLPI protein binds to annexin A2 of
the A2t heterotetramer receptor and blocks HPV interaction with A2t. B: under reduced
SLPI levels, after HSV-2 infection, HPV interaction with A2t occurs through L2 capsid
and S100A10 subunit of A2t. HPV is subsequently internalized. C: HPV-L1 only particles
cannot interact with A2t, and even under reduced SLPI concentrations, L2 capsid protein
is required for proper internalization of the virus.
and anti-microbial activities, SLPI constitutes a key molecular component during wound
healing, in
ammation and innate defense against microorganisms. In fact, addition of
SLPI protects epithelial cells against HSV-2 infection in vitro [27], and limited trans-
mission of HIV through the oral route has been linked to the high SLPI concentrations
in saliva (up to 24g/mL) [26, 38]. In the upper airways, with a concentration up to
10g/mL, SLPI is one of the most abundant antimicrobial agent found in the lung epithe-
lial lining
uid [48]. Additionally, SLPI concentration has been inversely correlated with
HPV infection in patients with HPV-associated head and neck cancers indicating that
lower SLPI levels in the upper respiratory tract correlate with high prevalence of HPV
50
in those areas [23]. In cervical mucus, SLPI levels vary with the menstrual cycle, but it
has been resported in concentrations as high as 70g/mL [33]. With the data presented
in this study we complemented and supported the protective role of SLPI against HPV
infection. Although initially we did not observe a signicant reduction in HPV-16 up-
take after adding rhSLPI at similar concentrations to those reduced after HSV-2 infection
(<160ng/mL), adding rhSLPI at higher concentrations but within a physiological range
(2.5-50g/mL), HPV infection was completely blocked. This intriguing observation that
only higher SLPI concentrations are able to block HPV binding and internalization is not
completely understood. Molecular dierences between native and recombinant SLPI, such
as disulde bond formation or post-translational modications absent in the recombinant
form, may stabilize and increase anity of native SLPI for Annexin A2, but this needs to
be examined in more detail. Additionally, since synthesized SLPI by HaCaT cell monolay-
ers binds to A2t on the cell surface immediately after being secreted, SLPI concentration
in cell supernatants may re
ect the surplus, or unbound SLPI, rather than the amount
bound to A2t. Therefore, a higher concentration of added rhSLPI is required in order for
SLPI to reach and to interact with its cellular receptor.
Surprisingly, no dual infection with HSV-2(G) and HPV-16 was observed. Since we
hypothesized that HSV infection enhances susceptibility to HPV infection and persistence,
our data indicate that increased HPV uptake only occurs in healthy non-HSV infected
cells. This observation adds relevance to the hypothesis that HPV uptake increases as
a consequence of immune down-modulation during HSV-2 infections. Although, there is
no epidemiological data to arm that in vivo HPV acquisition follows HSV infection in
vivo, co-infection of those in the same host is possible. In fact, both HSV-2 and hr-HPV
51
DNA have been detected in patients with cervical carcinoma [61], and HSV-2 seropositivity
increases the risk of developing cervical intraepithelial neoplacia (CIN) (11.1-fold increased
odds) [42]. Being that these pathogens are two of the most prevalent sexually transmitted
viruses, there is a high possibility to initially acquire HPV rather than HSV. In this case,
HSV would also increase the rate of HPV re-infection by the same mechanism of SLPI
down-regulation.
To conclude, the results shown here not only corroborate the protective role of SLPI
against HPV infection, but also demonstrate that HSV-2 interferes with SLPI expression
resulting in an enhanced susceptibility to HPV-16 infection. This molecular synergism
between HSV-2 and hr-HPV constitutes the rst evidence that points to herpes genital
infections as a risk factor for the acquisition of oncogenic HPV, and as a possible con-
tributor for the persistence of HPV. Therefore, HSV-2 genital infections may potentially
contribute to cervical cancer development. Our results form the basis to explore SLPI-
based therapeutic alternatives to be used in combinations with current anti-HSV agents
as an action to prevent hr-HPV infections and cervical cancer.
52
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57

Abstract (if available)

Abstract Despite the fact that Human Papillomavirus (HPV) is responsible for almost all cases of cervical cancer around the world, the epidemiological link between Herpes Simplex Virus (HSV) and cervical cancer has persisted for decades. Although both HSV-2 and HSV-1 cause genital herpetic ulcerative disease, HSV-2 remains the main causative agent of ano-genital infections. Host immune evasion strategies are crucial to facilitate pathogen infection and persistence, and HSV-mediated down-regulation of the Secretory Leukocyte Protease Inhibitor (SLPI) constitutes one of those mechanisms. SLPI, ubiquitously found in body fluids, is known for its an anti-microbial, anti-inflammatory and anti-protease properties. SLPI also constitutes the natural cellular ligand for annexin A2/S100A10 or A2t, which is the preferred cellular receptor for the internalization and infection of HPV. Therefore, we hypothesized that reduced SLPI protein levels after an HSV-2 infection would facilitate HPV interaction with A2t, resulting in an enhanced internalization and infection of HPV in a human keratinocyte cell line. We showed a significant increase in HPV internalization in epithelial cells previously infected with HSV-2 compared to HSV negative cultures. Restoring SLPI concentrations by adding human recombinant SLPI (rhSLPI) to HSV-2-infected cultures, HPV infection decreased confirming the protective role of SLPI against HPV infection. Additionally, we provided evidence that proper HPV binding and internalization through the A2t receptor requires interaction with the HPV L2 capsid protein. We concluded that HSV-2 intermittent reactivations and reinfections would create the perfect SLPI-depleted microenvironment for HPV to infect and persist. Our findings not only contribute to understanding the contribution of HSV-2 in the increased susceptibility to HPV infection, but they also provide the basis to explore SLPI-based anti-viral therapeutic alternatives to use against HPV as a strategy to prevent cervical cancer.

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Asset Metadata

Creator Porras, Tania B. (author)

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

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 01/29/2015

Defense Date 12/09/2014

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

Tag cervical cancer,Herpes Simplex,human papillomavirus,OAI-PMH Harvest,secretory leukocyte protease inhibitor,SLPI,viral co-infection,viral synergism

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Kast, W. Martin (committee chair)

Creator Email porrastb@gmail.com,tporrasc@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-526431

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Legacy Identifier etd-PorrasTani-3147.pdf

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Rights Porras, Tania B.

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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