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

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

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THETA DEFENSINS INHIBIT HIGH-RISK HUMAN
PAPILLOMAVIRUS INFECTION THROUGH CHARGE-DRIVEN
CAPSID CLUSTERING
by
Joseph Gary Skeate

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

December 2020

Copyright 2020 Joseph Gary Skeate
ii
Dedication
This work is dedicated to my wife, Cierra Mantz, and my father, Gary Rivet.

iii
Acknowledgements
I owe an incalculable amount of gratitude to Dr Martin Kast. Throughout the years I’ve
found that he absolutely embodies what a mentor should be. Your guidance, knowledge,
thoughtfulness, and ability to selflessly invest part of your life into the development of
others is unparalleled, and it is because of individuals like you that the world is a better
place.

I also offer my sincerest appreciation for Dr Diane Da Silva who has been an unwavering
source of support and my favorite person to interrupt while working. We’ve had quite the
run over the last decade, and I am so fortunate to have had the opportunity to learn from
you.

To my committee members Dr Andre Ouellette and Dr Omid Akbari: thank you for your
career guidance and constructive feedback throughout my time at USC. I also want to
thank members of the laboratory of Dr Michael Selsted, specifically Justin Schaal, Dat
Tran, and Patti Tran for their thoughtful discussions in the lunchroom.

Additionally, I would also like to recognize all of the lab mates that I have had the fortune
of spending part of my life with: Ruben, DJ, Kim, Andy, Tania, Elena, Suma, Wouter, Mikk,
Mauricio, and Féline… Each one of you contain an unlimited potential to do great things
for the world.

Finally, I want to thank my family: Mom, Dad, Milo, Pearl, Justin, Jeff, Jordan, Tim, Traci,
Hank, and everyone else that has supported me through this journey.
iv

TABLE OF CONTENTS
Dedication ............................................................................................................................. ii
Acknowledgements ............................................................................................................. iii
List of Tables ..................................................................................................................... viii
List of Figures ...................................................................................................................... ix
Abbreviations ...................................................................................................................... xi
Abstract .............................................................................................................................. xiv
Chapter 1 - Human papillomavirus and associated disease ............................................ 1
HPV infection and viral trafficking ................................................................................ 2
HPV viral replication ..................................................................................................... 7
HPV viral integration with host genome leads to oncogenic transformation .............. 11
HPV immune evasion drives persistent infection ....................................................... 12
Defensins ................................................................................................................... 14
a-defensins ................................................................................................................ 17
b-defensins ................................................................................................................. 18
q-defensins ................................................................................................................. 19
Anti-viral efficacy of defensin family members ........................................................... 19
v
Current understanding of defensin-mediated inhibition of HPV infection ................... 22
Rhesus theta-defensin 1 ............................................................................................. 23
Chapter 2 – Theta-defensins inhibit high-risk human papillomavirus infection
through charge-driven capsid clustering .............................................................. 25
Abstract ....................................................................................................................... 25
Introduction ................................................................................................................. 26
Results ........................................................................................................................ 29
Pretreatment of hrHPV Pseudovirions (PsV), not cells, with RTD-1 results in dose-
dependent inhibition of infection. ............................................................................. 29
RTD-1 significantly reduces uptake of HPV16 by inhibiting initial binding of
defensin-coated virions to the cell surface ............................................................... 32
RTD-1 interacts with the viral capsid and causes capsid clustering. ....................... 37
RTD-mediated inhibition of HPV infection is dependent on the innate charge of the
q-defensin isoform. .................................................................................................. 40
RTD-1 is able to inhibit other hrHPV genotypes, furin pre-cleaved (fpc) HPV16 PsV,
and ability to inhibit is influenced by the HSPG-status of virions. ............................ 43
Langerhans cells are not activated by RTD-1 generated viral aggregates of HPV16,
but still become active with PolyI:C in the presence of RTD-1. ............................... 46
Discussion ................................................................................................................... 50
Materials and Methods ............................................................................................... 56
vi
Cell lines .................................................................................................................. 56
Antibodies ................................................................................................................ 56
HPV pseudovirus (PsV) and virus like particle (VLP) production ............................ 57
Production of defensins ........................................................................................... 58
HPV PsV infection assays ....................................................................................... 58
HPV16-pHrodo VLP uptake assays ......................................................................... 59
NanoSight imaging of viral particles ........................................................................ 60
Confocal imaging and colocalization analysis ......................................................... 60
Transmission electron microscopy imaging ............................................................. 61
Western blot analysis of binding .............................................................................. 61
Langerhans cell generation ..................................................................................... 62
LC activation assay and flow cytometry ................................................................... 62
Statistical analysis ................................................................................................... 63
Chapter 3. Future directions for this research .......................................................... 64
Where to go from here ................................................................................................ 64
Vaginal challenge optimization ................................................................................ 65
RTD-1 topical dose escalation ................................................................................. 66
Immunogenicity studies ........................................................................................... 67
Other θ-defensins that may be considered .............................................................. 67
Potential pitfalls during translation ........................................................................... 68
vii
Research Summary ................................................................................................. 70
Chapter 4. Other projects and publications completed during PhD work .............. 72
References .................................................................................................................... 77

viii
List of Tables
Number Title Page
Table 1 High-risk HPV gene transcripts and associated functions 8
Table 2 Number of defensin genes by relevant species 17

ix
List of Figures
Number Title Page
Figure 1 HPV viral entry and infectious trafficking 6
Figure 2 Viral life cycle of human papillomavirus 11
Figure 3 Defensin genes, disulfide bond pairings, and tertiary structure 16
Figure 4 Summary of anti-viral effects of different defensin classes on both 21
enveloped and non-enveloped viruses
Figure 5 RTD-1 inhibits HPV16 PsV infection in a dose-dependent manner 30
driven by an interaction between viral capsid and θ-defensin
Figure 6 Dialysis experiment layout 32
Figure 7 RTD-1 treatment of HPV16 results in significantly reduced uptake 34-36
and localization of virions to early endosomes highlighted by
sparse detection of large aggregates at cell surfaces and reduced
cell binding capacity of RTD-1 treated virions
Figure 8 RTD-1 interacts with HPV16 VLP to form viral aggregates and 39
can be prevented with the addition of excess divalent cations
during incubation
Figure 9 The viral inhibitory concentration of RTD-1 is similar to HD5 and 42
is dependent on the charge of the RTD isoform
Figure 10 RTD-1 is able to inhibit furin pre-cleaved (fpc) HPV16 PsV, 45
capacity to inhibit is partially influenced by the HSPG-status of
virions, and RTD-1 has cross hrHPV genotype efficacy
Figure 11 Schematic diagram of LC generation and activation experiment 47
x
Number Title Page
Figure 12 Patient derived LC are not activated by RTD-1 induced viral 48
aggregates but can still be stimulated by PolyI:C in the
presence of RTD-1
Figure 13 Updated summary of anti-viral effects of different defensin 69
classes on both enveloped and non-enveloped viruses.

xi
Abbreviations
A2t AnnexinA2 S100A10 Heterotetramer
APC Antigen Presenting Cell
ATCC American Type Culture Collection
CCL20 C-C Motif Ligand 20
cGAS Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase
CIN Cervical Intraepithelial Neoplasia
DENV Dengue Virus
DMEM Dulbecco’s Modification of Eagle’s Medium
EGFR Epidermal Growth Factor Receptor
ELISA Enzyme-Linked Immunosorbent Assay
FBS Fetal Bovine Serum
fpc Furin pre-cleaved
GFP Green Fluorescent Protein
HAdV Human Adenovirus
HD Human Defensin
HDP Host Defensive Peptide
HIV Human Immunodeficiency Virus
HNP Human Neutrophil Peptide
HPIV Human Parainfluenza Virus
HPV Human Papillomavirus
hrHPV High-risk Human Papillomavirus
HSIL High-grade squamous intraepithelial neoplasia
xii
HSPG Heparin Sulfate Proteoglycan
HSV Herpes Simplex Virus
IMDM Iscove’s Modified Dulbecco’s Medium
LC Langerhans Cell
LSIL Low-grade squamous intraepithelial neoplasia
MHC Major Histocompatibility Complex
mRNA Messenger Ribonucleic Acid
MVB Multivesicular Bodies
NTA Nanoparticle Tracking Analysis
OBSL1 Obscurin-like 1
OSCC Oropharyngeal Squamous Cell Carcinoma
PCR Polymerase chain reaction
PML Promyelocytic Leukemia
pRb Retinoblastoma protein
PRR Pattern Recognition Receptor
PsV Pseudovirus
RSV Respiratory Syncytial Virus
RTD Rhesus Theta-Defensin
SEM Standard Error of the Mean
SD Standard Deviation
SLPI Secretory Leukocyte Protease Inhibitor
TAP Transporter Associated with Antigen Processing
TEM Tetraspanin Enriched Microdomains
xiii
TGN Trans Golgi Network
TLR Toll Like Receptor
VLP Virus Like Particle

xiv
Abstract

Human papillomavirus (HPV) continues to be the most prominent sexually
transmitted virus in humans. It is estimated that over 80% of all sexually active individuals
will become infected with HPV within their lifetimes. While the majority of these infections
will be cleared naturally and not progress to advance-stage disease, persistent infection
with high-risk human papillomavirus (hrHPV) genotype results in the development of
epithelial cancers in the skin and mucosa. While the previous paradigm focused on
cervical cancers in women, there now is an increasing incidence of HPV-driven head and
neck cancers occurring in men, highlighting the continued importance to study and
understand the spectrum of HPV-driven disease. The most common hrHPV genotype,
HPV16, is frequently used to study all aspects of the HPV viral life cycle from viral entry,
propagation, immune evasion, to carcinogenic transformation of host cells and resulting
cancer phenotypes. Although prophylactic vaccination exists and coverage has improved
in the United States, there still remains significant hurdles to achieving herd immunity
through vaccination strategies; leaving a need to develop methods that inhibit viral
transmission and can prevent the spread of HPV-driven disease to those at risk.
Defensins are a class of host defense peptides produced by cells and tissues in
both plants and animals that both protect from infection by pathogens such as HPV and
have immunomodulating properties. Previous work utilizing α and β defensins from
humans has demonstrated that α-defensins are effective at inhibiting the most common
high-risk genotype, HPV16. What remains to be explored is the third class, θ-defensins.
This group consists of small, 18-amino acid cyclic peptides found in old-world monkeys
xv
whose unique structure makes them both highly cationic and resistant to degradation.
The focus of this study is to examine the anti-viral effects of the prototype θ-defensin,
RTD-1, on hrHPV infection and characterize the mechanism of inhibition. Through this
investigation I laid the groundwork needed to develop this into a durable translational
product that can be used to effectively prevent hrHPV infection of at-risk individuals.
1
Chapter 1 - Human papillomavirus and associated disease

Although it was first identified in cervical samples by virologist Harold Zur Hausen
through PCR in the 1980’s, human papillomavirus (HPV) has always been a significant
human pathogen that causes disease in both women and men (1). In the United States it
is still the number one sexually transmitted virus, and the last estimated annual cost of
preventing and managing HPV-related morbidities was $8 billion in 2008 (2). Currently
there are over 200 HPV genotypes that have been identified based on genomic DNA
differences. Of these, 40 preferentially infect the genital tract and are classified into
different risk groups based on their ability to transform epithelial cells and severity of
associated disease.
Low risk HPV, which includes genotypes 6, 11, 40, 42, 43, 44, 53, 54, 61, 72, 73,
and 81 cause genital warts, low-grade histological abnormalities in cervical epithelium,
and recurrent respiratory papillomatosis within oropharyngeal tissue of both adolescents
and adults (3). Nearly all disease caused by this risk group is driven by genotypes 6 and
11, but does not lead to carcinogenic transformations. High risk HPV (hrHPV) genotypes,
however, each have a capacity to cause low to high-grade cellular abnormalities and
induce oncogenic transformation. As of June 2020, there are 14 hrHPV genotypes that
have U.S. Food and Drug Administration-validated tests (16, 18, 31, 33, 35, 39, 45, 51,
52, 56, 58, 59, 66, and 68) (4). Of these, HPV16 is the dominant genotype found in HPV-
positive cervical, oropharyngeal, anal, penile, vulva, and vaginal cancers (5, 6).
2
While historically associated with the development of cervical cancer, the overall
trends of hrHPV-associated cancers are shifting. Preventative measures such as routine
cancer screenings through cytological analysis of Papanicolaou smear tests and
campaigns to increase rates of prophylactic vaccination in adolescent women has
resulted in a steady decline in patients presenting with precancerous transformations that
precede cervical cancer, however the incidence of HPV+ cancer at other sites are on the
rise (7). Nowhere is this more apparent than in HPV+oropharyngeal squamous cell
carcinomas (OSCC). Strikingly, between 2012-2016 the average yearly number of OSCC
overtook those of cervical cancer (19,000 vs 12,015). Moreover, these cases are
predominantly occurring in males (15,540 of 19,000) (8). Several factors such as changes
in sexual behavior/preference, cessation of smoking, and an aging population have been
posited to contribute to this shift. But given that there is no active screening of head and
neck sites nor an intervention strategy in place to treat pre-cancerous lesions, it is
expected that these numbers will continue to increase for some time given the non-
existent vaccination rate in the middle to older age populations and the duration of time
needed for HPV infection to progress to cancer.

HPV infection and viral trafficking

Propagation of HPV requires uptake by basal cells within stratified epithelial
tissues. Infection of this cell population is necessary for the viral replication cycle as the
messenger ribonucleic acid (mRNA) profile of progressively maturing keratinocytes
3
dictates gene expression of the HPV genome (9). The infectious uptake pathway of HPV
is still not fully characterized, however throughout my time in this field it has grown into a
more and more complicated dance of cell surface proteins, signaling factors, and
endocytic pathways. A point that I would like to stress here is that infection by HPV is
possible within many cell types that are not basal cells, however the effects a non-
productive HPV lifecycle has on these cells and the surrounding cell population has yet
to be fully characterized or explored.
Given that basal cells are localized to the basement membrane of epithelial tissue
and normally inaccessible, it takes an event the HPV field has termed a micro-wound for
HPV virions to gain access to this cell population. This micro-wounding event is caused
by physical or chemical disruptions, typically through sexual activity, that allows
immediate access to all layers of the epithelium. Through this fissure HPV virions gain
access to the lower layers of the epithelium and associate with factors such as laminins,
growth factors, heparin sulfate proteoglycans (HSPG, such as syndecan-1 and 4), and
tetraspanins found on target cells and the basement membrane. Of note, several of these
proteins are directly involved in anchoring basal cells to the basement membrane through
hemidesmosome structures, and given that microwounding is thought to also disrupt
these anchoring proteins and activate the wound healing process, it is no surprise that
these factors have been found to be important in the context of viral infection.
Initial interactions with these basement and basal-cell associated proteins lead to
a decoration of the HPV viral capsid with HSPGs and growth factors. Additionally, during
this time, extracellular viral processing events post association with HSPG are required
for “priming” virions for infectious uptake and trafficking (10, 11). The term priming is used
4
to describe two distinct changes to the viral capsid that occur in both L1 and L2 proteins.
First, HSPG-bound L1 is proteolytically processed by kallikrien-8 to allow enhanced
exposure of L2 (minor capsid protein) (12). Second, exposed L2 is then stabilized by
cyclophilin B to allow cleavage by furin, a proprotein convertase (13). Cleaved L2 was
originally thought to be part of the affinity change that occurs during these priming steps;
however, it has been determined that its role is limited to downstream endosomal escape
of the viral DNA-L2 complex (14-16).
Once priming is complete, affinity of the virions shifts from the primary binding
partners over to cofactors involved in viral entry. The process of HPV viral uptake has
been shown by multiple groups to be asynchronous as demonstrated by the differing
times groups have reported (5 min to 4 hours post viral addition), suggesting that there is
a complex series of events that needs to occur before entry proceeds (15, 17, 18). Initial
signaling from the cell surface during viral priming through HPV-associated growth factors
results in the assembly of what is now known as an HPV viral entry platform or tetraspanin
enriched microdomains (TEMs). This platform is made up of a growing list of components
that include tetraspanins (CD151 and CD63), integrin complexes (a6b4), growth factor
receptors, and the annexin a2/s100a10 heterotetramer (A2t). The most recent study
defining these TEMs indicated that HPV virions sit on top of CD151, integrin-a3, and -a6
colocalized clusters (19). Post platform assembly, HPV is taken up into the cell. As it
stands, the current model defines HPV infectious uptake as a micropinocytosis-like
mechanism that is clathrin, caveolin, dynamin, cholesterol, flotillin, and lipid raft-
independent (20, 21). This macropinocytosis-like process hinges upon actin-cytoskeleton
rearrangements, cytoskeletal adaptor obscurin-like 1 (OBSL1), and tetraspanin
5
interactions with other tetraspanins as well as integrins (a3 and a6) (19, 20, 22, 23). It has
been shown that A2t plays a role in this process as well given its membrane bending
capabilities, actin binding potential, and that HPV16 exposure results in translocation of
A2t to the outer leaflet of lipid membranes from epidermal growth factor receptor (EGFR)-
Src signaling cascades (24, 25). Previous knockout studies have shown that virions are
still able to enter cells when either annexin A2 or S100A10 are eliminated, however this
may be due to a shift of viral entry into a non-infectious pathway (26).
Post uptake into early endosomes, virions are then trafficked to multivesicular
bodies (MVBs) via an L1 association with the tetraspanin CD63. This L1-CD63 interaction
function as an adaptor and allows HPV-containing endosomes to be trafficked via
syntenin-1 and members of the ESCRT machinery (ALIX and VPS4, specifically) (27).
Inside MVBs, virions begin to disassemble due to lower pH and the L2 minor capsid
protein embeds into the endosomal membrane while still associated with the viral
genome. Once the C-terminal end of L2 is exposed within the cytosol, it associates with
the retromer complex to facilitate trafficking to the trans-Golgi network (TGN) via TBC1
Domain Family Member 5 (TBC1D5) driving Rab7 GTPase activity (14, 28).
Fragmentation of TGN during G2 to early prophase of mitosis then allows the L2-vDNA
complex to travel along spindle fibers towards the minus end and accumulate with
centrosomes during prometaphase (29). Once the cell passes into early metaphase, there
is a directionality shift and the L2-vDNA complex begins travel towards and associates
with chromatin (16). These localized sites of L2+vDNA within chromatin are referred to
as promyelocytic leukemia (PML) bodies. Once residing in these bodies, they begin the
process of early gene expression to push basal cells into a state of continuous renewal
6
so that the viral genome may be amplified and maintained. How the L2-vDNA complex is
translocated outside of its transport vesicle and whether this occurs while in association
with chromosomes remain unknown.
In an effort to provide a visual representation of the current state of HPV viral entry
and lay out this process I have drawn a graphical summary of this viral entry section
(Figure 1).

Figure 1. HPV viral entry and infectious trafficking. Current model of HPV viral entry
that now highlights tetraspanin-enriched microdomain formation (otherwise known as
viral entry platform assembly), trafficking partners for early endosomes OBSL1 and A2t,
7
syntenin and ESCRT machinery for trafficking to MVB, and L2-retromer association for
TGN transport.
HPV viral replication
The HPV genome is double-stranded, circular DNA moiety that is approximately 8
kB in size and comprised of three distinct regions: early, late, and non-coding regulatory
regions. HPV utilizes viral gene transcripts, differential nucleosome locations within the
viral genome, histone modifications, and DNA methylation as methods to control early
and late gene transcripts. It is posited that these complex regulation methods are due to
co-evolution of HPV with humans and play a role in evading immune detection as they
tightly regulate gene transcription to the maturation process of keratinocytes. Within the
three regions there are 8 open reading frames that express early (E1, E2, E4, E5, E6,
and E7) and late (L1 and L2) genes. A summary table of these genes and their role in the
viral life cycle can be found in Table 1.

8

Table 1. High-risk HPV gene transcripts and associated functions. Updated and
expanded from (30).

hrHPV
transcript
Function
E1
Adenosine triphosphatase (ATPase) and DNA helicase; recognizes and
binds to the viral origin of DNA replication as a hexameric complex;
necessary for viral DNA replication.
E2
Main regulator of viral gene transcription; binds the viral transcriptional
promoter as a dimer; involved in viral DNA replication; interacts with
and recruits E1 to the origin.
E4
Acts late in the viral life cycle; interacts with the keratin cytoskeleton
and intermediate filaments; induces G2 arrest; believed to aid in virus
assembly and release.
E5
Induces cell proliferation; interacts with 16k subunit c of vacuolar
ATPase; activates growth factor receptors and other protein kinases;
inhibits apoptosis; inhibits traffic of major histocompatibility complexes
to the cell surface.
E6
Induces DNA synthesis; induces telomerase; prevents cell
differentiation; interacts with four classes of cellular proteins:
transcriptional co-activators, proteins involved in cell polarity and
motility, tumor suppressors and inducers of apoptosis, primarily p53,
and DNA replication and repair factors.
E7
Induces cell proliferation; interacts with histone acetyl transferases;
interacts with negative regulators of the cell cycle and tumor
suppressor pRb.
L1
Major viral structural protein; assembles in capsomeres and capsids;
interacts with L2; interacts with cell receptors
L2
Minor viral structural protein; interacts with DNA; believed to facilitate
virion assembly; required for viral trafficking, interacts with viral
receptors
9
Upon successful infection of basal keratinocytes, the first viral transcripts
expressed are E1 and E2. These two work in tandem to maintain a low copy number of
the viral genome within basal cells (~50-100 copies per cell) (31). E1 functions as an ATP-
dependent DNA helicase that is required for establishing base copies of episomal DNA
and amplification of the genome post basal cell differentiation (32). E2 is a critical
component that tethers the viral genome to chromatin of infected cells through
bromodomain protein 4, acts as a loading site for E1 helicase activity during replication,
as well as regulates progressive viral gene transcripts during keratinocyte development
through its dual binding sites immediately upstream of E6 and E7 (33-35). After
establishment of initial infection and maintenance of episomal copies of the HPV genome,
the E5, E6, and E7 proteins come into play as basal cells begin differentiating into upper
layer of the epithelium.
These three proteins function as host modifiers to make the cellular environment
more conducive to the viral replication. E5 functions to make the differentiating
keratinocytes more motile by augmenting the signaling from EGFR, which is a critical
component during wound healing. It carries out this function by manipulating the relative
levels of EGFR mRNA and receptor trafficking to the cell surface (36). Additionally, it has
the ability to interfere with apoptotic pathways normally activated in aberrant cells (37).
This function coincides with the oncogenic E6 and E7 proteins, which bind and degrade
p53 and the retinoblastoma protein (pRb) respectively, forcing infected cells to renter S-
phase and amplify the viral genome (38). Additional targets for E7-mediated degradation
include those that control cell cycle entry in the basal layers (p105 and p107), and upper
epithelial layers (p130) (39, 40). All of these factors essentially work in parallel with the
10
keratinocyte differentiation process, causing a physical expansion of the suprabasal
epithelial layers and development of HPV+ lesions.
As infected cells make their way to the cornified layer there is a shift in viral mRNA
splicing that favors E4, L1, and L2 protein transcripts to be translated (41, 42). The
abundance of viral genome found in the infected cells then begin to associate with L2,
while pentamers of L1 start self-assembling (43). Together these begin to form immature
capsids within the nucleus of the cell and undergo L1-L1 disulfide “maturation” as the
keratinocytes shift to an oxidative state due to the loss of mitochondrial activity (44).
Transmission to a new host or reinfection is then carried out by a release of matured
virions from the upper layer of HPV+ lesions, and E4 is believed to play a role in this
process through the disruption of the keratin network in HPV+ cells (45) (Figure 2).
For cervical cancer, sites of the early stages of HPV infection lead to disruptions
in normal cytology and are classified as low-grade squamous intraepithelial neoplasia
(LSIL), which is also known as cervical intraepithelial neoplasia grade 1 (CIN1). If the host
fails to clear the infected cells the lesions persist and develop into CIN2 (moderate cellular
dysplasia) or CIN3 (severe cellular dysplasia), both of which are classified high-grade
squamous intraepithelial neoplasia (HSIL). Patients that maintain HSIL for 5 years have
an estimated 20% chance of developing cancer, which then increases to a 50% risk once
the 30 year mark is reached (46, 47).

11

Figure 2. Viral life cycle of human papillomavirus annotated with progressive gene
expression.

HPV viral integration with host genome leads to oncogenic transformation
While it’s expected that almost all sexually active individuals will be infected with a
hrHPV genotype at some point in their lives, the majority will clear these infections (48).
It is only when hrHPV persist and not cleared by the immune system that issues arise.
12
Persistent infection with a hrHPV genotype leads to the increased risk of viral genome
integration, especially at transition zones within epithelial tissues (49). While detrimental
to the viral life cycle, hrHPV integration causes unchecked expression of E6 and E7,
which alone are able to cause oncogenic transformation. The major effect of this
integration event is the loss of E2 expression and/or function, which directly regulates E6
and E7 through repressive binding upstream their transcriptional start sites. Proposed
mechanisms disrupting E2’s regulatory role include direct disruption of coding sequences
due to integration, methylation of E2 binding motifs which prevent repressor binding,
tandem repeats of viral genomes, E6/E7-host fusion protein generation, or a combination
of these scenarios (50).
HPV immune evasion drives persistent infection
HPV employs several mechanisms to avoid immune detection and persist within
epithelial tissue. First, and inherent to the viral life cycle, very few viral antigens are
produced during the genome amplification that occurs in the basal keratinocytes and as
cells differentiate into the cornified layers. Viral proteins that are transcribed are brought
to the nucleus and function to regulate gene expression (as described in the previous
section). Late gene products, such as L1, are highly immunogenic yet are only expressed
in layers of the epithelium that are almost devoid of antigen presenting cells (APC).
Additionally, innate mechanisms to detect pathogen infection such as toll-like receptors
(TLRs), other pattern recognition receptors (PRRs), and even the ability to produce
inflammatory cytokines and present antigens are manipulated by high-risk HPV E5, E6,
and E7 proteins.
13
TLR9, which recognizes double-stranded viral DNA, is directly manipulated by
HPV16 E7 through methylation of the TLR9 promotor region and suppression of
transcription (51). Other cytosolic sensors of viral DNA such as cyclic guanosine
monophosphate-adenosine monophosphate synthase (cGAS) are prevented from
signaling through STING by direct E7 blocking the binding motif (52). E6 and E7 are also
able to deregulate NF-kB signaling by interactions with upstream factors in the cytosol
and sequestration of coactivators of the NF-kB within the nucleus, leading to a signaling
failure and non-production of IL-1b and subsequently IL-8 (53-55). Even in the instance
of HPV-specific effector cell generation by the host, HPV16 E7 is able to reduce the level
of major histocompatibility complex I and transporter associated with antigen processing
(TAP) at the transcriptional level, making it so less HPV-associated antigens are loaded
onto major histocompatibility complex (MHC) class I and expressed on the surface of
infected cells (56, 57). It is of note that these last two mechanisms have only been shown
in HPV-transformed cervical cancer cells, therefore they may not truly play a role in HPV
persistence and are only functional in HPV-driven cancer immune evasion.
Outside of the infected cell population, hrHPV also directly manipulate Langerhans
cells (LC), the primary APC that surveil epithelial tissue (58). The minor capsid protein,
L2, has been shown to be directly responsible for the inability of LC to become active and
migrate towards lymphoid tissues through an interaction with the annexin A2 S100A10
heterotetramer, which in itself also suppresses LC activation (59). Additionally, there is
evidence of HPV-infected tissues becoming devoid of LC over time through a
downregulation of C-C motif ligand 20 (CCL20) (once again driven by the disruption of
NF-kB signaling), but it has yet to be established if this occurs during the infectious
14
process or after oncogenic transformation (60). While this immature phenotype can be
overcome through the usage of strong immunostimulants such as PolyI:C or cytokine-
based biologics (IRX-2), it remains that this mechanism is conserved across both a and
b papillomavirus genotypes (61-63). Together, these modifications to host cells and the
surrounding tissue hamper immune responses against HPV and allow the virus to persist,
giving rise to a greater chance of developing HPV-driven cancers.
Cells are not, however, completely susceptible to infection by HPV as whole
classes of cationic host defensive peptides (HDP) still function as innate immune barriers
for cells and can function to guide immune responses. In my previous work I had shown
that one such HDP, secretory leukocyte protease inhibitor (SLPI), was able to inhibit
hrHPV infection through interaction with A2t, one of the critical components involved in
viral trafficking (25, 59, 64). This opened up the possibility of examining the roles of other
HDPs and their effects on HPV infection, which lead to this dissertation project that
focused on defensins.

Defensins
Defensins are class of HDP that have anti-microbial, anti-viral, anti-fungal, and
immunomodulatory properties. While they share certain characteristics such as a cationic
charge & b-sheet dominated structures, they are split into a-, b-, and q-classes based on
their amino acid sequence, disulfide bond patterns, secondary and tertiary folded
structures. Expression of different members of each class is variable and oftentimes
inducible upon inflammation or tissue injury. All defensins are initially produced as
15
prepropeptides that undergo proteolytic processing, however these propeptides from
each class are then subject to different localization and secondary processing into their
mature forms (Figure 3).

16

Figure 3. Defensin genes, disulfide bond pairings, and tertiary structure. Left side
of figure indicates gene exon and splicing regions. Right side indicates disulfide pairings
via amino acid number of mature defensin. 3D images are rabbit a-defensin RK-1 (top),
human b-defensin-1 (middle), and q-defensin RTD-1 (bottom). Image from (65).

17
Defensin Genes α enteric α myeloid β θ
Human
2 3 ~37 Ψ
Mouse
~26 Ψ ~49 -
Rhesus Macaque
6 6 ~37 3
Table 2. Number of defensin genes by relevant species. Ψ indicates pseudogene.
Summary table modified from (66).
a-defensins
Humans produce a-defensins that are between 29-35 amino acids in length and
fall into the subgroups myeloid and enteric. Pro-a-defensins contain an additional 40
amino acid anionic sequence that neutralizes the overall charge of the molecules and
reduces intracellular toxicity during production, but this is proteolytically removed during
the maturation process. Myeloid defensins include human neutrophil peptides (HNP) 1
through 4. HNP1, 2, and 3 are found at high concentrations within neutrophil azurophilic
granules while HNP4 is relatively less abundant. Primary usages of this subclass of
defensins occur within intracellular phagolysosomal compartments where endocytosed
microbes or virions have been directed. It has been shown that extracellular myeloid
defensins can be detected when neutrophils infiltrate damaged or infected tissue, but
these levels remain at very low concentrations in circulating plasma. Enteric a-defensins
include human defensin 5 (HD5) and 6 (HD6). These defensins are produced and
secreted by Paneth cells and epithelial cells found in male and female genitourinary
tracts. Unlike myeloid defensins, the propeptide forms of a-defensins are packaged with
18
a trypsin zymogen and does not take on its active form until it is within the extracellular
environment. Because of their constitutive expression and secretion, these defensins has
been detected in high concentrations within vaginal lavage fluid and the lumen of
intestinal crypts.

b-defensins
Humans have approximately 37 genes that code for this class of defensin, however
very few have been studied at the protein or transcriptional levels. Cells that primarily
produce human b-defensins (HBD) include keratinocytes and epithelial cells of the
reproductive and respiratory tracts (67). Primary function of this defensin class has been
linked to fertility in mice, however many studies have highlighted anti-bacterial and anti-
viral functions that suggest they bridge a role between innate and adaptive immunity
(reviewed in (66)). Additional evidence for this role comes with the transcriptional
regulation of HBD (except HBD-1) being largely driven by innate immune signaling
factors, such as TLRs (68). Interestingly, the anti-microbial activity of prominent members
of defensin class is lost under physiological salt concentrations (HBD-1 and 2) and can
be highly sensitive to the reducing or oxidizing extracellular environment, suggesting that
there is post-secretion regulation of activity that may limit function to certain tissues or
organs (67, 69).
19
q-defensins
The final class of defensins are produced in the leukocytes of Old World Monkeys such
as rhesus macaques, olive baboon, vervets, as well a member of the ape family,
orangutans (65). While humans express q-defensin ortholog genes (termed retrocyclins),
premature stop codons within the open reading frame prevent their translation and
production (70). These peptides are made from two separate 9 amino acid sequences
from truncated a-defensin precursors that are cyclized from N to C terminus to form a
unique 18-amino acid circular peptide stabilized by three cysteine bond pairs (71).

Anti-viral efficacy of defensin family members
Studies that have focused on the biology of defensins have largely found that the
dominant characteristics are that of anti-microbial and immune-modulating activities.
Within the realm of viral inhibition, different mechanisms span the entire viral life cycle
and include disruptions to the lipid bilayer and associated factors that are found on
enveloped viruses, inhibiting viral glycoprotein interactions with host receptors, inhibiting
viral fusion with the host cell for capsid delivery, neutralizing aggregation of viral particles,
inhibition of viral proteins, direct inhibition of viral gene expression, and modifications to
viral trafficking and viral uncoating (reviewed in (66, 72)). For the enveloped virus family
including Herpes simplex virus 1 & 2 (HSV), human immunodeficiency virus (HIV),
influenza A virus (IAV), Baculovirus, and Sindbis virus, the primary driver of interaction is
the attraction of the cationic defensin peptides to the negative phospholipids that are
20
found on the viral coats, however there are members of the alpha-defensin group that
can also function as lectins and bind to viral glycolipids and glycoproteins (70, 73-84).
The combination of these two modes of action have shown that enveloped viruses are
primarily halted at either surface receptor interactions or during viral fusion with the host
cell by all three classes of defensins. Other mechanisms have included the theta defensin
retrocyclin-1 inhibiting viral proteases from Dengue Virus (DENV), human beta defensin
2 disrupting the viral envelopes of respiratory syncytial virus (RSV), human parainfluenza
virus (HPIV), and interfering with HIV reverse transcriptase enzyme, the alpha defensins
HNP-1 preventing HIV viral transcripts from entering the nuclease, and finally both HNP-
1 and HD5 binding directly to HSV-2 vDNA to prevent transcription of the genome (77,
85-88)
Nonenveloped viruses such as HPV, BK and JC polyomavirus, and human
adenovirus (HAdV) have also been studied, but to a lesser extent (89-94). For this group
beta-defensins have shown little to no neutralizing effects on this family while alpha
defensins often show inhibitory characteristics through interactions with the viral capsid
and a common mechanism of improper trafficking and failure to deliver genetic material
to the host. Additionally, viral aggregation of BK polyomavirus has been shown to be
neutralizing (91). Interestingly, studies using q-defensins within this family have been
severely lacking and have yet to be examined, alternatively negative datasets may have
not been published or commented on. In an effort to better visualize the current state of
viral inhibition studies that have been documented with the different classes of defensins
I have made a visual summary that was inspired by Wilson et al 2013 (ref 64), but is an
21
original illustration that improves on the original by including q-defensins, new research
studies, and new viruses.

Figure 4. Summary of anti-viral effects of different defensin classes on both
enveloped and non-enveloped viruses. Defensins from each class have shown direct
anti-viral efficacy on many enveloped viruses. On the other hand, studies looking at non-
enveloped viruses have found little antiviral activity of b-defensins while q-defensins have
largely been unexplored or unreported.

22
Current understanding of defensin-mediated inhibition of HPV infection
Buck et al originally determined that human beta defensins had little to no
detectible activity against HPV infection, however several alpha defensins showed
promise as long as they were used in the absence of serum (90). After screening HNP1,
2, 3, and HD5, they found that HD5 specifically was able inhibit hrHPV infection at low
concentrations. HPV16 virions that were pretreated were unable to escape late
endosomal compartments. A follow-up study found that HD5-treated HPV16 virions were
unable to undergo furin cleavage, which had been found to be critical to both viral
uncoating and endosomal escape (92, 95). It became apparent in the next few years
however that virions isolated from primary raft cultures came furin pre-processed and did
not require processing at the basement membrane/on basal cells after seeding in
microwounds, therefore the overall mechanism proposed could have just been an artifact
of HPV16 PsV production. The same group then published a detailed follow-up study in
which they showed HD5 was able to inhibit furin pre-cleaved HPV16 virions from infecting
cells through direct interactions of HD5 with the viral capsid. These interactions stabilized
the viral capsid in late endosomal compartments and prevented viral uncoating, which
then resulted in their redirection to the lysosome and rapid degradation (93).
Mechanistically, this was found to be driven by HD5 binding to disorganized regions of L1
and L2 that were protruding from the viral capsid which in turn tightens up associations
of the L2-viral genome complex to the L1 capsid (94). While these studies outlined the
ways in which a naturally produced defensin, HD5, could protect against HPV infection
and transmission, they did not propose future studies to examine whether supplementing
23
defensins could be examined in a translational manner. Furthermore, these groups did
not consider examining whether members of the q-defensin class could be used as an
anti-viral against HPV, which in itself would be a novel method to prevent transmission.
Rhesus theta-defensin 1
Rhesus monkeys contain 3 separate genes that code for these q-defensin
precursors (RTD1a, RTD1b, and RTD1c). As was stated before, q-defensins are
generated from head to tail splicing of two precursor peptides. Theoretically this makes it
possible for the production of 6 different isoforms (RTD-1 through 6), however in a screen
of leukocyte granule content from 10 adult rhesus macaques approximately 50% of q-
defensin content was made up of the RTD-1 (96). Given its prominence, RTD-1 is the
prototype q-defensin used in studies has been shown to have broad spectrum
antimicrobial activity against multi-resistant bacteria, fungi, and even HIV (97-99).
Additional effects include immunomodulating activities in the setting of bacterial sepsis,
systemic candidiasis, acute lung injury and chronic lung infection, and is currently being
examined as a therapeutic for patients with rheumatoid arthritis (NTC04286789) (100-
103). In vivo studies that have examined toxicity have found it is well tolerated in mice in
doses up to 160 mg/kg through sub cutaneous delivery or 50 mg/kg via intraperitoneal
injection (100, 101). While toxicity is a highly important factor, another promising feature
in its usage as an anti-viral comes from the stability. Given the cyclic nature and tri-
disulfide bridge stabilization, RTD-1 remains properly folded and active after heating to
100°C for 30 min, does not break down in serum after a 72h incubation, and stays intact
when stored at extreme pH (2.0) (100, 104). This collectively suggests that if RTD-1 and
24
other q-defensin isoforms have HPV-inhibiting properties they would be promising
candidates for translation into an anti-HPV agent that could be included in sexual barrier
lubricants or used as a standalone topically applied braod-spectrum anti-viral agent. With
this in mind, the following chapters in this thesis outline my investigation into this central
question and outline future directions for the continuation of this research.

25
Chapter 2 – Theta-defensins inhibit high-risk human papillomavirus
infection through charge-driven capsid clustering

** note this chapter is taken from a submitted publication
Abstract

Persistent infection with high-risk human papillomavirus (hrHPV) genotypes
results in a large number of anogenital and head and neck cancers worldwide. Although
prophylactic vaccination coverage has improved, there remains a need to develop
methods that inhibit viral transmission towards preventing the spread of HPV-driven
disease. Defensins are a class of innate immune effector peptides that function to protect
hosts from infection by pathogens such as viruses and bacteria. Previous work utilizing α
and β defensins from humans has demonstrated that the α-defensin HD5 is effective at
inhibiting the most common high-risk genotype, HPV16. A third class of defensin that has
yet to be explored are θ-defensins: small, 18-amino acid cyclic peptides found in old-
world monkeys whose unique structure makes them both highly cationic and resistant to
degradation. Here we show that the prototype θ-defensin, rhesus theta defensin 1, inhibits
hrHPV infection through a mechanism involving capsid clustering that inhibits virions from
binding to cell surface receptor complexes.
26
Introduction

Human papillomavirus (HPV) remains the most common sexually transmitted
infection within the human population and it is estimated that over 80% of all sexually
active individuals will be infected at one point in their lifetimes (105). Persistent infection
with a high-risk HPV (hrHPV) genotype is causally associated with the development of
both anogenital and oropharyngeal cancers (106, 107). Even though prophylactic HPV
vaccination has increased over the years, the United States still had an estimated 13,170
new cervical cancer cases diagnosed and approximately 4,250 deaths due to disease in
2019 (108). Additionally, the number of hrHPV-positive head and neck cancers occurring
in men has seen a dramatic increase, underscoring that infection with hrHPV is a health
burden on all sexually active individuals (109, 110). In both cases, the primary genotype
associated with either cervical cancer (CC) or oropharyngeal squamous cell carcinoma
(OPSCC) was HPV16, which is found in >50% of CC and >90% OPSCC (111, 112). As
such, there is a continuous need to develop and test novel methods that may be used to
prevent the transmission of hrHPV that can work alongside prophylactic vaccination and
protect at-risk populations. A potential candidate to fill this need may be a unique member
of antimicrobial peptides known as defensins.
Defensins are small cationic peptides of the innate immune system that are rich in
cysteine and arginine residues (113). Originally studied because of their antibacterial
activity, defensins have exhibited antiviral activity against both enveloped and non-
enveloped viruses through interactions with both cellular receptors, viral anchoring
27
proteins, direct binding to viral capsids, cell membrane fusion inhibition, and direct
modulation of host responses to infection (for full review see Wilson et al. 2013 and
Mayumi et al. 2017) (66, 72). While genetically similar, defensins are classified into α, β,
and q subgroups based on the three disulfide bond pairings and amino acid sequence
similarities shared within each group (114). Uniquely, the θ-defensin family is the only
known group of cyclic polypeptides expressed in the animal kingdom and exclusively
found in old-world monkeys (115). Of the 6 different isoforms found in rhesus macaque
neutrophils, the Rhesus Theta Defensin-1 (RTD-1) isoform is the most abundant and
accounts for over 50% of RTD content (96). While humans also have genes that code for
q-defensins (termed retrocyclins), a premature stop codon within the sequences prevents
their translation (70, 116). Structurally, this class of defensin is formed by the excision
and binary ligation of two nonapeptides that create the mature 18-mer cyclic peptide (97,
117). This highly-cationic moiety is further stabilized by a core of three disulfide bonds,
making it highly resistant to proteolytic cleavage and stable in blood, plasma, and serum
(100).
In the context of hrHPV biology, the anti-viral efficacy of both α and β defensins
have been explored. Buck et al. screened 6 different naturally produced defensins for
their ability to inhibit HPV infection and found that β defensins showed minimal anti-viral
activity while a-defensins, specifically HD5, were highly effective in blocking mucosal
targeting hrHPV genotypes in vitro (90). Similar to a-defensins, θ-defensins have been
shown to also exhibit anti-viral activities against several human pathogens including
herpes simplex virus (HSV), human immunodeficiency virus (HIV-1), and influenza A virus
28
(IAV), however have not been screened for efficacy against hrHPV (80, 81, 99). Given
this, the purpose of our study was to examine whether the q-defensin RTD-1 could inhibit
prominent hrHPV genotypes 16, 18 and 31, and then characterize whether this inhibition
was through viral capsid or host cell interactions.

29
Results

Pretreatment of hrHPV Pseudovirions (PsV), not cells, with RTD-1 results in dose-
dependent inhibition of infection.

HPV16 pseudovirions (PsV) were used to examine whether the q-defensin RTD-1
could inhibit infection, as measured by gene transduction of the reporter plasmid
pCIneoGFP within HPV PsV, resulting in GFP expression by target cells. Initial screening
involved using serial dilutions of RTD-1 in serum-free IMDM and combining them with
PsV for 1h prior to addition to cells. 48h post infection GFP+ cells were assessed by flow
cytometry. Through this method we were able to clearly see a dose-dependent inhibition
on infection rates with significant reductions starting at 2.5µg/mL of RTD-1 (Figure 5A).
To address whether the inhibition was resultant of RTD-1 interacting with cell surface
proteins or virions, we used several approaches. First, we found that pretreating cells
directly for 1h with increasing concentrations of RTD-1 prior to PsV addition resulted in
no significant change in infection levels (Figure 5B). Next, we found that the presence of
serum during the PsV+RTD-1 pre-incubation steps resulted in a failure of RTD-1 to inhibit
PsV infection (Figure 5C), suggesting that competing serum proteins can interfere with
the initial interaction between RTD-1 and the virus capsid. It is worth noting that this
“serum effect” has also been documented in other defensin studies and was used as an
indirect way to show defensin-pathogen interactions (88, 90, 118).

30
Figure 5. RTD-1 inhibits HPV16 PsV infection in a dose-dependent manner driven
by an interaction between viral capsid and θ-defensin. (A) HPV16 PsV pre-incubated
with RTD-1 in serum-free medium, (B) HeLa cells pre-incubated with RTD-1, or (C)
HPV16 PsV pre-incubated with RTD-1 in the presence of serum were added to cells for
48h prior to measurement of GFP expression resulting from PsV gene transduction. (D)
Alternatively, excess RTD-1 was dialyzed out of PsV+RTD-1 (d(PsV+RTD-1)
combinations, with separately dialyzed PsV (d(PsV)) combined with dialyzed RTD-1 (20
µg/mL) to indicate removal of RTD-1 from dialysis cassettes (Ctrl). Each panel shows the
31
percentage of GFP+ (PsV infected) cells ± SD as analyzed by flow cytometry. All
experiments were carried out in triplicate. Results shown are representative data from 3
independent experiments. (ns=not significant, *p<0.05, ****p<0.0001, one-way ANOVA
followed by Dunnett’s multiple comparisons test against PsV within groups)

To investigate whether “free” RTD-1 in the PsV+RTD-1 mixture contributed to
inhibition of HPV16 infection, we used dialysis as a method to take advantage of the size
discrepancies between RTD-1 (~ 2 kDa) and PsVs (>20 megaDa) (119). After the initial
1h pre-incubation of virions and RTD-1 we used a large molecular weight membrane to
dialyze out unbound RTD-1 prior to PsV collection and addition to cells. Post dialysis the
PsV+RTD-1 combinations were collected, diluted in IMDM containing 10% FBS, and
added to cells for the infection assay. Separately dialyzed PsVs were used as a control
to verify there were no effects of the dialysis procedure on baseline infectivity. To provide
evidence that excess RTD-1 passes through the membrane, we dialyzed the highest
concentration of RTD-1 (20 µg/mL) in a separate container in the same manner, collected
the dialyzed solution, and combined it with the dialyzed PsV for 1h in serum-free IMDM
before adding it to the cells in complete media for the infection assay (labeled as Ctrl).
Significant, dose-dependent decreases in the PsV infection rate were seen in the
PsV+RTD-1 combinations while there was no change in infection rate with the control
dialyzed RTD-1 combined with PsVs (Figure 5D). A diagrammatic outline of this
procedure is seen in Figure 6. Taken together, this set of data suggests that the infection
inhibition occurs through interactions between the PsV and RTD-1 directly and not
through binding of RTD-1 to cell surface virus entry receptors.
32

Figure 6. Dialysis Experiment Layout. Schematic of the dialysis experiment from figure
5D indicating how the different groups were created.

RTD-1 significantly reduces uptake of HPV16 by inhibiting initial binding of
defensin-coated virions to the cell surface

Evidence suggests that the α-defensin HD5 inhibits HPV infection through direct
capsid interactions that prevent viral uncoating, which redirects the trafficking of virions
to lysosomes for degradation (92, 93). Because our data similarly suggested a direct
33
interaction between RTD-1 and the viral capsid, we wanted to examine the effects of HPV
uptake using HD5 inhibition as a comparison. As we have previously reported, pHrodo-
labeled HPV16 virus like particles (VLPs) can be used to evaluate uptake as the
fluorescence intensity of the label increases as particles are trafficked to successively
lower pH endosomal compartments (26, 64). After pre-incubating the pHrodo-labeled VLP
with increasing concentrations of RTD-1 or HD5, complexes were added to HeLa cells
and hourly changes in fluorescence intensity were measured to assess viral uptake. We
found that pre-incubating HPV16 pHrodo-labeled VLP with 2.5 µg/mL of RTD-1 resulted
in significant decreases in pHrodo-particle signal beginning at 3h post addition, and that
the higher concentrations of RTD-1 reduced the pHrodo-particle signal to near
background levels, suggesting an elimination of viral uptake (Figure 7A). In contrast,
pretreatment of pHrodo-labeled VLP with HD5 resulted in significant increases in pHrodo-
VLP signal throughout the time course (Figure 7B), consistent with augmented
redirection of virus particle to acidic compartments. These findings are in support of the
previous study by Wiens et al, who demonstrated that HD5 stabilizes the capsid and
redirects it to the lysosome (93).

34

35

Figure 7. RTD-1 treatment of HPV16 results in significantly reduced uptake and
localization of virions to early endosomes highlighted by sparse detection of large
aggregates at cell surfaces and reduced cell binding capacity of RTD-1 treated
virions. Time course of pHrodo-HPV16 VLP internalization and trafficking to low pH
endosomal compartments in the presence of (A) RTD-1 or (B) HD5. Shown is the mean
pHrodo signal intensity of triplicate wells ± SD. Background signal from untreated cells
included. (C) Immunofluorescence imaging of internalized HPV16 PsV untreated (NTr) or
36
incubated with 5.0 µg/mL RTD-1 or HD5. (Left) Representative images from each field of
view per treatment group (scale bar = 10 µm). (Right) Quantitation of HPV16 and EEA-1
colocalization was performed using ImageJ with the JaCOP plugin using 10 individual
fields of view (10-20 cells/field) from 2 independent experiments. (D) Immunofluorescent
image of bound untreated PsV (Left) or RTD-1 treated PsV (Right) on the surface of HeLa
cells. (E) Western blot of cell surface bound HPV16 L1 capsid protein in the presence of
RTD-1. Membrane fractions are shown, probed for HPV16 L1 (green) and actin (red).
ns=not significant, **p<0.01, ****p<0.0001 two-way ANOVA followed by Dunnett’s
multiple comparison test against untreated VLPs at each timepoint for uptake
experiments. One-way ANOVA followed by Dunnett’s multiple comparison test against
untreated PsV in colocalization studies.

Given that pHrodo-labeled VLP signal is not a direct quantitative measure of uptake
but rather a combination of the quantity of particles and their endosomal location, we next
sought to examine whether there were changes in the localization of HPV virions to early
endosomal compartments. Using immunofluorescence imaging we found that, after 3
hours of uptake, there was no significant difference between the number of untreated and
HD5-treated HPV16 PsV localized to EEA1+ early endosomes, however RTD-1-treated
virions had a significant reduction in colocalization events (Figure 7C). Interestingly, there
were very few cells that had positive signal for HPV16 when PsV were pre-incubated with
RTD-1, however the ones that did, showed large, irregular areas of bright staining (Figure
7D). These data suggest that RTD-1 may be driving viral particles to cluster/aggregate
and stay at the surface of specific cells while failing to bind to others.
37
Since our pHrodo data indicated a limited amount of virus particle uptake (confirmed
by colocalization analysis), we decided to examine whether RTD-1-treatment of virions
reduced their capacity to bind to the cell surface, which is a requisite step to initiate
infectious viral entry (10, 25, 120). After initial pretreatment of HPV16 VLP with increasing
concentrations of RTD-1, virions were bound to pre-cooled HeLa cells for 1h at 4°C.
Excess, unbound virions were gently removed with cold PBS washes and cells were
harvested via scraping into separate 1.5 mL siliconized tubes. Membrane and cytosolic
factions were extracted to quantify the relative amounts of virions bound to the cell surface
between groups. Western blot of membrane fractions clearly indicated a dose-dependent
decrease in the amount of L1 capsid protein recovered from the cell surface (Figure 7E).
This indicates that as the concentration of RTD-1 treatment increases, the ability of virions
to effectively bind to the cell surface is lost.

RTD-1 interacts with the viral capsid and causes capsid clustering.

Our data to this point provides evidence that RTD-1 is able to inhibit HPV16 infection
through reducing uptake potentially driven by an inability of RTD-1 treated virions to
interact with the cell surface, however it was not known whether the q-defensin treatment
of virions was disrupting individual capsid structures or causing viral aggregates. To
examine this, we utilized nanoparticle tracking analysis (NTA) to assess whether the size
and quantity of particles was being modified with RTD-1 treatment. With this light
scattering method, we noted that the overall number of particles that flowed through the
NS300 flowcell were reduced as the concentration of RTD-1 was increased (Figure 8A).
38
Concurrently, we mounted HPV16 VLP and RTD-1 treated VLP on formvar-treated grids
and performed TEM imaging. Untreated VLP show the characteristic 55 nm virus
structure whereas no individual virus particles could be seen in the presence of RTD-1
(Figure 8B). RTD-1 by itself did not form large negatively stained protein aggregates.
Histograms of particle size based on quantity clearly showed a demarcated increase in
larger particles as the concentration of RTD-1 increased (Figure 8C, i-v). This quantified
increase in particle size can also be clearly visualized from the individual frames from
videos taken with the NS300 (histogram inserts). Together, these three observations
suggest that viral particles are aggregating in the presence of RTD-1. Finally, in an effort
to establish whether these capsid clusters are forming due to the highly cationic nature of
RTD-1, we added in an excess of divalent cations (100 mM CaCl2) to the PsV+RTD-1
mixture and observed that the histogram distribution of particle sizes nearly returned to
non-RTD-1 treated samples (Figure 8C, vi). These findings suggest that by saturating
the environment with positive charged ions RTD-1 is unable to interact with the virions
and induce aggregation.

39

Figure 8. RTD-1 interacts with HPV16 VLP to form viral aggregates and can be
prevented with the addition of excess divalent cations during incubation. HPV16
VLP were incubated with RTD-1 at indicated concentrations and run on a NanoSight
NS300 to quantify number of particles or mounted on formvar-coated copper grids and
imaged via TEM. (A) Shown is the mean quantification of particles/mL as analyzed by
nanoparticle tracking analysis (NTA) through 5 separate video segments (± SD), insert (i)
shows background signal from RTD-1 without VLP present. (B) TEM imaging of HPV16
VLP, VLP incubated with 1.5 µg/mL RTD-1, or RTD-1 alone (scale bar = 100 nm). (C)
40
Representative histograms generated by nanoparticle tracking analysis (NTA) showing
mean particle sizes and quantity ± SEM (red bars). Inserts on each show a single frame
from the videos the particle analysis was performed on.

RTD-mediated inhibition of HPV infection is dependent on the innate charge of
the q-defensin isoform.

After noting that excess positive charge within RTD-1+VLP combinations inhibited
the formation of aggregates seen in the nanoparticle tracking analysis, we wanted to
confirm whether the inherent charge of the θ-defensin had an effect on infection inhibition
and viral uptake. To test this, we compared RTD-1 (+5 charge) to two different RTD
isoforms: RTD-2 (+6 charge) and RTD-4 (+3 charge) (Figure 9C). As a positive control
for defensin-mediated inhibition of HPV infection we included HD5 in our experiments.
Importantly, we found that RTD-1 and HD5 were both able to inhibit HPV16 infection
within the µM-range with RTD-1 having an IC50 at approximately 14 µM while the HD5
IC50 was 26 µM (Figure 9A). RTD-4, with almost half the charge of RTD-1, had an IC50
of 101 µM, suggesting that reduced charge leads to reduced infection blocking (Figure
9B). This reduced capacity to inhibit infection was also seen in the HPV16 pHrodo-labeled
VLP uptake assay as a greater concentration of RTD-4 was needed to significantly inhibit
uptake in HeLa cells (Figure 9E). RTD-2 showed a nearly identical inhibitory IC50 to
RTD-1 (Figure 9B) and showed a concentration-dependent effect on viral uptake (Figure
9D) which was much more effective than RTD-4 at matched concentrations. These results
41
suggest that the inherent charge of the RTD-1 isoform dictates the ability of the θ-defensin
to inhibit VLP uptake and HPV16 PsV infection.

42

Figure 9. The viral inhibitory concentration of RTD-1 is similar to HD5 and is
dependent on the charge of the RTD isoform. (A, B) HPV16 PsV were pretreated with
indicated concentrations of HD-5, RTD-1, RTD-2, or RTD-4 prior to addition to HeLa cells.
Infection rates were normalized to untreated PsV infection and inhibition curves
generated for each defensin. Shown is a non-linear regression curve fit with IC50 values
shown. Individual dots are the mean of triplicate infection rates values ± SD. (C) Cyclic
43
amino acid sequence diagram of RTD-1, RTD-2, and RTD-4 with positively charged
sidechains indicated in red and disulfide bridges in yellow. (D & E) pHrodo-labeled HPV16
VLPs were pretreated with indicated concentrations and added to HeLa cells. Mean
fluorescent intensity of pHrodo signal was measured hourly to assess uptake and is
shown as the average of three separate wells at each time point ± SD. All figures are
representative of data from at least 3 independent experiments. ns= not significant,
**p<0.01, ***p<0.001, ****p<0.0001 two-way ANOVA followed by Dunnett’s multiple
comparison test against untreated VLPs at each timepoint for uptake experiments.

RTD-1 is able to inhibit other hrHPV genotypes, furin pre-cleaved (fpc) HPV16
PsV, and ability to inhibit is influenced by the HSPG-status of virions.

Given that furin-processing at the cell surface is requisite for successful infection
and partially dictates the asynchronous uptake of HPV virions (18, 121), we sought to
assess whether RTD-1 would still be able to inhibit fpc HPV16 PsV. We found that fpcPsV
are just as sensitive to RTD-1 inhibition as regular PsV (Figure 10A). Interestingly,
removal of HSPG moieties that may co-purify during normal PsV production resulted in a
PsV (HSPG-) that was slightly more resistant to RTD-1 inhibition as the IC50 was found
to be roughly twice the amount needed to inhibit unmodified PsV (Figure 10A).
Additionally, while HPV16 is the dominant genotype found in both cervical and head and
neck cancers, we still wanted to examine the effects of RTD-1 on infection by other
prevalent hrHPV to confirm a multi-hrHPV subtype effect (122). Using genotype-specific
neutralizing antibodies as positive controls for inhibition, we found that RTD-1 was able
44
to significantly reduce infection of HPV18 and HPV31 PsV in a dose-dependent manner,
similar to HPV16 (Figure 10 B & C, 2.5µg/mL shown), suggesting that RTD-1 inhibition
can be applied across many hrHPV genotypes.
45
Figure 10. RTD-1 is able to inhibit furin pre-cleaved (fpc) HPV16 PsV, capacity to
inhibit is partially influenced by the HSPG-status of virions, and RTD-1 has cross
hrHPV genotype efficacy. Furin pre cleaved (fpc) HPV16 PsV, HPV16 PsV that had
been treated with heparinase and trypsin before purification (HSPG-), and hrHPV
genotypes 18 and 31 shown. (A) RTD-1 is able to inhibit fpc HPV16 PsV and has a
reduced capacity to inhibit HSPG-free HPV16 PsV. Infection with non-treated HPV16
46
PsV, fpc PsV, or HSPG- PsV were used to normalize infection. Individual points on graph
are means of triplicate wells ± SD with a non-linear regression curve. IC50 for each curve
are shown. RTD-1 inhibits infection of HeLa cells by both (B) HPV18 and (C) HPV31 high-
risk genotypes. Shown is the mean %GFP+ cells of triplicate wells ±SD. Genotype
specific antibodies were used for neutralization controls. (C) ****p<0.0001, one-way
ANOVA followed by Dunnett’s multiple comparisons test against PsV-only group.

Langerhans cells are not activated by RTD-1 generated viral aggregates of
HPV16, but still become active with Poly I:C in the presence of RTD-1.

One of the methods that HPV16 utilizes to evade immune detection is through the
generation of tolerogenic Langerhans cell (LC) phenotypes (58, 59, 61). As was stated
before, LC are the first line defenders against epithelial targeting pathogens, and the
ability to overcome the negative signaling by hrHPV has been a proposed method to clear
persistent infection (59, 123). Given this, I sought to examine whether LC derived from
healthy patient blood would be stimulated by the RTD-1 driven HPV16 viral aggregates
(experimental outline in Figure 11). Additionally, given that RTD-1 has been shown to
have immunosuppressive effects on circulating leukocytes, I wanted to see whether the
higher concentrations of RTD-1 has any negative signaling effects on LC activation
through the TLR-3 agonist PolyI:C, which we have used in the past to overcome HPV-
induced LC tolerance (61, 124-127). Treatment of LC with HPV16 viral aggregates did
not change the status of LC as measured by surface MHCII and costimulatory CD86
47
expression, however, activation of LC by Poly I:C treatment was still successful (Figure
12A and B).
Figure 11. Schematic diagram of LC generation and activation experiment. Briefly,
LC were derived from patient PBMC and treated with HPV16 VLP, HPV16 VLP+RTD-1,
or PolyI:C/PolyI:C+RTD-1 combinations prior to assessment of activation state via flow
cytometry.

48

Figure 12. Patient derived LC are not activated by RTD-1 induced viral aggregates
but can still be stimulated by PolyI:C in the presence of RTD-1. HPV16 VLP or HPV16
49
pretreated with indicated concentrations of RTD-1 to induce viral aggregates were added
to LC. Alternatively, PolyI:C or PolyI:C + indicated concentrations of RTD-1 were added
to LC. 24h post addition, LC were collected and stained for surface Langerin, CD86, and
MHCII. Neither MHCII (A) or CD86 (B) expression is significantly upregulated by the
addition of HPV16 viral aggregates. PolyI:C and PolyI:C+RTD-1 results in significant
upregulation of both (**p<0.01, ***p<0.001, One-way ANOVA followed by Dunnett’s
multiple comparisons test to NTr LC).

Taken together, the overall results provide evidence that RTD-1 and other cyclic θ-
defensin isoforms are able to inhibit hrHPV infection through charge-based capsid
clustering and inhibition of infectious uptake in target cells.

50
Discussion

Antimicrobial peptides play essential roles in defending epithelial tissues against
infection by viruses and bacteria (114). Defensins are a subclass of these innate immune
components and have been found in both plants and animals (128). Previous work has
demonstrated that α defensins, specifically HD5, were most effective in inhibiting HPV
infection out of all human-produced defensins screened (90). Within this study we have
now expanded the list of defensins that act against HPV to include q-defensins by
characterizing how the prototype q-defensin, RTD-1, inhibits HPV16 (our model high-risk
genotype). Similar to HD5, we found evidence that HPV viral inhibition by RTD-1 is
facilitated directly through defensin-viral capsid interactions (90). This observation initially
came from the combination of approaches taken that show infection inhibition is lost when
serum is present during incubation or cells are pretreated with RTD-1. Additionally, we
showed that dialyzing out unbound RTD-1 from RTD-1+PsV combinations still results in
significant infection reductions, further supporting this conclusion.
While both HD5 and RTD-1 interact directly with HPV virions, their inhibitory
mechanisms differ. Alpha defensin HD5-mediated inhibition comes from the stabilization
of the HPV viral capsid through charge-driven interactions with the C-terminal tail of the
L1 major capsid protein and dimerized HD5. This directly prevents viral uncoating in
endosomal compartments and redirects virion trafficking to the lysosome (93, 94). We
show through our data that RTD-1 mediated inhibition of HPV is driven primarily through
capsid clustering, which limits the capacity of virions to bind to the cell surface and
51
prevents virion uptake. This mechanism of viral inhibition by q-defensins has been
documented before with IAV, where it was hypothesized that the defensins were able
self-associate once bound to viral surfaces (80). Unlike the study with IAV, however, we
found that the inherent cationic charge of the q-defensin isoform had an impact on how
well RTD inhibited HPV uptake and infection. This difference may potentially be explained
by the affinity of q-defensins to self-associate when bound to enveloped vs non-enveloped
viruses or the availability/quantity of interacting partners on the viral surface and their
changes in affinity when interacting with q-defensins. One such candidate for our study
was viral-associated HSPGs.
Infectious uptake of HPV is a highly complex dance of virion associations with
factors found on the cell surface and surrounding basement membrane. One of the key
interactions that has been found is that binding of HPV virions to HSPG and growth factors
can cause intracellular signaling prior to endocytosis of virions (120, 129). Given that the
production of HPV PsV requires overnight maturation of a whole cell lysate, our purified
PsV may have been pre-decorated with these factors. Because of this we utilized HPV
PsVs treated with trypsin and heparinase in an effort to eliminate HSPG-decorated
particles prior to purification. The HSPG-negative particles were more resistant to RTD-
1-mediated inhibition, however the effect was not significant enough to suggest RTD-1
interactions with viral-associated HSPG are the primary drivers of capsid clustering.
Importantly, we also found that RTD-1 is able to inhibit furin pre-cleaved HPV16 PsV.
Furin pre-cleaved PsVs are able to bypass the initial HSPG binding interactions at the
cell surface that are hypothesized to precede infectious uptake through a non-canonical,
52
clatherin-independent, macropinocytosis-like mechanism that utilizes tetraspanins
CD151 and CD63, integrins, growth factor receptors, the annexin A2 heterotetramer,
actin, and obscurin-like protein 1 (reviewed in chapter 1)(120, 121, 130, 131). Given that
it is still not known whether WT virions come furin pre-cleaved, uncleaved, or in a mixed
population as seen in WT preps from organotypic raft cultures (132), it is reassuring to
note that fpc PsV were just as susceptible to inhibition by RTD-1 when compared to our
normal PsV preparations.
HPV16 is currently found in a majority of HPV+ head and neck cancer cases (5),
however is not the only genotype associated with HPV-driven carcinogenesis. Because
of this it was important to examine the cross-genotype inhibitory effects of RTD-1 on
HPV18, and 31, which together with HPV16 make up the three most prominent genotypes
associated with cervical cancer (133, 134). We found the same inhibitory capacity of RTD-
1 on each genotype tested, making it is reasonable to assume that RTD-1 may be
considered as a prophylactic measure against hrHPV infection with genotypes most
commonly associated with HPV+ cancers. These findings open the door to considering
the usages of q-defensins as a way to reduce the infection and spread of HPV within the
population.
Previous groups have proposed the application of human defensins as broad-
spectrum antiviral agents that could be used to stop the transmission of viral and bacterial
infections (80, 90). There are two significant limitations to this however: 1) The non-cyclic
nature of α and β defensins makes them less stable and susceptible to degradation and
2) Physiological concentrations of NaCl, Ca
2+
, and Mg
2+
can modulate the anti-microbial
53
activity of certain defensins (65, 135, 136). While we had shown in our data that excess
divalent cations in solution (in the form of Ca
2+
) were able to prevent the viral aggregating
effects of RTD-1, these concentrations were at 100 mM, which is not found physiologically
(137). Given that a previous group has demonstrated that RTD-1 can withstand heating
to 100°C for 30 min, is not toxic to cells in vitro or to mice in doses up to 160 mg/kg, does
not break down in serum after a 72h incubation, does not induce inflammation, and
remains intact when stored at extreme pH (2.0), we propose that RTD-1 is a superior
candidate for future development into a topical prophylactic agent (100, 104). This could
be added to lubricants currently used in condoms or a stand-alone product to prevent the
spread of HPV and potentially other sexually transmitted infections in high-risk
populations who are either unvaccinated or not eligible for prophylactic vaccination.
Furthermore, because q-defensins are not naturally produced in humans it would be much
easier to interpret efficacy as you would not have to contend with variable endogenous
production of human defensins between study participants. An intriguing question,
however, is whether topical application of q-defensins would lead to dysbiosis of the
microflora of vaginal and oral cavities as it is shown to have significant antimicrobial
effects against Escherichia coli, Staphylococcus aureus, and Candida albicans through
enhancement of host directed mechanisms (96, 101).
Future studies examining the interactions between HPV and q-defensin family
members should explore whether human retrocyclin isoforms or those occurring in olive
baboons also have anti-viral efficacy, and whether RTD-1 viral aggregates show changes
in opsonization rates by macrophages or dendritic cells (DC) (117). Previous work
54
examining IAV aggregates formed by q-defensin interactions showed enhanced uptake
by macrophages and neutrophils, however it has yet to be explored as to whether DC
antigen uptake and priming of anti-viral responses would be impacted (80, 138). Towards
this goal, we examined whether the RTD-1+HPV16 viral aggregates were immunogenic
to Langerhans cells, the APC of the epithelium. Unfortunately, we found no differences in
LC maturation or activation state when HPV16+RTD-1 complexes were compared to WT
HPV16 treated LC. While it would have been exciting to find that we could both inhibit
viral transmission and overcome the tolerant phenotype LC taken on after exposure to
HPV virions (59, 63), it appears as though tolerogenic interactions by HPV viral
aggregates are maintained. Future studies looking to use RTD-1 mediated viral
aggregates as an attempt to generate antiviral responses in APC populations other than
LC could consider the addition of adjuvants that enhance humoral responses to bolster
the production of neutralizing antibodies against the viral vector (139). In the case of this
study it did not appear that RTD-1 inhibited LC stimulation by PolyI:C treatments, which
shows promise for future development.
Overall, we have documented that q-defensins from rhesus macaques can be
utilized to inhibit hrHPV infection. Through an aggregation of virions that is contingent
upon the inherent charge of the peptide, q-defensins are able to prevent virions from
binding to cell surfaces and subsequently reduce HPV uptake into target epithelial cells
in a dose-dependent manner. This insight into the molecular interactions between q-
defensins and non-enveloped viruses may translate into a prophylactic strategy against
hrHPV that can be used within populations where vaccination is not a viable option and
55
informs future developments of anti-viral therapies against epithelial targeting pathogens
through the usage of defensins.
56
Materials and Methods
Cell lines
The cervical cancer cell line HeLa (CCL-2, ATCC) was maintained in Iscove’s
Modified Dulbecco’s Medium (IMDM) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) (Omega Scientific, Tarzan, CA) and gentamycin (Lonza,
Walkersville, MD). HaCaT cells, spontaneously immortalized keratinocytes, were cultured
in Dulbecco’s Modification of Eagle’s Medium (DMEM) with 4.5g/L glucose, L-glutamine,
and sodium pyruvate (Corning, 10-013CV, NY) supplemented with 10% FBS (Omega
Scientific), and gentamycin (Lonza). 293TT and 293TTF cells (kind gifts from John
Schiller (NIH) and Richard Roden (Johns Hopkins University), respectively) were
maintained in IMDM supplemented with 10% FBS (Omega Scientific) and gentamycin
(Lonza). Episomal plasmids coding for additional SV40 large T antigen (293TT and
293TTF) and furin (293TTF) were maintained by the inclusion of 250 µg/mL hygromycin
B (ThermoFisher) and 1 µg/mL puromycin (MilliporeSigma). All cells were grown in a
humidified incubator at 37°C with 5% CO2 and passaged when confluency was ~80%.

Antibodies
The HPV16 L1 antibody H16.V5, HPV18 L1 antibody H18.G10, and HPV31 L1
antibody H31.A6, used as neutralization controls within hrHPV infection studies, were
gifts from Neil Christensen (The Pennsylvania State University). For western blot analysis
of HPV surface binding the HPV16 L1 mAb CAMVIR-1 (550840, BD Bioscience), β-actin
(4970, Cell Signaling Technologies), goat-anti-mouse IRDye 800CW (925-322, LI-COR),
57
and goat-anti-rabbit (H + L) Alexa Fluor 680 (A27042, Thermo Fisher) were used.
Immunofluorescent imaging was carried out using the HPV16 L1 antibody 56E.L1 (a kind
gift from Martin Sapp, Louisiana State University), early endosomal antigen 1 (EEA-1)
(Ab109110, Abcam), rabbit isotype control (02-6102, Thermo Fisher), mouse isotype
control (03001D, BD Biosciences), TRITC goat-anti-rabbit (ab6718, Abcam), and DyLight
488 goat anti-mouse (405310, Biolegend).

HPV pseudovirus (PsV) and virus like particle (VLP) production
HPV16, 18, and 31 PsVs were prepared as previously described with the ripcord
modification (140). Briefly, 293TT cells were co-transfected with codon-optimized L1 and
L2 plasmids for HPV16 (p16L1L2), HPV18 (p18L1L2), and HPV31 (p31L1L2), along with
a pCIneoGFP reporter plasmid (all gifts from John Schiller). Following a 2-day production,
PsVs were matured overnight and purified on an iodixanol gradient (OptiPrep,
MilliporeSigma, Burlington, MA). For bulk PsV preparations, the self-packaging p16L1L2
plasmid was utilized and the same purification method used. Infectious titer was
determined by flow cytometric analysis of green fluorescent protein (GFP)+ 293TT cells
at 48 h post-addition of serially diluted PsV stock and calculated as infectious units
(IU)/mL. Non-reporter plasmid containing PsV preps were quantified via coomassie blue
staining with known BSA concentrations. To remove heparin sulfate proteoglycans
(HSPG) that co-purify attached to the virion surface, a PsV prep was subjected to a brief
trypsin and heparinase treatment prior to the addition of soybean trypsin inhibitor and
purification on the iodixanol gradient and is referenced as “HSPG-“ within the manuscript.
Furin pre-cleaved HPV16 (fpc) PsV were generated using 293TTF cells using the protocol
58
outlined by Wang et al (141). Assessment of L2 cleavage by furin was carried out by
western blot.
HPV16 virus-like particles (VLP) were produced using a recombinant baculovirus
expression system as previous described (142). Western blot analysis confirmed the
presence of HPV L1 and L2, while intact particles were verified by an enzyme linked
immunosorbant assay (ELISA) using conformationally-dependent L1 antibodies.
Coomassie Blue staining was performed to determine the concentration of HPV L1
protein within VLP preparations.

Production of defensins
The hydrochloride salts of RTD-1, 2, 4, and HD5 were prepared as previously
reported producing pure (>98%) material verified by analytical reverse-phase ultra-
performance liquid chromatography and tandem mass spectrometry (97, 115). Sterile
stock solutions of were dissolved in saline and stored at 4°C. Defensin stocks were a kind
gift from Michael Selsted (University of Southern California).

HPV PsV infection assays
Stock PsV were titrated for each genotype so that the multiplicity of infection (MOI)
given for experiments resulted in approximately 30% gene transduction at 48h post
addition. HeLa or HaCaT cells were seeded at 2x10
4
cells/well in 24-well plates and
incubated overnight at 37°C prior to infection assay. PsV were diluted in IMDM without
FBS and combined with indicated concentration of RTD-1, RTD-2, RTD-4, HD5, or
neutralizing antibody in a total volume of 100 µL for 1 hour at 37°C and then added to
59
cells. Alternatively, cells were pre-incubated with indicated concentration of RTD-1 for 1
hour at 37°C, excess, unbound RTD-1 was washed away with warm PBS, and HPV16
PsV added.
For dialysis experiments, RTD-1, HPV16 PsV, or RTD-1+PsV combinations in
IMDM with phenol red were loaded into Pierce 96-well Microdialysis cassettes (88260,
Thermo Fisher) and dialyzed against 2 L of PBS in separate containers for 4 hours at
room temperature (RT) (4 x 500 mL exchanges, 1 per hour). Dissociation of phenol red
out of cassettes was used as a visual indicator of complete exchange. Post dialysis, PsV
and RTD-1+PsV were removed from cassettes and added to HeLa cells for 48h.
Separately dialyzed PsV and RTD-1 were combined for 1 hour at 37°C before addition to
cells and served as a control to show RTD-1 was dialyzed out of cassettes.
For all infection readouts, cells were trypsinized, collected, and analyzed on a
Cytomics FC500 using CXP software (version 2.2) (Beckman Coulter). Infection was
defined as % of GFP expressing cells within treatment groups. Viability was assessed
concurrently through the use of a propidium iodide stain.

HPV16-pHrodo VLP uptake assays
HPV16 VLP were labeled with pHrodo-red iFL (ThermoFisher) according to
manufacturer’s instructions at a dye to L1 protein ratio of 20:1. Excess dye was removed
from VLP preps through agarose bead column filtration. The pHrodo labeled VLPs were
re-quantified through coomassie blue staining with known BSA concentrations.
HeLa cells were seeded at 2.0x10
4
cells/well in a 48-well plate and incubated
overnight before uptake assays. pHrodo-labeled VLPs were mock treated or incubated
60
with indicated concentrations of RTD-1, RTD-2, RTD-4, or HD5 for 1 hour at 37°C. After
treatment of VLP, media was removed from cells and replaced with VLP or VLP+defensin
combination at a concentration of 1.25 µg L1/1x10
6
cells. Cells were incubated at 37°C
for duration of uptake assay with intensity of pHrodo-red signal read every hour for the
duration of the experiment on a CLARIOstar Plus microplate reader (BMG Labtech).

NanoSight imaging of viral particles
HPV16 VLP were diluted in PBS or treated with indicated concentrations of RTD,
HD5, or RTD+VLP combination in 100mM CaCl2. After incubation, samples were run on
a NanoSight NS300 (Malvern Panalytical) and particle size/number was quantified using
Nanoparticle Tracking Analysis (NTA) software (version 3.2).

Confocal imaging and colocalization analysis
For HPV16 colocalization with EEA1, HeLa cells were seeded into 8-well chamber
slides (ibidi) at 1x10
4
cell/well and grown overnight. HPV16 PsV were diluted in PBS or
treated with indicated concentration of RTD-1 or HD5 for 1h at 37° prior to addition to
cells. Cells were treated with 0.5 μg PsV/1x10
6
cells for 2h at 37 °C. After treatment, cells
were gently washed with PBS to remove unbound excess virions and then fixed with 4%
paraformaldehyde. Cells were permeabilized with triton X-100 and stained overnight in a
humidified chamber at 4°C for HPV16 (H16.56E, 1:200) and EEA1 (1:250). After
secondary antibody incubation for 30 minutes at RT, excess antibody was removed, and
slides were coverslip mounted with ProLong Gold Antifade Mountant with DAPI (Thermo
61
Fisher). Immunofluorescent (IF) images were captured on a Nikon Eclipse Ti-E laser
scanning confocal microscope running Nikon Elements software (version 4.0). For
colocalization analysis, 15 images from each treatment group with >20 cells/image were
analyzed with Fiji (a distribution of ImageJ, NIH) (143). Using the JACoP plugin thresholds
were automatically set, and the extent of colocalization was measured and reported as
Mander’s colocalization coefficient (144).

Transmission electron microscopy imaging
HPV16 PsV were diluted in PBS or treated with indicated concentration of RTD-1
for 1h at 37° prior to mounting on Formvar-coated copper support grids (FF200-Cu,
Electron Microscopy Sciences). Grids were washed by floating on sterile PBS followed
by counterstaining with uranyl acetate (22400, Electron Microscopy Sciences). Images of
VLP were collected on a JEOL JEM-2100 LaB6 electron microscope running Gatan
imaging software (JEOL Ltd., Akishima, Tokyo).

Western blot analysis of binding
HeLa cells were seeded at 5.0x10
5
cells/well in 6-well plates and incubated
overnight at 37°C prior to binding assay. VLP were diluted in IMDM without FBS and
combined with indicated concentrations of RTD-1, HPV16.V5 antibody, or PBS for 1h at
37°C. Tissue culture plates were cooled to 4°C, media was removed, and cells were
gently washed with cold PBS. VLP or treated VLP were then added to cells and bound
for 1 hour at 4°C followed by removal of excess unbound virions through 2 washes with
62
PBS. Cells were then collected through scraping and membrane fraction isolated with
Mem-PER reagent (89842, Thermo Fisher) using the manufacturer protocol. Protein
concentrations were quantified between samples through a Bradford assay and 20µg of
cellular protein was run on a NuPAGE 10% Bis-Tris gel (Thermo Fisher) in MOPS buffer.
Gels were transferred to nitrocellulose using the iBlot2 system (Thermo Fisher), blocked
for 1h in StartingBlock buffer, and then stained overnight at 4°C for HPV16 L1 (CAMVIR,
1:2500) and β-actin (CST, 1:1000). The following day blots were washed, secondary
antibodies were added for 45 min at RT, then blots were imaged on an Odyssey imaging
system (LI-COR). Band intensity within images were then quantified using Image Studio
Lite software (version 5.2.3, LI-COR).

Langerhans cell generation
Monocytes were isolated from peripheral blood mononuclear cells and incubated
in complete media (RPMI 1640 supplemented with 10% FBS, 1X Pen/Strep, 1X Non-
essential amino acids, and 1X 2-mercaptoethanol) with the addition of 1000 U/mL (~180
ng/mL) GM-CSF, 1000 U/mL (~200 ng/mL) IL-4, and 10 ng/ml TGF-β for 7 days. LC
phenotype was confirmed via flow cytometry as CD1a
+
Langerin(CD207)
+
.

LC activation assay and flow cytometry
LC were treated with HPV16 VLP, HPV16 VLP+RTD-1 (viral aggregates formed 1
hr prior to addition to LC), PolyI:C, or PolyI:C+RTD-1 and surface markers were detected
via flow cytometry. Briefly, 10
6
LC were seeded in a 6-well plate and either left untreated,
63
treated with 2.5 μg HPV16 VLP, 2.5 µg HPV16 VLP+RTD-1 pre-incubated at indicated
concentrations, or PolyI:C/PolyI:C+RTD-1 combinations for 24 hr in 2 ml complete
medium with periodic mixing for the first 1 hr (37°C, 5% CO2). After 24 hr, the cells were
harvested, washed, stained for surface Langerin, MHC II, and CD86 and analyzed by flow
cytometry.

Statistical analysis
For this study analysis was performed using GraphPad Prism (version 8.4.2,
GraphPad Software) with a minimal threshold set at p£0.05 for significance. Details for
statistical analysis for each individual experiment can be found within figure legends.

64
Chapter 3. Future directions for this research
Where to go from here
As was highlighted in chapter 1, hrHPV-driven cancers account for the a
continually growing number of head and neck cancers, majority of cervical cancer cases,
and other cancer types within anogenital regions. Since oncogenic transformation by the
virus requires the establishment of infection and evasion of immune detection within
stratified epithelial tissue at mucosal sites, there remains a need to develop prophylactic
measures that inhibit initial infection events as well as treatments to alert the immune
system to persistent infections. While vaccination with Gardasil is safe and highly effective
at generating antibody-based protective immunity from infection by the most common
hrHPV genotypes, it faces barriers to widespread adoption. First, delivery of Gardasil
requires cold-chain transport from sites of manufacture to vaccine clinics (145). This alone
prevents low- to middle-income countries, which account for the greatest incidence of
HPV-driven disease, from obtaining and distributing dosages to the population (146).
Additionally, there remains a growing resistance to vaccinations within developed
countries such as the United States which found new momentum after Andrew Wakefield
suggested a connection between the measles, mumps, and rubella vaccine and the
development of autism in children (147). Together these create difficult obstacles to
obtaining herd immunity. Brisson et al came to the conclusion that even with 90%
vaccination rates and drastically improved screening it would take over 100 years to
eliminate cervical cancer alone and suggested that no single method to combat HPV-
driven disease would be sufficient (148).
65
With that said, the long-term objective of this research is to develop a topically
applied compound containing θ-defensins that can be used to prevent initial infection by
HPV at target epithelial sites. This anti-viral method would not only provide the inherent
storage and stability that θ-defensins needed for delivery to low-vaccination countries, but
also provide a non-vaccination-based method to combat the transmission of HPV to at-
risk hosts. Within this study I have demonstrated that RTD-1 has the ability to prevent
multiple hrHPV genotypes in vitro at µM concentrations (chapter 2). In order to bridge
these in vitro findings, we must test the ability of RTD-1-mediated inhibition of HPV16 in
vivo.
To carry out these studies it will be necessary to implement an HPV vaginal
challenge model in immunocompetent mice and utilize a combination of in vivo imaging
paired with immunofluorescent imaging of histological sections of infected tissue to
assess relative levels of infection between treatment groups. I began work on this model
prior to the novel coronavirus-driven shutdown and am including a brief discussion of the
proposed work and experiments needed to move this project closer to translation.
Vaginal challenge optimization
The purpose of the HPV vaginal challenge model is to roughly replicate anogenital
infection with HPV and quickly assess infection rates. While the original published
procedure for this model proposes using BALB/c mice, follow up studies have found
success with C57BL/6 mice, showing this can be done in mice with a fully functional
immune systems (149, 150). Initial experiments need to focus on optimizing the vaginal
challenge procedure and titrating infectious units of HPV16 PsV so that a detectable
signal is obtained while not overloading the environment with viral particles. Mice should
66
be synchronized into estrous cycle by the subcutaneous injection of hydroxyprogesterone
several days before vaginal challenge with PsV as this will normalize the histology of
vaginal tissue for infection analysis. Ideally, a single PsV prep should be used for all mice
across experiments as this will minimize variation, and carbon methyl cellulose in
phosphate buffered saline should be used as the inoculum vehicle.
To successfully replicate chemical microwounding of the epithelium mice should
be pretreated briefly with 4% nonoxynol-9 (N-9, common ingredient in lubricants that acts
as a spermicide) prior to PsV instillation (151). 48h post infection, live imaging via an IVIS
Spectrum or similar device will allow infection assessment in live mice. Alternatively, if
this method does not work, mice should be sacrificed, reproductive tract harvested and
embedded in O.C.T. compound for sectioning and staining. Immunofluorescent imaging
from sections with DAPI counterstaining will allow infection assessments as well as any
changes to histology.

RTD-1 topical dose escalation
Once vaginal challenge methods are optimized, the same workflow should be used
to study HPV16 PsV pre-incubated with different concentrations of RTD-1. Given the
preliminary in vitro data it is expected that the higher the RTD-1 concentration, the more
effective the inhibition. Matched concentrations of RTD-1 without PsVs should be
included for controls to make sure that the N-9 induced wound healing of the vaginal tract
is not compromised. Immunofluorescent analysis of infection differences should also
include detection of capsid proteins using minor-capsid protein recognizing antibodies as
67
this will verify if virions/capsid clusters that failed to infect cells remain within the
environment.

Immunogenicity studies
A potential secondary outcome of RTD-1 treatments may be that neutralized
virions will be processed by resident immune cells and generate anti-HPV immunity,
conferring protection against viral rechallenge in addition to protecting from primary
infection. While it is clear that viral aggregates do not active human Langerhans cells
(chapter 2), the combination of inflammation from wound-healing and agitation created
by normal movement post viral challenge may be sufficient to induce maturation. To
assess this, the optimal dosage(s) from the RTD-1 concentration studies should be used
for initial challenge and rechallenge with non-RTD-1-treated HPV16 PsV should occur
14-21 days afterwards. Control groups would include HPV-naïve mice and mice pre-
vaccinated (i.p.) with HPV16 VLP (antibody generation control). Anti-HPV antibody
production would be assessed through serum collection pre and post challenge followed
by a previously developed ELISA (152).

Other θ-defensins that may be considered
The class of θ-defensins is not just limited to the ones tested in this study. Other
species such as olive baboons (Papio anubis) contain 4 different defensin genes which
make up to 10 different variants (117). Additionally, human retrocyclins could also be
investigated. Retrocyclins from humans show promise as they have inhibited infection by
HIV, HSV-1 and 2, as well as IAV. Once again, HPV has yet to be studied with
68
retrocyclins, which leaves another area for furthering this work. Optimization of RTD-1 by
single amino acid substitution and screening, as was done to create a more potent analog
against anthrax lethal factor protease, could also be explored (119). Finally, if future data
suggests certain θ-defensins are only effective against specific human pathogens,
combinations of different θ-defensins may be created to achieve a truly broad spectrum
anti-viral product.

Potential pitfalls during translation
There are two areas that may be pitfalls for the translation of this work: dysbiosis
of epithelial sites, and suppression of ongoing anti-viral responses. Both of these
concerns stem from the primary roles of defensins: maintaining physiological
homeostasis between naturally occurring bacterial and fungal flora while also working to
regulate immune responses (153). Dysbiosis occurs when there is a compromising of the
diversity, stability, resistance, and resilience of natural microbial flora that either results in
a bloom of pathology-associated microbes or loss of commensal colonies (154). Given
that previous work has specifically shown anti-microbial activity against pathogenic
bacteria, the concern is more about directly impacting the normal flora and allowing
opportunistic pathogens to take over in sites that were exposed to θ-defensin treatment.
Because of this, additional pre-translational studies in mice should also assess whether
there are changes to vaginal and gut microflora through 16S rRNA sequencing pre- and
post-treatments. If changes are found, modifications to the dosage/formulation of the
compound as well as the composition/usage of θ-defensin analogues should be the first
factors examined in follow-up experiments.
69
Systemic suppression of ongoing anti-viral immune responses is another potential
pitfall that is unlikely but should be kept in mind. RTD-1 has been used to mitigate
inflammatory cytokines in sepsis and systemic candidiasis models, which resulted in
improved survival, however the delivery methods involved daily i.p. or i.v. injection of 5
mg/kg RTD-1 (100, 101). Topical applications of compounds present the possibility of
transdermal delivery to blood stream, but that being recognized, whether systemic or local
θ-defensin treatment would even interfere with the generation of anti-viral immunity or
inhibit ongoing antiviral responses has yet to be explored.

70

Figure 13. Updated summary of anti-viral effects of different defensin classes on
both enveloped and non-enveloped viruses. This updated figure includes research
outlined in chapter 2.
Research Summary
Within this dissertation I have provided an up-to-date summary of HPV viral
uptake, defensin-mediated inhibition of viral infection in both enveloped and non-
enveloped viruses (Figure 13), characterized how the third class of defensins (q) can be
used to inhibit hrHPV infection, and have outlined future directions for this research so
that it may one day yield a broad spectrum antiviral product. The summaries and results
71
contained within this writing and other projects I’ve been part of represent steps towards
truly understanding HPV biology, associated diseases, and gives insight into how one
day we may be able to just spread love and not HPV.
72
Chapter 4. Other projects and publications completed during PhD
work

Publications by year enrolled in PhD program

Year 1 (2015-2016)

1. Skeate JG, Porras TB, Woodham AW, Jang JK, Taylor JR, Brand HE, Kelly TJ,
Jung JU, Da Silva DM, Yuan W, Kast WM (2015). Herpes Simples Virus
downregulation of secretory leukocyte protease inhibitor enhances human
papillomavirus type 16 infection. Journal of General Virology. 97(2):422-34

2. Da Silva DM, Woodham AW, Skeate JG, Rijkee LK, Taylor JR, Brand HE,
Muderspach LA Roman LA, Yessaian AR, Pham H, Matsou K, Linn Y, McKee
GM, Salazar AM, Kast WM (2015). Langerhans cells from women with cervical
precancerous lesions become functionally responsive against human
papillomavirus after activation with stabilized Poly-I:C. Journal of Clinical
Immunology. 161(2):197-208

3. Da Silva DM, Woodham AW, Rijkee LK, Skeate JG, Taylor JR, Koopman ME,
Brand HE, Wong M, McKee GM, Salazar AM, Kast WM (2015). Human
73
papillomavirus-exposed Langerhans cells are activated by stabilized Poly-I:C.
Papillomavirus Research. 1:12-21

4. Woodham AW, Yan L, Skeate JG, van der Veen D, Brand HE, Wong MK, Da
Silva DM, Kast WM (2016). T cell ignorance is bliss: T cells are not tolerized by
Langerhans cells presenting human papillomavirus antigens in the absence of
costimulation. Papillomavirus Research. 2:21-30

Year 2 (2016-2017)

5. Skeate JG, Woodham AW, Einstein MH, Da Silva DM, Kast WM (2016).
Current therapeutic vaccination and immunotherapy strategies for HPV-related
diseases. Human Vaccines and Immunotherapies. 12(6):1418-29

6. Woodham AW, Sanna AM, Taylor JR, Skeate JG, Da Silva DM, Kast WM
(2016). Annexin A2 antibodies but not inhibitors of the annexin A2
heterotetramer impair productive HIV-1 infection of macrophages in vitro.
Virology Journal. 13(1):187

7. Wu BW, Graff R, Badash I, Skeate JG, Lane C, Mansour I, Rao R, Yi A,
Marriman J, Hatch III GF, Dorr L, Gilbert P, Schroeder ET (2017). The effect of
tourniquet use during total knee arthroplasty on global cytokine changes
74
associated with ischemia reperfusion injury. Journal of Applied Physiology.
2(1):1-10

Year 3 (2017-2018)

8. Skeate JG, Da Silva DM, Chavez-Juan E, Anand S, Nuccitelli R, Kast WM
(2018). Nano-Pulse Stimulation induces immunogenic cell death in human
papillomavirus-transformed tumors and initiates an adaptive immune response.
PLOS ONE. 13(1): e0191311

9. Woodham AW, Cheloha RW, Ling J, Rashidian M, Kolifrath SC, Mesyngier M,
Duarte JN, Bader JM, Skeate JG, Da Silva DM, Kast WM, Ploegh HL (2018).
Nanobody-antigen conjugates elicit HPV-specific anti-tumor immune
responses. Cancer Immunology Research. 6(7): 870-880

10. Taylor JR, Fernandez DJ, Thornton SM, Skeate JG, Lühen KP, Da Silva DM,
Langen R, Kast WM (2018). Heterotetrameric annexin A2/S100A10 (A2t) is
essential for oncogenic human papillomavirus trafficking and capsid
disassembly, and protects virions from lysosomal degradation. Scientific
Reports. 8(1): 116420

11. Taylor JR, Skeate JG, Kast WM (2018). Annexin A2 in virus infection.
Frontiers in Microbiology. 9:2954
75

Year 4 (2018-2019)

12. Da Silva DM, Skeate JG, Chavez-Juan E, Lühen KP, Wu JM, Wu CM, Kast
WM, KinKai H (2019). Therapeutic efficacy of human pappilomavirus type 16
E7 bacterial exotoxin fusion protein adjuvanted with CpG or GPI-0100 in a
preclinical mouse model for HPV-associated disease. Vaccine. 37(22): 2915-
2924

Year 5 (2019-2020)

13. Skeate JG, Segerink W, Garcia M, Fernendez DJ, Prins RP, Lühen KP, Voss
FO, Da Silva DM, Kast WM. Theta-defensins inhibit high-risk human
papillomavirus through charge-driven capsid clustering. Frontiers in
Immunology. Accepted August 2020.

14. Skeate JG, Otsmaa ME, Prins R, Fernendez DJ, Da Silva DM, Kast WM
(2020). TNFSF14: LIGHTing the way for effective cancer immunotherapy.
Frontiers in Immunology. 11:922

15. Thornton SM, Samararatne VD, Skeate JG, Buser C, Lühen KP, Taylor JR, Da
Silva DM, Kast WM (2020). The essential role for anxA2 in Birbeck Granules
formation. Cells. 9(4): 974
76

16. Da Silva DM, Enserro DM, Mayadev J, Skeate JG, Matsuo K, Pham H, Lankes
HA, Moxley K, Ghamande S, Lin TG, Schilder RJ, Birrer MJ, Kast WM. Immune
activation in patients with locally advanced cervical cancer trated with
ipilimumab following definitive chemoradiation (GOG 9929). Clinical Cancer
Research. Accepted July 2020.

77
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Abstract (if available)

Abstract Human papillomavirus (HPV) continues to be the most prominent sexually transmitted virus in humans. It is estimated that over 80% of all sexually active individuals will become infected with HPV within their lifetimes. While the majority of these infections will be cleared naturally and not progress to advance-stage disease, persistent infection with high-risk human papillomavirus (hrHPV) genotype results in the development of epithelial cancers in the skin and mucosa. While the previous paradigm focused on cervical cancers in women, there now is an increasing incidence of HPV-driven head and neck cancers occurring in men, highlighting the continued importance to study and understand the spectrum of HPV-driven disease. The most common hrHPV genotype, HPV16, is frequently used to study all aspects of the HPV viral life cycle from viral entry, propagation, immune evasion, to carcinogenic transformation of host cells and resulting cancer phenotypes. Although prophylactic vaccination exists and coverage has improved in the United States, there still remains significant hurdles to achieving herd immunity through vaccination strategies

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Asset Metadata

Creator Skeate, Joseph Gary (author)

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

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Medical Biology

Publication Date 09/18/2020

Defense Date 07/17/2020

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

Tag alpha-defensins,human papillomavirus,infection,innate-immunology,OAI-PMH Harvest,sexually transmitted infection (STI),theta-defensins

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Ouellette, Andre (committee chair), Akbari, Omid (committee member), Kast, Martin (committee member)

Creator Email skeate@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-373364

Unique identifier UC11666312

Identifier etd-SkeateJose-8974.pdf (filename),usctheses-c89-373364 (legacy record id)

Legacy Identifier etd-SkeateJose-8974.pdf

Dmrecord 373364

Document Type Dissertation

Rights Skeate, Joseph Gary

Type texts

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...

Repository Name University of Southern California Digital Library

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