University of Southern California Dissertations and Theses

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

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

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Content Copyright 2023 Emma Antoinette Martinez
INVESTIGATION INTO THE USE OF REPURPOSED INFLUENZA VACCINES FOR IMMUNOTHERAPY
OF HPV-INDUCED TUMORS
by
Emma Antoinette Martinez
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
MOLECULAR MICROBIOLOGY AND IMMUNOLOGY
2023 May

ii
APPENDIX B

Table of Contents
ACKNOWLEDGMENTS ..................................................................................................................................... III
LIST OF TABLES ............................................................................................................................................... IV
LIST OF FIGURES ...............................................................................................................................................V
ABSTRACT ....................................................................................................................................................... VI
CHAPTER 1: INTRODUCTION ............................................................................................................................. 1
1.1 CANCER .................................................................................................................................................................. 1
1.2 HUMAN PAPILLOMAVIRUS......................................................................................................................................... 2
1.3 HPV VACCINATIONS................................................................................................................................................. 8
1.4 IMMUNOTHERAPIES.................................................................................................................................................. 9
1.5 TUMOR MICROENVIRONMENT ................................................................................................................................. 10
1.6 T-CELL MEMORY .................................................................................................................................................... 11
1.7 DEVELOPMENT OF C3.43 HPV16 TUMOR CELLS ......................................................................................................... 12
CHAPTER 2: BACKGROUND STUDIES ON REPURPOSING VIRAL VACCINES TO STIMULATE TUMOR IMMUNITY .... 12
2.1 IMMUNOLOGICALLY COLD TUMORS VERSUS HOT TUMORS ............................................................................................. 12
CHAPTER 3: SPECIFIC AIMS AND HYPOTHESIS .................................................................................................. 17
3.1 HYPOTHESIS .......................................................................................................................................................... 17
3.2 SPECIFIC AIMS ....................................................................................................................................................... 18
CHAPTER 4: METHODS AND MATERIALS ......................................................................................................... 21
4.1 MICE AND CELL LINES ............................................................................................................................................. 21
4.6 ENZYME-LINKED IMMUNOSPOT ASSAY (ELISPOT)....................................................................................................... 24
CHAPTER 5: RESULTS ...................................................................................................................................... 26
CHAPTER 6: DISCUSSION ................................................................................................................................ 41
CHAPTER 7: CONCLUSION ............................................................................................................................... 46
REFERENCES .................................................................................................................................................. 48

iii
Acknowledgments

First and foremost, I would like to thank my research advisor and thesis committee chair, Dr.
Martin Kast, whose assistance and dedication to every step through the process have made this
thesis possible. Thank you for the last two years and your undying support.

I would also like to my fellow research thesis committee members, Dr. Weiming Yuan and Dr.
Peter Mullen. Dr. Weiming Yuan, one of my first-year professors at the University of Southern
California, introduced me to my current research advisor in Recent Microbiology Advances. His
energetic and uplifting teaching style was infectious and will always be remembered.
Furthermore, during the initial planning phase of my project, Dr. Peter Mullen offered
invaluable insight into the metabolism of cancer and possible assays I could use. Finally, I want
to thank Dr. Diane Da Silva, whose endless patience and excellent teaching style made
collaboration easy.

In August 2016, I attended California State University, Fullerton, where Dr. Alison Miyamoto
gave me my first exposure to the research world. My time at California State University,
Fullerton, was highly productive, and working with Dr. Alison Miyamoto was such an
enlightening experience. So many of the skills I brought forth with me were honed in her lab,
and I am eternally grateful for that.

Getting through my thesis required more than just academic support, and I have many people I
would like to thank for listening and, at times, tolerating my absence over the past two years. I
cannot express my gratitude and unwavering support for their friendship. Likewise, Kevin
Somoza, Stephanie Hernandez, Rachel Mulondo, and Stephanie Wong have tirelessly supported
me at USC.

More importantly, none of this could have happened without the support of my family. To my
mother, who continually offered her words of encouragement, especially when I felt it was an
insurmountable obstacle. To my father, thank you for listening to me ramble and providing
advice during our many late-night talks; to my cousin, Sylvia Martinez, who continued to make
me laugh during our many lengthy phone calls and who shares the same humor as me. Finally, I
would like to thank my sister and my brother, who, in the end, pushed me to finish this project.
Thank you!

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List of Tables

TABLE 1: SURVIVAL OF MICE RECEIVING DIFFERENT PRIME-BOOST REGIMENS. MICE WERE CHALLENGED WITH C3.43 TUMOR CELLS TEN
DAYS AFTER THE LAST VACCINATION. LOG-RANK TEST, UNVACCINATED MICE WERE USED AS CONTROL. ..................................... 37

v
List of Figures

FIGURE 1 HALLMARK OF CANCER WITH UPDATED CHARACTERISTIC TRAITS IN 2022. ( FROM HANAHAN, 2022) ................................... 2
FIGURE 2 HUMAN PAPILLOMAVIRUS GENOMICS. ( FROM PORTER, V.L. & MARRA, M.A., 2022) ..................................................... 3
FIGURE 3: INTRATUMORAL INJECTION OF SEASONAL INFLUENZA VACCINE RESULTS IN A REDUCTION OF TUMOR GROWTH. (MODIFIED FROM
NEWMAN ET AL., 2020) ........................................................................................................................................... 14

FIGURE 4: I.T. INJECTION OF SEASONAL INFLUENZA VACCINE ELICITS AN INCREASE IN ANTIGEN-PRESENTING CELLS AND TUMOR-SPECIFIC
CD8
+
T-CELL POPULATION. (MODIFIED FROM NEWMAN ET AL., 2020) ............................................................................ 14

FIGURE 5: SEASONAL ADJUVANTED INFLUENZA VACCINE FAILS TO REDUCE MELANOMA TUMORS COMPARED TO NAÏVE MICE.
(MODIFIED FROM NEWMAN ET AL., 2020). ................................................................................................................. 16

FIGURE 6: ILLUSTRATION OF SCHEDULED PRIME-BOOST INTERVALS.( FROMDA SILVA, D., MARTINEZ, E., ET.AL, 2022) ....................... 23
FIGURE 7: MICE CHALLENGED WITH 1 X 10
5
B16-F10 MELANOMA TUMOR CELLS OR C3.43 CELLS WERE TREATED WITH PBS (CONTROL)
OR 2021-2022 UNADJUVANTED INFLUENZA VACCINE (N=40, 10/GROUP). CREATED WITH BIORENDER. ................................ 28

FIGURE 8: MICE CHALLENGED WITH 1 X 10
5
C3.43 HPV 16 CELL LINE WERE TREATED WITH PBS (CONTROL) AND HPV 16 L1 VLP
(N=16, 8/GROUP). . ................................................................................................................................................ 31

FIGURE 9: INTRATUMORAL INJECTION OF PFIZER’S COVID-19 MRNA VACCINE FAILS TO REDUCE HPV-INDUCED TUMORS IN VIVO (N=20,
10/GROUP).. .......................................................................................................................................................... 32

FIGURE 10: ANALYSIS OF DIFFERENT PRIME-BOOST INTERVALS ON T-CELL RESPONSE.( FROM DA SILVA, D., MARTINEZ, E., ET AL., 2022)
............................................................................................................................................................................ 35
FIGURE 11: ENHANCED MEMORY RECALL AND DIFFERENTIAL INDUCTION OF MEMORY T-CELL PHENOTYPES CORRESPONDING WITH
LONGER PRIME-BOOST INTERVALS POST-IMMUNIZATION. ( FROM DA SILVA, D., MARTINEZ, E., ET AL, 2022) ........................ 38

FIGURE 12: LONGER PBIS PRODUCE A GREATER QUANTITY OF EFFECTOR MEMORY T-CELLS. ( FROM DA SILVA, D., MARTINEZ, E., ET AL,
2022) ................................................................................................................................................................... 39
FIGURE 13: PRIME-BOOST INTERVALS DO NOT IMPACT THE EFFICACY OF VRP-BASED THERAPEUTIC VACCINES. ( FROM DA SILVA, D.,
MARTINEZ, E., ET AL, 2022) ..................................................................................................................................... 41

vi
Abstract

The progression of cervical cancer from persistent Human Papillomavirus (HPV)
infection remains a global health concern. The standard of care for cervical cancer is surgical
resection, chemotherapy, or radiation, which is suitable for patients in the early stages of
cancer. However, developing a treatment for recurrent, persistent, and metastatic cervical
carcinoma remains challenging. Therefore, the investigating and targeting of the tumor
microenvironment with various immunotherapies in the hopes of converting immunologically
cold tumors to hot tumors have been at the forefront of research. Newman et al. hypothesized
that repurposing a seasonal Influenza vaccine and injecting it into the tumor could elicit a
proinflammatory immune response, thus overcoming the highly immunosuppressive tumor
microenvironment seen in HPV-induced tumors. However, in this thesis, the data suggests that
this conversion from an immunologically cold tumor to a hot tumor does not occur with the
intratumoral injection of a repurposed Influenza vaccine. Instead, tumor reduction and
complete regression are dependent on the length of time between the priming and boosting
(PBIs) of a therapeutic vaccine in tumor models. This study therefore also used an HPV16E7E6
Venezuelan equine encephalitis virus replicon particle (VRP) vaccine to determine the effects of
six different PBIs in an HPV model. Analysis of the data demonstrates that sufficient HPV 16 E7-
specific CD8
+
T-cells formation and proliferation was achieved with a minimum of a one-week
prime-boost interval. Furthermore, longer PBIs resulted in higher differentiation of memory T-
cells and increased cytotoxicity. Our studies suggested a novel approach to apply therapeutic
vaccines to tumor microenvironment and stimulate effective immunotherapies for cervical
cancer.

1
Chapter 1: Introduction

1.1 Cancer

Cancer is one of the leading causes of death and is considered a significant public health
issue globally [1,2]. The increase in incidence and mortality rates has seemingly corresponded
with the socioeconomics of developing countries where disease burden is more prevalent [2].
This is evident as half of all cancer cases, and 58.3% of cancer deaths are estimated to occur in
Asia compared to 20.9% of incidence rates and 14.2% of mortality rates in America [2].
According to the World Health Organization in 2019, cancer was considered the 1st or 2nd
cause of death before 70 years old in 112 of 183 countries [1]. These rates are estimated to
increase in 2022, with 1,918 030 new cancer cases diagnosed, leading to about 5,250 new cases
each day [2]. The commonality of the occurrence of this disease requires novel techniques and
therapies to conquer it.
Cancer is defined as a disease of mutations within somatic cells that elicit uncontrolled
proliferation with the possibility of metastasis. Decades ago, scientists had collectively
characterized cancer with six distinct hallmarks. More recently, two additional hallmarks were
added and are comprised of: the ability to sustain proliferative signaling, evading growth
suppressors, avoiding immune destruction, enabling replicative immortality, tumor-promoting
inflammation, activate invasion and metastasis, inducing or accessing vasculature, genome
instability, and mutation, resisting cell death, and deregulating cellular metabolism (seen in
Figure 1) [3]. These hallmarks provided targets that scientists and physicians could target for
novel therapies. However, recent studies have suggested that tumor and tumor

2
microenvironment heterogeneity significantly augment the antitumor effects seen in standard
drug therapy or immunotherapies. In addition, virus-induced carcinogenesis, such as seen with
the human papillomavirus, also needs to be considered, for persistent infection with an
oncogenic virus could sustain cancer and metastasis despite antitumor therapeutics.

Figure 1 Hallmark of Cancer with updated characteristic traits in 2022. Left- The classical hallmarks of cancer were
proposed in 2000, with the addition of two emerging hallmarks discovered in 2011. ( From Hanahan, 2022)

1.2 Human Papillomavirus

Human papillomavirus, or HPV, are small, nonenveloped, double-stranded DNA viruses
that primarily infect stratified epithelium in cutaneous and mucosa tissues. These
epitheliotropic viral particles are about 50-55nm in size and are characterized by their 72
pentameric capsomers in the viral capsid. HPV contains a circular genome of about 8kbps[4-6].
One strand of HPV DNA contains eight open reading frames or ORFs. The ORFs are categorized
into the early, late, and long control regions [6]. The early region encodes for six regulatory
proteins, E1-E2 and E4-E7 (Figure 2). The first protein, E1, is a DNA helicase, an enzyme needed
to uncoil the double-stranded DNA into single-stranded DNA, which is necessary for viral

3
transcription through the hosts’ cellular machinery [7]. The second early protein, E2, is another
factor that recognizes the viral origin of replication and is the primary regulator of the viral gene
transcription [6]. E2 can act as an accessory protein, helping the E1 protein find the origin of
replication and uncoil HPV’s double-stranded DNA. E2 also is present in the equal distribution
of chromatins during mitosis of the epithelia by binding to the mitotic spindles [8]. The E4
protein can induce arrest between the G2 and M phases of the cell cycle and help facilitate the
amplification of HPV’s oncogenes, E6 and E7 [9]. Recent studies of HPV’s E5 protein have
illustrated that it helps with the oncogenicity of the virus. Ren et al. has shown that HPV16’s E5
can collaborate with E7 in the cellular transformation of epithelia and the immune suppression
seen in these viral infections [9]. The final two proteins located in the early region of the
genome is the most crucial for persistent infection and the oncogenicity of HPV.

Figure 2 Human papillomavirus genomics. HPV is a small uneveloped double-stranded DNA virus that can infect
stratified epithelium in cutaneous and mucosal tissues. The genomics of HPV is categorized into early and late
regions depending on the timing of replication and assembly of HPV viral particles. There are six early genes (E1-E2,
E4-E7), each one important for the replication of HPV in host cells. The two late genes, L1 and L2, are primarily
targeted by prophylactic vaccines and commonly encode for the structural proteins responsible for the maturation
and assembly of HPV’s capsid ( From Porter, V.L. & Marra, M.A., 2022)

4
The first of two oncoproteins encoded by HPV’s genome is the E6 protein. The E6
protein is about 151 amino acids in length and contains two zinc finger domains [7]. As
mentioned previously, one hallmark of cancer is the ability to have transformed cells
immortalized. However, studies have shown that E6 alone cannot induce the immortalization of
primary foreskin keratinocytes but can elicit the immortalization of human mammary epithelial
cells [7]. This suggests that E6’s role is defined by the nature of the cells that HPV infects.
E6 is a necessary protein that helps transform the cellular environment suitable for viral
replication. It achieves this by degrading p53, a protein required for the apoptosis of cells with
aberrant DNA replication. E6 degrades p53 by binding to E6-associated protein (E6-AP), a
ubiquitin ligase. Another way E6 augments the cellular environment to make it adequate for
viral replication is by altering the transcription of cellular genes by binding p300 [7]. Recent
studies have also shown that E6 interacts with cellular telomerase, an enzyme that shortens
linear chromosomes and maintains a telomeric DNA [4,6,7]. When a cell replicates, telomerase
shortens the linear chromosomes, effectively limiting the number of replications somatic cells
can endure. When the linear chromosomes become too compressed, the cell stops replicating
and enters a senescence phase. E6 effectively prevents the shortening of the telomeric DNA,
allowing for uncontrolled proliferation. This effect seems cell-specific, in which specific cells
cannot be immortalized with E6 alone and requires a second hit to the host genome via HPV’s
second oncoprotein, E7.
The E7 protein is the primary way HPV infection immortalizes human epithelial cells. E7
is commonly known to bind to retinoblastoma (Rb) in high-risk HPV genotypes [4]. The
retinoblastoma protein is a significant regulator of cell cycle progression from the G1 phase to

5
the S phase. Rb achieves this by inhibiting E2F transcription factors, which encode for cyclins A
and E. Cyclins A and E bind to cyclin-dependent kinase 2 (CDK2), promoting the cell cycle's
progression from the G1 phase to the S phase [4,6]. HPV E7 maintains Rb’s phosphorylated
state, allowing the continuous and unregulated passage of the cell cycle, which in turn
increases the ability of the cell to proliferate and generate viral genomes [4]. A secondary
response to inhibit Rb, E7 binds to retinoblastoma family members, Rb1, RBL1, and RBL2,
known as pocket proteins, and tags them for degradation [5]. This effectively allows the release
of E2F. Once the viral genome is replicated and proteins expressed, assembly and maturation
are needed before the departure of the viral particles.
The late region of HPV’s ORFs encompasses genes that encode for two structural
proteins, L1 and L2. HPV icosahedral capsids contain 360 copies of the L1, arranged in the
characteristic 72 pentamers with a single L2 protein in the center [4]. These structural proteins
are the last to be expressed, primarily responsible for the virus's assembly, maturation, and
release to the outermost epithelial layer [4]. The L1 ORF is one of the most conserved regions of
the genome between HPV genotypes, which is helpful in the development of prophylactics and
helps identify HPV isolates in biopsies and culture [6].
Papillomaviruses have coevolved with their hosts, and it is estimated that they shared
an ancestor about 423 million years ago [10]. They are species-specific and have been identified
in reptiles, mammals, birds, and fish [10]. These ancient papillomaviruses shared a conserved
exoskeleton consisting of E1, E2, L1, and L2 proteins [10]. The arrival of E5, E6, and E7
oncoproteins propagated papillomaviruses into their primate hosts about 6-8 million years ago
[10]. With the onset of genotyping and the advent of next-generation sequencing, more than

6
200 genotypes of HPV have been identified. To achieve this, researchers created a regulation
for the taxonomy of HPV genotypes based on the conserved L1 capsid protein sequence.
Studies indicate a new genotype is identified if the L1 sequences differ at least 10% from any
previously identified genotype [4-6,10]. This percent difference in the L1 sequences also
conferred a 10% difference in genomic DNA between genotypes. These differences led to the
classification of human papillomaviruses such as the alpha-papillomaviruses, beta-
papillomaviruses, and gamma-papillomaviruses [10].
The alpha-papillomaviruses are characterized by inflicting mucosal and cutaneous
lesions in humans and primates. Most high-risk and low-risk HPV serotypes are founded in this
genus of papillomaviruses [6,10,11]. Within this genus, the HPV genotypes contain a conserved
E5 open-reading frame of about 300-500 bp within the early region of the genome [6,10,11]. This
genus is responsible for HPV’s ability to immortalize human keratinocytes. Beta-
papillomaviruses are classified by their ability to cause cutaneous lesions in humans.
In contrast to the alpha-papillomavirus genus, HPV genotypes in the beta-papillomaviruses are
found to be in the latent stage of infection within the human population. These genotypes are
only activated when an individual is immunosuppressed [6,10,11]. These genotypes are also
classified into this genus when the early region of the genome is less than 100 nucleotides, and
the E5 ORF is missing. The gamma-papillomavirus genus is the last clinically relevant genus that
infects the human population. The genotypes within this genus cause cutaneous lesions in
humans and are histologically distinguishable by the intracytoplasmic inclusions compared to
beta-papillomaviruses. Classification of genotypes into this genus consists of HPV genomes with
an early region less than 100 nucleotides long. Similar to the beta-papillomaviruses, gamma-

7
papillomaviruses have an E5 ORF absent in their genomes [6]. This study will focus on the alpha-
papillomaviruses, which contain high-risk HPV genotypes.
High-risk human papillomaviruses (HR-HPV) are the causative agent of cervical, penile,
vulvar, vaginal, anal, and oropharyngeal cancers. The genotypes in this classification are HPV
16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66, and 68. HPV 16 and 18 cause 55% and 15% of all
cervical cancers, respectively. In contrast, low-risk HPV genotypes are responsible for benign
growths, such as genital warts. While these genotypes do not cause carcinogenesis, they are
commonly treated as a cosmetic nuisance [12].
Cervical cancer is the fourth most common cancer in women worldwide [12,13]. As
previously mentioned, HPV 16 and 18 are responsible for 55% and 15% of cervical cancer cases
worldwide, respectively. In 2018, approximately 569,847 new cases of cervical cancer were
diagnosed, and 311,365 deaths occurred worldwide [13]. In 2020, an estimated 630,000 cancers
were attributable to HPV, including 570,000 cancers in women [14]. HPV has been identified in
89% of low-grade squamous intraepithelial lesions (LSIL) and more than 90% of high-grade
squamous intraepithelial lesions (HSIL) [10]. HPV lesions do not initially begin as cancer; it is a
sustained persistance of infection that leads to cancer.
The progression of HPV infection to cancer depends on the infection persisting and the
evasion of the immune system. HPV infects basal cells within stratified epithelial found in
cutaneous and mucosa tissues [4,5,7,15-17]. The virus enters the stratified epithelium through
microtears produced during sexual activity. Once within the stratified epithelium, it will
migrate down to the basement membrane, where basal cells are present. It is these cells that
allow the virus to replicate due to these cells constantly differentiating. As these cells progress

8
through the cell cycle, the oncoproteins E6 and E7 will be expressed. As mentioned, E6 and E7
proteins can push the basal cells through the cell cycle by degrading or inhibiting tumor-
suppressing proteins, p53 and Rb. As these cells migrate to the surface, integration of the viral
genome is incorporated into the host genome, leaving the assembly and production of L1 and
L2 capsid proteins to occur near the surface. Once the cell is at the surface, the newly replicated
viral particles will exit the cells, waiting for another individual to infect. Due to the prevalence
of HPV-induced cancers within the global population, a way to prevent the disease was needed.
1.3 HPV Vaccinations

In the year 2006, two biotech companies released prophylactic vaccines.
GlaxoSmithKline released a bivalent preventive vaccine, Cervarix, that targeted HPV16 and
HPV18 L1 proteins, which was popular in the European Union. In the United States, Merck
released a quadrivalent prophylactic vaccine, Gardasil, that targeted two HR-HPV genotypes,
HPV 16 and 18, and two LR-HPV genotypes, HPV 6 and 11 L1 proteins. In 2014, Merck released
an updated prophylactic vaccine called Gardasil 9. This vaccine incorporates the four genotypes
seen in the original vaccine but includes 5 additional HR-HPV genotypes: 31,33,45,52 and 58.
Both Gardasil and Gardasil 9 utilize the differences in genetic sequences of the L1 proteins in
the different genotypes of HPV as targets for antibody production. These complex multivariant
vaccines remain extremely expensive to procure and implement into national vaccine programs
[18]. To combat this rising cost of vaccines, many countries are researching the development of
a broad-spectrum HPV prophylactic vaccine by targeting a more conserved capsid protein L2.
However, due to its location within the capsid and its low immunogenicity, multiple doses of
the vaccine are needed to reach comparable titer levels seen with the multivariant L1 VLP

9
vaccines [18]. Furthermore, persistent infection with HPV requires decades (10-25 years) before
progressing to cancer, making it difficult to measure both Gardasil and Gardasil 9’s efficacy [19].
Regardless of its availability, vaccination rates remain low, allowing incidence rates to increase.
There are many reasons why vaccination rates for the HPV prophylactic vaccines are
low. The COVID-19 pandemic has impacted vaccination initiation and completion in the last two
years due to stay-at-home orders [20]. This pandemic emphasized prior hindrances to
vaccination uptake, including socioeconomic disparities in access to healthcare, national
budgets for low-income countries, parents' education status, physicians' recommendations,
vaccine safety concerns, and marketing tactics for the prophylactic vaccines. Low uptake in HPV
vaccination was seen primarily in the US Southern states compared to the Northeastern states
[20]. The pandemic also illustrated statistical associations between vaccination initiation and
completion between the sexes of adolescents. It was suggested that the cause of these
differences was biotechnology companies marketing the HPV vaccine as a preventive for
cervical cancer, thus determining it as a “woman’s disease vaccine” [20]. Regardless of the type
of barriers to immunization, low uptake of the HPV vaccine in adolescents allows the disease to
continue to propagate throughout the population, prompting research into future
immunotherapies to conquer HPV-induced cancers.
1.4 Immunotherapies

For many years the standard of care for cancer was surgery, radiation and chemotherapy for all
cancers. However, in recent years, there has been a push for optimizing care for solid tumors
due to their ability to resist chemotherapies and radiation. One direction physicians and
scientists researched was the use of immunotherapies for these solid tumors. The first case of

10
immunotherapies was the development of monoclonal antibodies [21]. Though monoclonal
antibodies seem to revolutionize the treatment of blood-based cancers, such as leukemias, they
failed to cross the threshold of being effective in solid tumors [21]. The next revolutionary
immunotherapies developed were the checkpoint blockade inhibitors such as PD-1/PDL-1 and
CTLA-4. The checkpoint blockade, PD-1 is expressed on tumor-infiltrating T cells (TILs) to help
the immune system maintain self-tolerance, while its ligand is found on the tumor itself [22].
Anti-PD-1has been proven effective in a myriad of cancers consisting of both hematologic
malignancies and solid tumors, such as Hodgkin’s lymphoma, head and neck squamous cell
carcinoma, and melanoma [23]. Unfortunately, this checkpoint blockade is overexpressed on
CD8
+
T cells, which allows the propagation of cancers [22]. The other checkpoint blockade, CTLA-
4, expressed primarily on regulatory T cells, is another way for the immune system to maintain
its self-tolerance [22]. Studies have implicated dysregulation of CTLA-4 as the cause for several
autoimmunity disorders found in the lungs, heart, and eyes [24]. Anti-CTLA-4 drugs have been
used as a source of immunotherapy in melanoma, small cell lung cancer, and prostate to name
a few [24]. However, within the last decade, the role of the tumor microenvironment was
determined to play a significant role in immunotherapy resistance, as seen in these solid
tumors.
1.5 Tumor Microenvironment

As mentioned, the tumor microenvironment has been proven to be a determining factor
in the poor prognosis of HPV-associated cancers. This is due to the highly immunosuppressive
nature of the microenvironment. The immunosuppressive environment is a byproduct of a
higher level of immunosuppressive cell types found within, such as regulatory T cells (T regs),

11
regulatory B cells (B regs), myeloid-derived suppressor cells (MDSCs), and tumor-associated
macrophages (TAMs). These immunoregulating cells release immunosuppressive cytokines that
prevent the immune system from recognizing atypical cell proliferation. These cytokines, such
as interleukin-10 (IL-10), interleukin-6 (IL-6), interleukin-4 (IL-4), and TGF-ß, can neutralize
proinflammatory cytokines, such as IFN , interleukin-23 (IL-23), and interleukin-2 (IL-2) by
limiting their expression in the microenvironment [11,25]. TAMs are a more recent find in the
ability of HPV to suppress the immune system. TAMS are polarized into M1 TAMs, which
release proinflammatory cytokines, or M2 TAMs, which are immunosuppressive [11,25]. This
polarization is guided under the cues given by T-helper cells 1 (Th1) and T-helper cells 2 (Th2)
[11,25]. The number of immunoregulating cells found within the tumor microenvironment
correlates to the progression of cervical intraepithelial neoplasia into cervical carcinoma. These
cells also help scientists to discern whether the tumor is “cold,” or immunosuppressed, or
“hot,” where the tumor has proinflammatory cytokines dispersed within it [26]. A cold tumor is
harder to treat once established than a hot tumor, making developing a therapeutic vaccine
incredibly challenging. Therefore, therapeutic vaccines have been pushed to elicit memory T
cells to eradicate already established tumors in recent years.
1.6 T-cell memory

During prophylactic immunization, a piece of a virus is injected into the body, prompting
antibodies' differentiation and proliferation from naïve B cells. These antibodies provide
immune memory of antigens, thus allowing a quicker response in the event of repeated
exposure. However, in developing therapeutic vaccines for virally induced cancer, T-cell
activation to eradicate these infected cells is what biotechnological companies aim for [27-30].

12
Nonetheless, the effector T-cells (T eff), which have the most potent cytotoxic potential for
tumor cells, have a short-term effect. To combat this, a subset of T-cells called memory T-cells
determine the efficacy of the therapeutic vaccine [30]. The two main subtypes of T-cells are
central memory T-cells and effector memory T-cells. Central memory T-cells (T cm) are found
mainly in the lymph nodes. They are usually the first to become activated and differentiate into
effector T-cells that help combat antigens [30]. In contrast, effector memory T-cells (T em) are
found in the periphery of the human body but have a limited proliferative capability. However,
these cells can produce effector cytokines in response to the presence of antigens [30].

1.7 Development of C3.43 HPV16 tumor cells

The cell line, C3.43, is a derivative of the HPV 16 C3 line, which was transformed by a
pRSVneo-derived plasmid consisting of the HPV16 cell line [31]. This cell line expresses a murine
H-2Db-bound peptide, E7 49-57 [31]. This is important as T lymphocytes can recognize this peptide
and eradicate these transformed cells in vivo
Chapter 2: Background studies on repurposing viral vaccines to stimulate
tumor immunity

2.1 Immunologically cold tumors versus hot tumors

In 2020, Newman et al. published an article in the PNAS that illustrated the use of an
intratumorally injected repurposed influenza vaccine as an immunotherapy in B16-F10
melanoma tumors. The basis of their theory was that an immunologically hot tumor or
proinflammatory tumor microenvironment correlated with a better prognosis versus an
immunologically cold tumor or an immunosuppressive tumor microenvironment. Many

13
patients in the clinical settings display solid tumors with an immunosuppressive tumor
microenvironment due to a higher infiltration rate of T regs, B regs, and MDSCs and a low number
of CD8
+
T cell infiltrates. Newman argued that the tumor microenvironment could be targeted
via the injection of vaccine antigens to increase the infiltration of proinflammatory immune
cells, thus outcompeting immunosuppressive immune infiltrates [26].
Newman et al. hypothesized that a commercially manufactured seasonal Influenza
vaccine could be repurposed as an immunotherapy treatment for skin-based melanoma [26]. A
2017-2018 unadjuvanted seasonal Influenza vaccine (FluVx) resulted in a decrease in tumor
growth compared to naïve mice (Figure 3). Harvested tumors elucidated an increased T cell: B
cell ratio, demonstrating that a higher number of CD8
+
T cells was necessary for tumor
reduction. The tumors also showed an increase of both DCs and CD3
+
CD8
+
T cells in tumors
treated with FluVx compared to naïve mice (Figure 4E). Flow cytometry stained the
lymphocytes to determine which CD8
+
T-cells were specific to the B16-F10 melanoma by
reacting to the gp100 dextramer (Figure 4F).

14

Figure 3: Intratumoral injection of seasonal Influenza vaccine results in a reduction of tumor growth. A) A schematic
of experimental design. B) Tumors measured manually with calipers demonstrated a significant reduction of tumor
volume when injected with FluVx intratumorally compared with PBS (Modified from Newman et al., 2020)

Figure 4: I.T. injection of seasonal Influenza vaccine elicits an increase in antigen-presenting cells and tumor-specific
CD8
+
T-cell population. E) AN increase in DC presentation and tumor-specific CD8
+
T-cells in tumors treated with
FluVx compared to PBS F) An increase in CD3
+
CD8
+
T-cells compared to CD3+ lymphocytes. The ratio increase

15
suggests FluVx was successfully presented and activated in melanoma-specific T-cells. G) Flow cytometry T-cells
harvested from FluVx-treated tumors compared to PBS when stained with melanoma antigen, gp100 dextramer.
(Modified from Newman et al., 2020)

A comparative assay showed that adjuvanted influenza vaccines (AdjFluVx)
demonstrated a lack of tumor growth reduction (Figure 5A-B). In contrast to FluVx, AdjFluVx
failed to increase CD8+ T cells within the tumor. However, it increased the number of dendritic
cells (DC) among antigen-presenting cells (APCs). When the adjuvant was removed from the
AdjFluVx, it rescued the effect seen in the original FluVx (Figure 5 E left & F left). A surprising result
discovered by intratumorally injecting the seasonal Influenza vaccine was the protection it
provided against respiratory Influenza infection on top of the reduction of tumor volumes of
melanoma (Figure 5 B bottom-G).

16

Figure 5: Seasonal adjuvanted Influenza vaccine fails to reduce melanoma tumors compared to naïve mice. A)
Schematic experimental design of seasonal adjuvant Influenza vaccine. B) Seasonal adjuvant Influenza vaccine fails
to reduce melanoma tumors compared to PBS-treated mice. E left) Schematic of experimental design of FluVx,
adjuvant-sequestering agent (Adj), and AdjuFluVx vaccine treatment. F left) Graphically depict FluVx, Adj, Adj+FluVx,
and AdjFluVx treatments on mouse tumor volumes. Adj successfully rescued the effect of FluVx from AdjFluVx,
leading to a statistically significant reduction in tumor volumes measured. B
bottom
) AdjFluVx and FluVx increase
ratios of DCs among antigen-presenting cells (APC) in melanoma tumors when injected intratumorally compared to
PBS control. ) FluVx successfully increased antigen-activated CD8
+
T cells compared to AdjFluVx and PBS control. D)
Flow cytometry elucidates that FluVx increased melanoma-specific CD8
+
T cells compared to AdjFluVx and PBS
control. E-G) Increase in T cells to B cells and decreased Bregs ratios in tumors injected intratumorally with FluVx
compared to PBS control. In contrast, AdjFluVx had larger B cells to T cell ratios and increased in Bregs (IL10+)
compared to PBS control. Similarly, FluVx had a decrease in Tregs (FoxP3+) among CD4+ T cell populations
compared to both AdjuFluVx and PBS control. (Modified from Newman et al., 2020).

17
At the conclusion of the article, Newman et al. suggested that the seasonal Influenza
vaccine was responsible for the reduction of tumor growth via activation of dendritic cells (DCs)
and tumor-specific cytotoxic CD8+ T cells within the tumor microenvironment, independent of
previous exposure to the Influenza virus. These activations are responsible for overturning the
highly immunosuppressive or “cold” tumors tumor microenvironment, allowing the infiltration
of immune cells. An interesting effect seen within this article was observed when Newman et
al. compared adjuvanted Influenza vaccines versus the unadjuvanted Influenza vaccine. Under
normal circumstances, an adjuvant is added to a vaccine to increase effectiveness by creating a
substantial immune response to the antigen and adjuvant. Conversely, an adjuvanted Influenza
vaccine seemed to undo the effect of tumor growth reduction seen in the unadjuvanted
Influenza vaccine. The authors theorized that this effect was because the adjuvanted vaccine
maintained high levels of regulatory B cells (B regs), one of the cells responsible for the
suppressive tumor microenvironment when injected intratumorally [26].
Chapter 3: Specific Aims and Hypothesis

3.1 Hypothesis

The above information procured from an in-depth literature review, helped me develop
two hypotheses to help combat HPV-induced tumors. First, I hypothesized that the
intratumoral injection of Seqirus’ 2021-2022 Influenza vaccine, HPV 16 L1 VLP (like in
Gardasil), or Pfizer’s mRNA COVID-19 vaccine in C3.43 tumors could also convert the cold
tumors to hot tumors, which would reduce tumor growth and eventually lead to tumor
regression. Second, I hypothesized that prime-boost intervals would play a role in the
effectiveness of therapeutic cancer vaccines.

18
A comprehensive review of Newman’s PNAS article led to the discovery of a potential
experiment design flaw. Newman et al, primed C57BL/6J mice on day 7 and boosted on day 9 ,
which is considered a 2-day prime-boost interval (PBI). Historically, optimal differentiation and
proliferation of T-cells from a single exposure of antigens, such an initial vaccination or priming,
requires approximately 40 days before boosting with the same antigens [30]. Recent studies
have suggested that a short prime-boost interval may induce a detrimental cellular mechanism
called activation-induced cell death (AICD) of T-cells [32]. AICD is an apoptosis-like mechanism
that is regulated by the expression of Fas on activated T-cells and FasL on natural killer (NK)
cells. This process allows for the maintenance of peripheral immune homeostasis and prevents
peripheral T-cell autoreactivity [32]. However, overexposure to antigens within a short interval
of time, such as Newman’s prime-boosting schedule of the repurposed unadjuvanted seasonal
Influenza vaccine, may lead to the development of AICD, suggesting that prime-boost intervals
play a larger role in the efficacy of therapeutic cancer vaccines.
3.2 Specific Aims

AIM 1: Determination if intratumoral injection of Seqirus’s 2021-2022 Influenza
vaccine in C3.43 solid tumors will elicit a reduction of growth.
HPV solid tumors are highly immunosuppressive, making the development of
therapeutics challenging once tumors are established. To determine if this seasonal Influenza
vaccine can be used to treat C3.43 tumors. Tumor challenges will occur in 6–8-week-old
C57BL/6J mice from Jackson Laboratories. Once tumors reached a substantially palpable
volume, injecting the seasonal Influenza vaccine in the tumor (i.t.) will be done. Manual caliper

19
measurements will occur biweekly. This method has not been performed prior in the C3.43
tumor model.
AIM 2: Determine if converting cold tumors to hot tumors can be translated to other
vaccines, such as HPV 16 L1 VLP and Pfizer’s mRNA COVID-19 in C3.43 tumors.
Prophylactic vaccines, such as Gardasil, encompass the HPV 16 L1 VLP within the
vaccine. If this vaccine promotes tumor reduction in C3.43 HPV tumor cells, it will revolutionize
the industry since it is already in production. To elucidate if the HPV 16 L1 VLP can reduce
tumor growth in C3.43, tumor challenge in 6 to 8-week-old C57BL/6J from Jackson Laboratories
will occur. Similar to the seasonal Influenza vaccine, once tumors are palpable, intratumoral
injection of this VLP will be performed. This method has not been previously explored in this
HPV model.
The COVID-19 pandemic brought to light the availability of vaccines that have not been
previously manufactured. Several studies have demonstrated the high efficacy of Pfizer’s
mRNA COVID-19 vaccine since its production in 2020. Hagin et al, demonstrated the vaccine’s
high immunogenicity by analyzing PBMCs, production of interferon-gamma (IFN ), and
antibodies ability to detect and neutralize the receptor-binding domain of SARS-CoV-2,
suggesting the production of memory B cells and memory T cells to be vital to lessen the
symptoms of viral infection [33].To test this idea, 6 to 8-week-old C57BL/6J mice will be tumor
challenged with C3.43 cells in the right flank. Intratumoral injection of Pfizer’s mRNA COVID-19
vaccine in palpable tumors will be performed to determine if the inflammatory response seen
in humans can be translated to a murine model.
AIM 3: Uncover the type of immune responses elicited by the repurposed vaccines.

20
HPV is a cold tumor resistant to standard therapies such as radiation and chemotherapy.
In addition, the use of immunotherapies has been challenging in solid tumors due to the
hindrance of lymphocytes infiltrating the tumor. Flow cytometry of harvested tumors and
splenocytes will be performed to determine the type of immune responses elicited by the three
repurposed vaccines. In addition, ELISpot or Multiplex analysis of cytokines within the tumor
samples would help ascertain if the vaccine successfully converted the cold tumors to hot ones.
AIM 4: Demonstrate the effect of prime-boost regimens on HPV-specific CD8
+
T-cell
populations.
Prime-boost interval regimens of therapeutic cancer vaccines have not been
standardized. To ascertain if the intervals affect the population of HPV-specific CD8+ T-cells,
mice will be challenged with C3.43 tumor cells subcutaneously in the right flank. Mice will be
vaccinated at different schedules, ranging from one dose to two doses up to 4 weeks apart. The
functionality and quantity of tumor-specific CD8
+
T-cells will be analyzed by MHC tetramer and
IFN  ELISpot assays.
AIM 5: Uncover if the VRP vaccine protects against tumor challenge regardless of the
prime-boost intervals in HPV 16 tumor models.
Mice will be immunized at predetermined intervals to test if the VRP vaccine can
protect against subsequent tumor challenges. For example, ten days post-immunization, mice
will be challenged with C3.43 tumor cells. Mice will then be monitored for 60 days following the
tumor challenge.
AIM 6: Determine if the length of prime-boost intervals enhances or diminishes
memory T-cells in response to tumor exposures.

21
To investigate whether increasing the prime boosts' intervals affect the functionality
and quantity of memory T-cells, mice will be challenged with C3.43 tumor cells four months
after final vaccination to allow for contraction of the initial T-cell population.
AIM 7: Elucidate the effect of prime-boost interval regimens on the efficacy of
therapeutic VRP-based vaccines.
To demonstrate if the intervals of prime-boost affect the efficacy of therapeutic VRP-
based vaccines, mice will be immunized with HPV16E7E6 VRP 5 days post-tumor challenge in
different prime-boost intervals, ranging from a single dose to 4 weeks. Mice will be observed
until the endpoint is reached or the experiment is done.
Chapter 4: Methods and Materials

4.1 Mice and Cell Lines

Pathogen-free female C57 BL/6J mice, from 6 to 8 weeks old, were purchased from
Jackson Laboratories for use in the repurposed vaccination experiments. Additional pathogen-
free female C57BL/6 mice, from 6 to 8 weeks old, were purchased from Taconic Farms. Tumor
challenge studies were performed using the C3.43 cell line, an in vivo passaged derivative of the
C3 HPV16 transformed murine tumor cell line to use in both sets of experiments [30,34,35].
C3.43 cells were cultured in an Iscove’s modified Dulbecco’s medium supplemented with 10%
fetal bovine serum, 1% gentamicin, and 1% ß-mercaptoethanol. B16-F10, the melanoma cell
line was cultured in Dulbecco’s Modified Eagle Medium, 10% fetal bovine serum, 1%
gentamicin, and 1% ß-mercaptoethanol. All animal studies complied with and approved by the
University of Southern California Institutional Animal Care and Use Committee.

22
4.2 Tumor challenge

For tumor challenge experiments, C57BL/6J mice (n=10 per group, 3-4 groups, three
independent experiments) were inoculated with 100,000 cells/ml C3.43 or B16-F10 melanoma
cells intradermally on the right flank, unanesthetized. Tumor growths were measured using
manual calipers in 3 dimensions, length, width, and height, starting 10-14 days after the tumor
challenge. Measurements were taken twice a week. Endpoints were calculated when the total
tumor volume reached 1500mm
3
per IACUC policies.
4.3 Repurposed Vaccines

United States Food and Drug Administration approved 2021-2022 unadjuvanted Seqirus
cell-based Flucelvax Quadrivalent, an Influenza vaccine was given intratumorally in both C3.43
and B16-F10 tumors undiluted. In addition, laboratory-prepared HPV16 L1 VLP, diluted to 10
µg/ml, was given intratumorally in mice with C3.43-based tumors. FDA-approved 2021 Pfizer’s
COVID mRNA vaccine was given intratumorally in C3.43-based tumors undiluted at ten µg/ml.
4.4 Vaccine Regimens

The HPV16E7E6 was produced by using the incompetent Venezuelan equine
encephalitis virus replicon particles (VRP) as previously described by [30,36]. This project used
Vero cells as a GMP-grade quality batch for mouse and non-human primate studies [30]. An in
vivo titration of these cells was performed, and it determined that the effective dose of 1 x10
7

infectious units (IU) per immunization was used throughout this study [30]. Mice were
immunized with HPV16E7E6 VRP intramuscularly (i.m.) in each quadriceps with a dose of 10
7

IU/ 20 µl phosphate-buffered saline (PBS) at the indicated intervals [30]. All vaccination intervals

23
were planned, so the mice received their final immunization on the same day [30]. All intervals
performed in the studies are represented in Figure 6.

Figure 6: Illustration of scheduled prime-boost intervals. Five experimental groups of mice were used in this study to
examine six different vaccine regimens (n=5/group); a prime vaccination with HPV16 VRP, followed by a boost with
the same vaccine after intervals (denoted by black arrows). Only one group received an initial immunization and no
boosting (1 vax). Another group remained unvaccinated (naïve mice). Splenocytes were isolated ten days after the
final vaccination, or mice were challenged with C3.43 tumors to assess effector T cell responses via a myriad of in
vitro assays. Determination of memory T cells recall responses; mice were challenged four months after the final
vaccination without additional treatments. (From Da Silva, D., Martinez, E., et.al, 2022)

4.5 Peptides

The HPV 16 E7 (49-57) RAHYNIVTF, an H2-Db-binding peptide[30,34], and the control
PSCA (23-31) AQMNNRDCL, a prostate stem cell antigen [37], were synthesized at the University of
Chicago (Chicago, IL, USA). These peptides were purified by reverse-phase high-performance
liquid chromatography (HPLC). The purity was assessed using an analytical HPLC and was
determined to be >95% pure [30].

24
4.6 Enzyme-Linked Immunospot Assay (ELISpot)

Ten days after the final vaccination, IFN-producing tumor antigen-specific cells specified
for HPV 16 E7 (49-57) were detected [30]. A total of 5 x 10
6
isolated splenocytes were stimulated
with or without E7 peptide at a final concentration of 1 µg/ml and with 5 IU interleukin-2/ml in
culture medium for 24 hours (n=5 per group, three independent experiments) [30]. Multiscreen
HA plates (EMD Millipore, Burlington, MA, USA) were coated with 10 µg/ml IFN  antibody (BD
Biosciences, San Jose, CA, USA) [30]. All plates were washed and blocked with a culture medium
previously specified [30]. After washing and blocking, harvested splenocytes were added to the
plates in 2-fold serial dilutions, ranging from 5 x 10
5
to 6.25 x 10
4
cells/well [30]. After 20 hours,
each plate was washed and incubated with 1 µg/ml of biotinylated IFN  antibody (BD
Biosciences) [30]. Additionally, washed plates were incubated with 100 µl of streptavidin-
horseradish peroxidase diluted to 1:4000 (Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes until
the reaction was stopped with water [30]. All assays performed were in triplicates, and results
were calculated by spot-forming cells (SFC) per 10
6
splenocytes while subtracting the
background from the culture medium [30].
4.7 MHC Tetramer Analysis

To quantify and phenotype the HPV 16 E7 (49-57) specific CD8
+
T cells, splenocytes were
stained with 0.5 µg/ml of H2-Db tetramers consisting of HPV 16 E7 (49-57) peptide obtained from
the National Institute of Allergy and Infectious Diseases Tetramer Facility (Atlanta, GA, USA) and
with CD3 and CD8 antibodies (BD Biosciences) [30]. Gating was performed on CD3
+
CD8
+
T cells,
and the percentage of positive tetramer CD8
+
T cells was elucidated [30]. In one of the
experiments, splenocytes were additionally stained with antibodies for a subset phenotype of T

25
cells pertaining to memory (i.e., CD44, CD127, and CD62L; all BD Biosciences, San Jose, CA) [30].
Based on these memory subsets of T cells, gating was performed on CD8
+
Tet
+
T cells, and the
percentages of T EM (CD62L
-
CD127
+
), T CM (CD62L
+
CD127
+
), and T EFF (CD62L
-
CD127
-
) were
quantified [30]. In addition, tetramer-positive cells were identified as positive for the CD44
+

phenotype [30]. A minimum of 100,000 events were captured on the Beckman Coulter FC500
flow cytometer and were analyzed using CXP software [30].
4.8 In Vivo Cytotoxicity Assay

Undifferentiated splenocytes were incubated with 0.5 µg/ml of either an irrelevant
PSCA (23-31) peptide or the relevant HPV 16 E7 (49-57) peptide [30]. Cells containing the HPV 16 E7 (49-
57) peptide were additionally labeled with 10mM CFSE using Vybrant CDFA SE Cell Tracer Kit
(Life Technologies, Carlsbad, CA, USA) [30]. Cells containing the irrelevant PSCA (23-31) peptide
were labeled with 0.66 mM CFSE [30]. Cells containing relevant or irrelevant peptides were
mixed in a ratio of 1:1 [30]. Approximately 10 million CFSE-labeled cells were injected
intravenously (i.v.) into both immunized and control mice (n=5 per group, ten days after final
vaccination) [30]. Spleens were harvested the following day, and 5 x 10
6
splenocytes were
analyzed on the Beckman Coulter FC500 flow cytometer [30]. A minimum of 5,000 CFSE-positive
events were collected [30]. Cell lysis percentages were calculated as follows: [ 1- (% CFSE
hi
/ %
CFSE
low
population] x 100 [30].
4.9 In Vivo Tumor Studies

In studies examining the effect of prophylactic vaccinations of HPV 16 VRP, groups of
ten 8-week-old female C57BL/6 mice were immunized with 10
7
IU of the VRP [30]. Mice were
challenged intradermally (i.d.) in the right flank with 5 x 10
5
C3.43 HPV 16 tumor cells in a 100

26
µl PBS [30]. Tumor growth was measured twice weekly with manual calipers in three dimensions
[30]. To test recall of long-term memory T cells post-immunization, mice were vaccinated as
described previously and left untouched for 4 months before the tumor challenge [30]. In
studies examining the therapeutic effects of HPV 16 VRP, mice were challenged with 5 x 10
5

C3.43 tumor cells at day 0 [30]. Five days after the tumor challenge, mice were vaccinated
intramuscularly with VRP with increasing prime-boost intervals (PBI) [30]. Tumor growth was
measured for 70 days post-tumor challenge [30]. When tumor volume exceeded 1500mm
3
, mice
were euthanized per the University of Southern California Animal Care and Use guidelines [30].
4.9.1 Statistical Analysis

For the repurposed vaccine experiments, the total tumor volumes for each group were
analyzed by multiple unpaired t-tests using the Šídák-Bonerroni method. Concerning prime-
boosting experiments, ELISpot, flow cytometry, and in vivo cytotoxicity assays were analyzed by
One-Way ANOVA testing followed by individual group comparison using Tukey’s multiple
comparison tests [30]. Mice survival was analyzed by using the log-rank (Mantel-Cox) test [30].
Statistical analyses were performed using the GraphPad Prism version 9.5.0 software
(GraphPad Software, San Diego, CA, USA).
Chapter 5: Results

5.1 The effect of the 2021Influenza vaccine on B16- F10 melanoma and C3.43 solid tumors in
vivo

To test the effectiveness of the seasonal Influenza vaccine as a form of cancer
immunotherapy, as demonstrated in Newman et al. article, C57BL/6J mice were challenged
with B16-F10 melanoma tumor cells or C3.43 HPV16 tumor cells intradermally on the right

27
flank. Mice were injected intratumorally with 20 µl PBS (control) or 2021-2022 Seqirus
unadjuvanted Influenza vaccine once tumors reached 50 mm
3
. Mice challenged with B16-F10
melanoma failed to reduce tumor growth with the seasonal Influenza vaccine (Figure 7,
p=0.976). The graph indicates that there were no augmentation to growth rate throughout the
experiment regardless of treatment.

28

C)

Figure 7: Mice challenged with 1 x 10
5
B16-F10 melanoma tumor cells or C3.43 cells were treated with PBS (control)
or 2021-2022 unadjuvanted Influenza vaccine (n=40, 10/group). A) Schedule of B16-F10 tumor challenge and
intratumoral injections of PBS or Influenza vaccine (FluVax), denoted by red arrows. B) Schedule of tumor challenge
of C3.43 HPV16 and intratumoral injections of PBS or Influenza vaccine (FluVax) denoted by green arrows. C) Graph
of experiments described in A and B. Created with Biorender.

29

Mice challenged with C3.43 HPV16 tumor cells were intratumorally injected with 20 µl
of PBS (control) or 2021-2022 Seqirus unadjuvanted Influenza vaccine once tumor volumes
reached 50 mm
3
. The seasonal Influenza vaccine was unsuccessful in reducing tumor growth
among the mice (Figure 7, p> 0.999).
5.2 The effect of “Gardasil” (HPV 16 L1 VLP) vaccine when given intratumorally in C3.43
HPV 16 solid tumors in vivo

To test the hypothesis of “Gardasil” (HPV 16 L1 virus-like protein) as a possible
immunotherapy for HPV 16-induced cancer when given intratumorally, C57BL/6J mice were
challenged with C3.43 tumor cells intradermally in the right flank. This VLP vaccine was chosen
due to HPV 16 L1 VLP being present in Gardasil quadrivalent and nonavalent vaccines. The mice
were injected with 20 µl of PBS (control) or 20 µl of 10ug/dose of HPV 16 L1 VLP intratumorally
once tumors reached 30 mm
3
in volume for the vaccine's effectiveness to be seen. Mice
injected with PBS (control) had a spontaneous reduction in tumor growth and eventually
complete tumor regression. The HPV 16 L1 VLP vaccine was ineffective at reducing tumor
growth or regressing tumors completely by Day 34 (Figure 8). Multiple t-tests confirmed no
significant reduction in tumor growth or complete reduction (p= 0.775).

30

31
B)

Figure 8: Mice challenged with 1 x 10
5
C3.43 HPV 16 cell line were treated with PBS (control) and HPV 16 L1 VLP
(n=16, 8/group). Illustrated schedule of tumor challenge and vaccination of both PBS and HPV L1 VLP in C57BL/6J
mice. B) Graph of experiments described in A.

5.3 The effects of Pfizer’s COVID-19 vaccine on C3.43 tumors in vivo when injected
intratumorally

Since the Seqirus’s FluCelVax is a cell-based vaccine and the HPV 16 L1 VLP is a protein-
based vaccine, the Pfizer-BioNtech COVID-19 provided an opportunity to examine the effects of
an mRNA-based vaccine as a possible therapeutic agent for HPV-induced cancer when injected
intratumorally. The Pfizer-BioNtech COVID-19 mRNA vaccine was chosen for its apparent
immunogenicity in humans when given intramuscularly (i.m.). Twenty C57BL/6J mice were
challenged with C3.43 cells intradermally in the right flank. Mice were injected intratumorally
with 20 µl of PBS (control) or 20 µl of Pfizer’s COVID-19 vaccine at 10 µg/dose when tumors
reached a total volume greater than 30 mm
3
. Mice injected with Pfizer-BioNtech’s COVID-19

32
mRNA vaccine failed to significantly regress tumor growth when given intratumorally compared
to the control (Figure 9) ( p=0.832).

B)
Figure 9:
Intratumoral injection of Pfizer’s COVID-19 mRNA vaccine fails to reduce HPV-induced tumors in vivo (n=20,
10/group). A) Schematic of tumor challenge and vaccination schedule for in vivo experimentation. B) Graph of
experiments described in A.

33

5.4 Effect of Increasing Prime-Boost Intervals on Degree of HPV-Specific CD8+ T-cell
Population

The replication incompetent Venezuelan Equine Encephalitis (VEE) replicon (VRP)
expressing HPV16 E6 and E7 mutated genes are considered immunogenic and able to induce
CD8
+
T-cells that display anti-tumor effectiveness in several HPV-induced murine models as
previously reported [30,36,38,39]. These previous articles used a variety of prime-boost intervals
(PBIs) ranging from 5–7-day injection schedules during therapeutic studies to 2-week intervals
in a preventive setting. In these studies, it was unclear which interval induced the highest
tumor-specific CD8
+
T-cell response. To investigate the role of different PBIs in the effectiveness
of inducing tumor-specific CD8
+
T-cells, we vaccinated mice with HPV16E7E6 VRP, as shown in
Figure 6. We evaluated the functionality and quantity of resulting tumor-specific T-cells via
major histocompatibility complex (MHC) tetramer and interferon-gamma (IFN ) ELISpot
analysis. The VRPs successfully induced tumor-specific T-cells even after one immunization
(Figure 10). All immunized groups displayed significant increases in HPV16 E7-specific T-cells
responses by IFN  release compared to naïve mice (p < 0.05, naïve vs. 1-vax, 3-day; p < 0.001
naïve vs. 1 wk, 2 wk, 3wk, 4 wk intervals) (Figure 10A). Prime-boost intervals of 1 week (1 wk), 2
weeks (2 wk), 3 weeks (3 wk), and 4 weeks (4 wk) were significant compared to naïve mice as
measured by tetramer positivity (p < 0.0001, naïve vs 1 wk; p < 0.01, naïve vs 2 wk, 3 wk; p
<0.001, naïve vs 4 wk) (Figure 10B). The maximum number of HPV16 E7 49-57 specific T-cells
induced was with a PBI of 1 week, as shown by ELISpot (Figure 10A) and tetramer analysis
(Figure 10B). PBIs longer than one week (2 wk, 3 wk, and 4 wk) did not show a change in the

34
quantity of HPV-specific T-cells compared to the 1-week interval (Figure 10B). In C57BL/6 mice,
the E7 49-57 peptide is a subdominant epitope of CD8
+
T-cells [30,34]. The one-week boost interval
was the only interval that demonstrated H2-Db-E7 49-57 tetramer binding by CD8
+
T-cells,
suggesting that the 1-week boost was the minimum to induce a maximum number of effector
antigen-specific CD8
+
T-cells via a viral therapeutic vaccine [30].
We also investigated the potential of the induced HPV-specific cytotoxic CD8+ T-cells
(CTLs) to destroy HPV-induced tumor cells activated by the different prime-boost intervals [30].
Cytotoxic studies were performed by transferring naïve differentially labeled CFSE
+
splenocytes
ladened with relevant HPV16E7 49-57 peptide or the control PSCA 23-31 peptide to mice groups
vaccinated, as indicated in Figure 6. The loss of CFSE
hi
E7-pulsed cells was suggestive of the
effectiveness of the different PBIs in generating CTLs. Our results established that all PBIs,
including the single vaccination, were able to lyse 100% of HPV16 E7 49-57 loaded cells compared
to the naïve mice (p < 0.0001) (Figure 10C) [30]. Furthermore, during our comparison of
different PBIs, the PBI of 3 days demonstrated a significantly lower lysis rate (p <0.05 to p
<0.001), including the single injection of the vaccine. This suggests that shortened prime-boost
intervals are destructive to generating effector CTLs by initiating the cellular mechanism of
activation-induce cell death (AICD) due to overexposure of antigens [30].

35

Figure 10: Analysis of different prime-boost intervals on T-cell response. C57BL/6 mice ( n = 5/ group) were
vaccinated intramuscularly with 1 x 10
7
IU HPV16 E7E6 VRP, as shown in Figure 6. A) HPV16 E7 IFN  secretion via
ELISpot assay as shown by the number of spot-forming cells (SFC) per million splenocytes from three independent
experiments. The mean indicated by the horizontal bar demonstrates that all immunized mice significantly
increased compared to naïve mice ( p < 0.05 to p <0.0001 via one-way ANOVA). B) Splenocytes were analyzed for
the H2-Db MHC tetramer E7 49-57 peptide binding. The percentage of E7 tetramer binding CD8
+
T-cells for each group

36
from 3 independent experiments with mean indicated by the horizontal bar. Mice immunized at 4 wk, 3 wk, 2 wk,
and 1 wk intervals demonstrated a greater mean quantity of E7 tetramer-specific CD8
+
T-cells compared to naïve
mice ( p <0.01 to p <0.001) C) Cytotoxic assay. C57BL/6 naïve splenocytes were ladened with E7 49-57 peptide or
control PSCA 23-31 peptide, labeled with high dose E7 peptide or low dose of CFSE. Corresponding histogram plots
labeled cell and CFSE populations in naïve and immunized mice. E7-specific cytotoxicity calculated is shown for all
groups. Lysis significantly differs from naïve mice to mice in all vaccinated groups ( p < 0.0001). *p < 0.05, ** p <
0.01, *** p < 0.001 **** p < 0.0001 ( One-way ANOVA, Tukey’s post-test). Significant pairwise comparisons are
indicated in the figure. Not significant pairwise comparisons are not shown. (From Da Silva, D., Martinez, E., et al.,
2022)

5.5 Vaccination Offers Protection against Tumor Challenge Independent of Prime-Boost
Interval in Preventive HPV16 Tumor Model

As mentioned, in prime-boost interval regimens that resulted in the expansion of E7-
specific T-cells with potent cytolytic activity, we tried to determine whether these T-cells were
useful in their ability to lyse HPV 16-transformed, immune-compatible tumor cells [30]. To
discover if there was a difference in protection level between PBIs against tumor challenge,
groups of mice received VRP injections per Figure 6. These groups were then challenged with
C3.43 tumor cells ten days post-final vaccination. Regardless of the lower cytotoxicity
demonstrated in the 3-day PBI group against peptide-loaded cells, as shown previously, all
vaccination intervals, including the one immunization dose, resulted in 100% protection against
the tumor challenge (p < 0.0001). Furthermore, no immunized mouse developed any visual or
palpable tumors 60 days post-tumor challenge. In contrast, all naïve mice developed and
reached the endpoint resulting in euthanasia (Table 1). Our data demonstrated that the bulk of
tumor-specific T-cells generated by the VRP immunization could recognize and lyse HPV16-
expressing tumor cells independent of the PBIs. This result suggests more E7-specific effector T-
cells developed even in the single-dose vaccination [30].

37

5.6 Enhanced Memory Recall and Differential Induction of Memory T-cell Phenotypes
Correspond with Longer Prime-Boost Intervals

To investigate if increasing PBIs affect the bulk of memory T-cells developed and their
memory recall response to tumor cells, we challenged groups of mice with C3.43 cells four
months post-final vaccination [30]. This time range was picked as to when it was expected the
initially expanded effector T-cell populations had diminished, thus allowing for a small
percentage of memory T-cells to remain. The survival of mice was independent of different
prime-boost intervals compared to control mice (p < 0.0001) (Figure 11). However, concerning
tumor-specific T-cell memory recall, the longer the PBIs demonstrated a more remarkable
survival compared to shorter intervals (4 wk, 3 wk, 2 wk vs. 1 wk, 3-day, 1-vax). Furthermore,
the two and four-week intervals resulted in the most significant long-term protection compared
to the 1-week interval regarding mice survival (p = 0.0109 and p = 0.0157) (Figure 11).

38

Figure 11: Enhanced Memory Recall and Differential Induction of Memory T-cell Phenotypes Corresponding with
Longer Prime-Boost Intervals post-immunization. C57BL/6 mice ( n = 10/group) were vaccinated with HPV16 E6E7
VRP according to the schematic shown in Figure 6. Four months post-final vaccination, mice were challenged with 5
x 10
5
C3.43 cells subcutaneously in the right flank to determine T-cell memory recall responses generated after
various prime-boost intervals. Median survival of naïve is 29 days; 1 vax was 37 days; 3 days PBI was 39 days; 1 wk
PBI was 39 days; 2 wk PBI was 54.5 days; 3 wk was 42.5 days; 4 wk PBI was 49.5 days. Significant pairwise
comparisons are indicated in the figure. All other pairwise comparisons were not significant. (From Da Silva, D.,
Martinez, E., et al, 2022)

The overall objective of cancer vaccination is to develop long-lasting memory recall to
sustain substantial protective immunity. We phenotyped the HPV16 E749-57 CD8
+
T-cells further
to determine the ratio of effector memory T-cell (T em) and central memory T-cell (T cm) subsets
in response to varying prime-boost interval regimens [30]. The phenotyping demonstrated an
increase in the T em population compared to T cm in the 1-week PBI. Most of the T-cell population
was T em, with the bulk reaching upwards of 60% of the total HPV 16 tumor-specific CD8
+
T cells,
generating a T em/T cm ratio of 9.3. Contrary to the 1-week PBI, the 3-day PBI generated a T em/T cm
ratio of 1.9, similar to the single dose vaccination schedule, which produced a T em/T cm ratio of
2.4 (Figure 12). This data suggests that longer the PBIs generated more T em than T cm, which may
have provided an immediate response and protection to subcutaneous peripheral tumor
challenge regardless of the length of post-exposure to HPV antigens. In contrast, shorter PBIs,

39
such as 3-day and single-dose immunization, generated more tumor-specific cells T-cells with a
T cm phenotype, leading to suboptimal recall to tumor challenge four months post-exposure [30].

Figure 12: Longer PBIs, produce more effector memory T-cells. HPV 16 E7 49-57 specific T-cells determined by MHC
tetramer staining were phenotyped for T-cell effector and memory subsets. Isolated splenocytes from vaccinated
mice (n =5) 10 days after boost were stained with the E7 tetramer (tet), CD8, CD44, CD127 (IL-7R ), and CD62L. The
proportions of T em, T cm, and T eff phenotypes were determined via Boolean gating on CD3
+
CD8
+
Tet
+
T-cells. The pie
charts show the effector and memory phenotypes frequencies among the positive tetramer population. The PBIs of
4 wk, 3 wk, 2 wk, and 1 wk demonstrated similar percentages of each population but had larger numbers of T em.
The ratios of T em: T cm is shown on the bar graph. T em, effector memory T-cells; T cm, central memory T-cells; T eff,
effector T-cells. ( From Da Silva, D., Martinez, E., et al, 2022)

5.7 Analysis of Prime-Boost Intervals on Tumors Using VRP-based Vaccines as Therapeutic
Agents

Determination of the cancer therapeutic vaccines' prime-boost intervals (PBI) was
ascertained by the aggressiveness of the tumor type [30]. This is important as these vaccines
aim to elicit the activations and proliferation of T-cell memory subsets and prevent the
phenomenon of T-cell exhaustion. To confirm the effect of PBIs on the growth rate of tumors,
we challenged mice with C3.43 tumor cells and then vaccinated them five days after the

40
challenge with HPV16E7E6 VRP. The growth rate and clearance of tumors were observed for 70
days (Figure 13). As seen in the previous experiments, the single-dose immunization was
sufficient to eliminate tumors in 100% of mice compared to naïve mice (p < 0.0001). When
increasing the PBIs up to 4 weeks did not significantly alter growth rates or elimination of
tumors. However, in mice in the prime-boost interval of 3 days, 80% of mice developed visually
and palpable tumors compared to the other groups, with a smaller percentage of mice
developing a tumor. This suggests that short PBIs were inappropriate in a therapeutic setting;
however, all mice in the experiment could eliminate their tumors [30].

41

Figure 13: Prime-boost intervals do not impact the efficacy of VRP-based therapeutic vaccines. C57BL/6 mice ( n=
10/group) were challenged with 5 x 10
5
C3.43 cells intradermally in the flank. Mice were vaccinated
intramuscularly with HPV16E6E7 VRP at increasing PBIs 5 days after tumor challenge. Arrows indicate
immunizations. All groups are graphed individually against naïve/ unvaccinated mice. All immunized mice were
significantly protected compared to naïve mice (p < 0.0001, log-rank test). (From Da Silva, D., Martinez, E., et al,
2022)
Chapter 6: Discussion

My investigation into the repurposing of Seqirus’s unadjuvanted seasonal Influenza
vaccine, HPV 16 L1 VLP (Gardasil), and Pfizer’s mRNA COVID-19 vaccines and injecting them
directly into the tumor demonstrated the need for a standardized prime-boost regimen in
therapeutic cancer vaccines. The intratumoral route of injecting vaccines allows the immune
system to recognize the antigen and activate naïve T-cells. Newman et al. utilized this injection
route to repurpose the seasonal Influenza vaccine. They demonstrated that this route was
sufficient to elicit the activation and differentiation of naïve T-cells into tumor-specific CD8
+
T-
cells [26]. Using this data as the foundation of this project, I hypothesized that the intratumoral
injection of seasonal Influenza vaccines into HPV 16-induced solid tumors (C3.43) could convert
cold tumors to hot tumors. First, the determination that the seasonal Influenza vaccines are
sufficient to reduce tumor growth in C3.43 tumors when given intratumorally will need to be
proven. Second, dependent on the data gathered from the initial experiments, can this method

42
be translated to repurposing other vaccines, such as “Gardasil” and Pfizer’s COVID-19, if
injected intratumorally in C3.43 tumors? Finally, uncovering the types of immune responses
produced by each vaccine when given intratumorally in C3.43 tumors.
To address the first aim of this project, C57BL/6J mice were used to determine if the
2021 unadjuvanted seasonal Influenza vaccine was sufficient in reducing tumor growth in C3.43
solid tumors when injected intratumorally. However, the results indicated that the 2021
unadjuvanted 2021 seasonal Influenza vaccine was ineffective at reducing tumor growth in
C3.43 solid tumors. This data directly contradicts my hypothesis that the Influenza vaccine
would be effective. The data suggests that the Influenza vaccine could be sequestered and
eliminated by the mouse immune system as this was performed in cutaneous tissue rather than
mucosal. Newman et al. also came across this dilemma when initially starting with a heat-
inactivated Influenza strain vaccine and suggested that it was due to the presence of pathogen-
associated molecular patterns (PAMPs) being recognized by the mice’s toll-like receptors (TLR),
especially TLR-7 [26]. Secondly, active influenza viruses can bind through the sialic acid residues
found on the epithelium of the upper respiratory tract [26,40]. These residues are required for
the influenza virus to produce a proinflammatory microenvironment [40]. Also, stratified
epithelium found in cutaneous tissues does not have these residues, which hinder the virus
from initiating a substantial immune response. Thirdly, the year in which the Influenza vaccine
was manufactured might have played a significant role in the Influenza vaccine's efficacy in
reducing tumor growth. In Newman’s article, the 2017 unadjuvanted seasonal Influenza vaccine
manufactured by Seqirus contained four strains of Influenza: H1N1 (A/California/7/2009), H3N2
(A/Hong Kong/4801 2014), B/Phuket/3073/2013, and B/Brisbane/60/08-like virus [41]. In

43
contrast, the 2021 unadjuvanted seasonal Influenza vaccine was formulated to have the four
Influenza strains: H1N1/ pdm09-like virus (A/Washington/19/2020), H3N2-like virus
(A/Tasmania/503/2020), B/Darwin/7/2019 (a B/Washington/02/2019-like virus), and
B/Singapore/INFTT-16-0610/2016) ( a B/Phuket/3073/2013-like virus) [42]. These differences in
strains could hinder the vaccine's effectiveness despite being manufactured by the same
company.
To determine if the vaccine type was the variable prohibiting the tumor reduction in
C3.43 solid tumors, “Gardasil” (HPV 16 L1 VLP) and Pfizer’s COVID-19 vaccines were injected
intratumorally in C57BL/6J mice. The results indicate that both vaccines did not reduce tumor
growth in C3.43 solid tumors. This suggests that the vaccine type does not play a significant
role in whether it is effective in reducing tumor growth.
Since vaccine type is not significant in reducing tumor growth, a new research question
was formulated. Why didn’t the intratumoral injection of the seasonal Influenza vaccine,
“Gardasil,” and Pfizer’s COVID-19 vaccines work? To address this, C57BL/6J mice were
challenged with B16-F10 melanoma tumor cells, the original model used in the Newman et al.
PNAS article. Tumors were intratumorally injected with the 2021 seasonal Influenza vaccine
when tumors became palpable. The results indicated that the tumor cell type was not
impacting the effectiveness of the vaccines yet pointed to a possible flaw in the experimental
design (Figure 7C).
A possible experimental design flaw was that the spacing between vaccine priming and
the subsequent boosting was too short to elicit a potent immune response in these tumors.
Activated-induced cell death (AICD) occurs when CD8
+
T cells are subject to repeated antigen

44
exposure over a short interval of time [43]. This could explain why the vaccines did not reduce
tumor growth in both C3.43 cells and B16-F10 tumor cells. This concept led to a new
hypothesis: I hypothesized that prime-boost intervals (PBIs) could influence vaccine efficacy.
To ascertain if PBIs affect the frequency and functionality of antigen-specific CD8
+
T-
cells, C57BL/6 mice were injected with C3.43 tumor cells and subsequently vaccinated with VRP
at specific intervals. The results indicate that utilizing a VRP as a therapeutic vaccine, boosting
at one week is optimal compared to longer PBIs in generating antigen-specific CD8
+
T-cells [30].
However, immunizing with a single vaccination resulted in a more significant frequency of
functional CD8
+
T-cells via ELISpot, comparable to vaccination regimens that contained a
booster. Furthermore, the single dose vaccination and the 3-day prime-boost interval provided
protection in a prophylactic setting despite eliciting a smaller frequency of functional CD8
+
T-
cells with the characteristics of effector T-cells. These subsets of cytotoxic T-cells do not provide
ample protection against antigen exposure at peripheral locations where tumors might have
originated. The smaller frequency of functional CD8
+
T-cells during the 3-day PBI suggests that
shorter PBIs induce AICD compared to longer PBIs and should be avoided [30,43]. Additionally,
interpretations of human disease in murine models should be cautioned. Translation of murine
model data to humans is difficult to speculate due to the heterogeneity of human tumors,
genetics, and environmental exposures. On the other hand, the data indicate a correlation
between the length of PBIs and the effectiveness of the vaccine-induced immune response [30].
Depending on the location of tumors and tumor types, different proportions of effector
and memory T-cells may be required for immune protection [27,28,30,44,45]. The generation of
effector T-cells as immediate protection relies on chronic antigen exposure. This persistent

45
exposure is not optimal for generating long-term memory T-cells [30,46]. However, during viral
infections located within the lymph nodes, long-term protection is caused by the generation of
central memory T-cells (T cm). In contrast, viral infections in the periphery require the generation
of effector memory T-cells (T em) at significant numbers. During the initial stages of the disease,
T em is the predominant cell type in the population. It offers substantial immune protection due
to its significant presence in the periphery, where they are the first cells to contact the antigen
exposure [47]. This knowledge can be applied in a therapeutic setting for peripheral cancers,
such as cervical cancer. During the phenotyping of tumor-specific CD8
+
T-cells, the data suggest
that a minimum interval of 1 week is required to produce adequate T em populations in
peripheral locations [27]. PBIs shorter than one week prevent antigen-specific T-cells from
reaching their complete proliferation and can result in T-cell elimination or exhaustion.
However, repeated antigen exposure can push memory T-cells towards terminal differentiation,
which is optimal for effector T-cells responsible for clearing tumors at the periphery.
Additionally, repeated exposure to antigens, such as if the regimen requires more than three
vaccination or boosts, can lead to diminished Tcm populations [30,48]. The data suggests that
ten days after immunization showed phenotypic differences in memory T-cell populations;
however, the memory T-cell populations were monitored for only four months post-antigen
exposure [30].
With respect to in vivo experimentation, several limitations must be considered for the
differences seen in the Newman et al., PNAS article results and the results demonstrated here.
First, as mentioned previously, vaccine formulation of the unadjuvanted seasonal Influenza
vaccine could impact murine immune response. The mutation rate of animal Influenza strains

46
remains relatively high with an extensive change to their NS1 gene. This gene increases the
capability of the virus to infect and produce viable progeny by hindering the host cell from
eliciting an antiviral response [49]. Secondly, with respect to the murine model, differences in
housing facilities and feed could augment the composition of gut microbiota. One study
suggests that the composition of gut microbiota in mice could alter the responsiveness of the
innate and adaptive immune systems to the presence of antigens which could potentially
explain why the intratumoral injection of the unadjuvanted seasonal Influenza vaccine [50].
Finally, the conditions in which the B16-F10 melanoma cell line was stored and expanded in
media might have impacted the immune system's responsiveness in the mice.
Chapter 7: Conclusion

From the current study, it can be concluded that a repurposed Influenza vaccine, when
injected intratumorally, was unsuccessful at reducing tumor growth in both HPV 16 tumors
(C3.43) and B16-F10 melanoma. The inability to replicate data from Newman et al PNAS article,
suggested that there may be a problem with the design of the prime-boost regimen for the
2017 repurposed unadjuvanted seasonal Influenza vaccine. Potential explanations could
attribute to the difference in results from the Newman article and my own. First, with respect
to in vivo experimentation, there are variables that cannot be controlled. For example, immune
responses in murine models could differ individually due to environmental factors, such as
housing facilities of mice and feed that will directly affect gut microbiota composition.
However, my data suggest that the length between the initial priming and boosting should not
be shorter than one week to avoid tumor-specific T-cell elimination or exhaustion via repeated
antigen exposure, which may induce activated-induced cell death (AICD). In addition, when

47
studying memory T-cell formation in relation to long-term protection, prime-boost intervals can
be prolonged up to 4 weeks without compromising CD8
+
T-cell generation. Future studies
should aim to maintain optimal proliferation and differentiation of T-cells when developing
therapeutic cancer vaccines, while preventing AICD. New cancer immunotherapies should also
focus on augmenting the highly immunosuppressive tumor microenvironment to a
proinflammatory tumor microenvironment to make alternative therapies more widely available
and cost-effective than the standard treatments for HPV-induced solid tumors.

48
References

1. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F.
Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality
Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021, 71, 209-249,
doi:10.3322/caac.21660.
2. Siegel, R.L.; Miller, K.D.; Fuchs, H.E.; Jemal, A. Cancer statistics, 2022. CA Cancer J Clin
2022, 72, 7-33, doi:10.3322/caac.21708.
3. Hanahan, D. Hallmarks of Cancer: New Dimensions. Cancer Discov 2022, 12, 31-46,
doi:10.1158/2159-8290.CD-21-1059.
4. Cosper, P.F.; Bradley, S.; Luo, L.; Kimple, R.J. Biology of HPV Mediated Carcinogenesis
and Tumor Progression. Semin Radiat Oncol 2021, 31, 265-273,
doi:10.1016/j.semradonc.2021.02.006.
5. Crosbie, E.J.; Einstein, M.H.; Franceschi, S.; Kitchener, H.C. Human papillomavirus and
cervical cancer. Lancet 2013, 382, 889-899, doi:10.1016/S0140-6736(13)60022-7.
6. Cancer, I.A.f.R.o. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans;
International Agency for Research on Cancer: 150 cours Albert Thomas, 69372 Lyon
Cedex 08, France, 2007; Volume 90, p. 689.
7. McMurray, H.R.; Nguyen, D.; Westbrook, T.F.; McAnce, D.J. Biology of human
papillomaviruses. Int J Exp Pathol 2001, 82, 15-33, doi:10.1046/j.1365-
2613.2001.00177.x.
8. McBride, A.A. The papillomavirus E2 proteins. Virology 2013, 445, 57-79,
doi:10.1016/j.virol.2013.06.006.
9. Ren, S.; Gaykalova, D.A.; Guo, T.; Favorov, A.V.; Fertig, E.J.; Tamayo, P.; Callejas-Valera,
J.L.; Allevato, M.; Gilardi, M.; Santos, J.; et al. HPV E2, E4, E5 drive alternative
carcinogenic pathways in HPV positive cancers. Oncogene 2020, 39, 6327-6339,
doi:10.1038/s41388-020-01431-8.
10. Pina-Sanchez, P. Human Papillomavirus: Challenges and Opportunities for the Control of
Cervical Cancer. Arch Med Res 2022, 53, 753-769, doi:10.1016/j.arcmed.2022.11.009.
11. R, S.J. The Immune Microenvironment in Human Papilloma Virus-Induced Cervical
Lesions-Evidence for Estrogen as an Immunomodulator. Front Cell Infect Microbiol 2021,
11, 649815, doi:10.3389/fcimb.2021.649815.
12. Walboomers, J.M.; Jacobs, M.V.; Manos, M.M.; Bosch, F.X.; Kummer, J.A.; Shah, K.V.;
Snijders, P.J.; Peto, J.; Meijer, C.J.; Munoz, N. Human papillomavirus is a necessary cause
of invasive cervical cancer worldwide. J Pathol 1999, 189, 12-19, doi:10.1002/(SICI)1096-
9896(199909)189:1<12::AID-PATH431>3.0.CO;2-F.
13. Cohen, P.A.; Jhingran, A.; Oaknin, A.; Denny, L. Cervical cancer. Lancet 2019, 393, 169-
182, doi:10.1016/S0140-6736(18)32470-X.
14. Markowitz, L.E.; Schiller, J.T. Human Papillomavirus Vaccines. J Infect Dis 2021, 224,
S367-S378, doi:10.1093/infdis/jiaa621.
15. Yuan, Y.; Cai, X.; Shen, F.; Ma, F. HPV post-infection microenvironment and cervical
cancer. Cancer Lett 2021, 497, 243-254, doi:10.1016/j.canlet.2020.10.034.

49
16. Graham, S.V. Human papillomavirus: gene expression, regulation and prospects for
novel diagnostic methods and antiviral therapies. Future Microbiol 2010, 5, 1493-1506,
doi:10.2217/fmb.10.107.
17. Goodman, M.T.; Shvetsov, Y.B.; McDuffie, K.; Wilkens, L.R.; Zhu, X.; Thompson, P.J.;
Ning, L.; Killeen, J.; Kamemoto, L.; Hernandez, B.Y. Prevalence, acquisition, and
clearance of cervical human papillomavirus infection among women with normal
cytology: Hawaii Human Papillomavirus Cohort Study. Cancer Res 2008, 68, 8813-8824,
doi:10.1158/0008-5472.CAN-08-1380.
18. Huber, B.; Wang, J.W.; Roden, R.B.S.; Kirnbauer, R. RG1-VLP and Other L2-Based, Broad-
Spectrum HPV Vaccine Candidates. J Clin Med 2021, 10, doi:10.3390/jcm10051044.
19. Soliman, M.; Oredein, O.; Dass, C.R. Update on Safety and Efficacy of HPV Vaccines:
Focus on Gardasil. Int J Mol Cell Med 2021, 10, 101-113,
doi:10.22088/IJMCM.BUMS.10.2.101.
20. Abouelella, D.K.; Canick, J.E.; Barnes, J.M.; Rohde, R.L.; Watts, T.L.; Adjei Boakye, E.;
Osazuwa-Peters, N. Human papillomavirus vaccine uptake among teens before and
during the COVID-19 pandemic in the United States. Hum Vaccin Immunother 2022, 18,
2148825, doi:10.1080/21645515.2022.2148825.
21. Dobosz, P.; Dzieciatkowski, T. The Intriguing History of Cancer Immunotherapy. Front
Immunol 2019, 10, 2965, doi:10.3389/fimmu.2019.02965.
22. Dorta-Estremera, S.; Hegde, V.L.; Slay, R.B.; Sun, R.; Yanamandra, A.V.; Nicholas, C.;
Nookala, S.; Sierra, G.; Curran, M.A.; Sastry, K.J. Targeting interferon signaling and CTLA-
4 enhance the therapeutic efficacy of anti-PD-1 immunotherapy in preclinical model of
HPV(+) oral cancer. J Immunother Cancer 2019, 7, 252, doi:10.1186/s40425-019-0728-4.
23. Ai, L.; Xu, A.; Xu, J. Roles of PD-1/PD-L1 Pathway: Signaling, Cancer, and Beyond. Adv Exp
Med Biol 2020, 1248, 33-59, doi:10.1007/978-981-15-3266-5_3.
24. Rowshanravan, B.; Halliday, N.; Sansom, D.M. CTLA-4: a moving target in
immunotherapy. Blood 2018, 131, 58-67, doi:10.1182/blood-2017-06-741033.
25. Hanoteau, A.; Newton, J.M.; Krupar, R.; Huang, C.; Liu, H.C.; Gaspero, A.; Gartrell, R.D.;
Saenger, Y.M.; Hart, T.D.; Santegoets, S.J.; et al. Tumor microenvironment modulation
enhances immunologic benefit of chemoradiotherapy. J Immunother Cancer 2019, 7, 10,
doi:10.1186/s40425-018-0485-9.
26. Newman, J.H.; Chesson, C.B.; Herzog, N.L.; Bommareddy, P.K.; Aspromonte, S.M.; Pepe,
R.; Estupinian, R.; Aboelatta, M.M.; Buddhadev, S.; Tarabichi, S.; et al. Intratumoral
injection of the seasonal flu shot converts immunologically cold tumors to hot and
serves as an immunotherapy for cancer. Proc Natl Acad Sci U S A 2020, 117, 1119-1128,
doi:10.1073/pnas.1904022116.
27. Perret, R.; Ronchese, F. Memory T cells in cancer immunotherapy: which CD8 T-cell
population provides the best protection against tumours? Tissue antigens 2008, 72, 187-
194, doi:10.1111/j.1399-0039.2008.01088.x.
28. Muroyama, Y.; Wherry, E.J. Memory T-Cell Heterogeneity and Terminology. Cold Spring
Harb Perspect Biol 2021, 13, doi:10.1101/cshperspect.a037929.
29. Klebanoff, C.A.; Gattinoni, L.; Restifo, N.P. CD8+ T-cell memory in tumor immunology
and immunotherapy. Immunol Rev 2006, 211, 214-224, doi:10.1111/j.0105-
2896.2006.00391.x.

50
30. Da Silva, D.M.; Martinez, E.A.; Bogaert, L.; Kast, W.M. Investigation of the Optimal Prime
Boost Spacing Regimen for a Cancer Therapeutic Vaccine Targeting Human
Papillomavirus. Cancers (Basel) 2022, 14, doi:10.3390/cancers14174339.
31. Kanodia, S.; Da Silva, D.M.; Karamanukyan, T.; Bogaert, L.; Fu, Y.X.; Kast, W.M.
Expression of LIGHT/TNFSF14 combined with vaccination against human papillomavirus
Type 16 E7 induces significant tumor regression. Cancer Res 2010, 70, 3955-3964,
doi:10.1158/0008-5472.CAN-09-3773.
32. Arakaki, R.; Yamada, A.; Kudo, Y.; Hayashi, Y.; Ishimaru, N. Mechanism of activation-
induced cell death of T cells and regulation of FasL expression. Crit Rev Immunol 2014,
34, 301-314, doi:10.1615/critrevimmunol.2014009988.
33. Hagin, D.; Freund, T.; Navon, M.; Halperin, T.; Adir, D.; Marom, R.; Levi, I.; Benor, S.;
Alcalay, Y.; Freund, N.T. Immunogenicity of Pfizer-BioNTech COVID-19 vaccine in
patients with inborn errors of immunity. J Allergy Clin Immunol 2021, 148, 739-749,
doi:10.1016/j.jaci.2021.05.029.
34. Feltkamp, M.C.; Smits, H.L.; Vierboom, M.P.; Minnaar, R.P.; de Jongh, B.M.; Drijfhout,
J.W.; ter Schegget, J.; Melief, C.J.; Kast, W.M. Vaccination with cytotoxic T lymphocyte
epitope-containing peptide protects against a tumor induced by human papillomavirus
type 16-transformed cells. Eur.J.Immunol. 1993, 23, 2242-2249.
35. Smith, K.A.; Meisenburg, B.L.; Tam, V.L.; Pagarigan, R.R.; Wong, R.; Joea, D.K.; Lantzy, L.;
Carrillo, M.A.; Gross, T.M.; Malyankar, U.M.; et al. Lymph node-targeted
immunotherapy mediates potent immunity resulting in regression of isolated or
metastatic human papillomavirus-transformed tumors. Clin Cancer Res 2009, 15, 6167-
6176, doi:1078-0432.CCR-09-0645 [pii]
10.1158/1078-0432.CCR-09-0645.
36. Cassetti, M.C.; McElhiney, S.P.; Shahabi, V.; Pullen, J.K.; Le Poole, I.C.; Eiben, G.L.; Smith,
L.R.; Kast, W.M. Antitumor efficacy of Venezuelan equine encephalitis virus replicon
particles encoding mutated HPV16 E6 and E7 genes. Vaccine 2004, 22, 520-527,
doi:10.1016/j.vaccine.2003.07.003.
37. Garcia-Hernandez Mde, L.; Gray, A.; Hubby, B.; Klinger, O.J.; Kast, W.M. Prostate stem
cell antigen vaccination induces a long-term protective immune response against
prostate cancer in the absence of autoimmunity. Cancer Res 2008, 68, 861-869,
doi:10.1158/0008-5472.CAN-07-0445.
38. Eiben, G.L.; Velders, M.P.; Schreiber, H.; Cassetti, M.C.; Pullen, J.K.; Smith, L.R.; Kast,
W.M. Establishment of an HLA-A*0201 human papillomavirus type 16 tumor model to
determine the efficacy of vaccination strategies in HLA-A*0201 transgenic mice. Cancer
Res. 2002, 62, 5792-5799.
39. Velders, M.P.; McElhiney, S.; Cassetti, M.C.; Eiben, G.L.; Higgins, T.; Kovacs, G.R.;
Elmishad, A.G.; Kast, W.M.; Smith, L.R. Eradication of established tumors by vaccination
with Venezuelan equine encephalitis virus replicon particles delivering human
papillomavirus 16 E7 RNA. Cancer Res. 2001, 61, 7861-7867.
40. Smed-Sorensen, A.; Chalouni, C.; Chatterjee, B.; Cohn, L.; Blattmann, P.; Nakamura, N.;
Delamarre, L.; Mellman, I. Influenza A virus infection of human primary dendritic cells
impairs their ability to cross-present antigen to CD8 T cells. PLoS Pathog 2012, 8,
e1002572, doi:10.1371/journal.ppat.1002572.

51
41. Administration, U.S.F.a.D. Package Insert-FluCelVax2017. Available online: (accessed on
03/14/2023).
42. Administration, U.S.F.a.D. Package Insert-Flucelvax Quadrivalent. Available online:
(accessed on 03/14/2023).
43. Dhodapkar, M.V.; Krasovsky, J.; Steinman, R.M.; Bhardwaj, N. Mature dendritic cells
boost functionally superior CD8(+) T-cell in humans without foreign helper epitopes. The
Journal of clinical investigation 2000, 105, R9-R14, doi:10.1172/JCI9051.
44. Fernandez-Arias, C.; Arias, C.F.; Zhang, M.; Herrero, M.A.; Acosta, F.J.; Tsuji, M.
Modeling the effect of boost timing in murine irradiated sporozoite prime-boost
vaccines. PLoS One 2018, 13, e0190940, doi:10.1371/journal.pone.0190940.
45. Pettini, E.; Pastore, G.; Fiorino, F.; Medaglini, D.; Ciabattini, A. Short or Long Interval
between Priming and Boosting: Does It Impact on the Vaccine Immunogenicity?
Vaccines (Basel) 2021, 9, doi:10.3390/vaccines9030289.
46. Lanzavecchia, A.; Sallusto, F. Understanding the generation and function of memory T
cell subsets. Current opinion in immunology 2005, 17, 326-332,
doi:10.1016/j.coi.2005.04.010.
47. Liu, Q.; Sun, Z.; Chen, L. Memory T cells: strategies for optimizing tumor
immunotherapy. Protein Cell 2020, 11, 549-564, doi:10.1007/s13238-020-00707-9.
48. Sallusto, F.; Lanzavecchia, A.; Araki, K.; Ahmed, R. From vaccines to memory and back.
Immunity 2010, 33, 451-463, doi:10.1016/j.immuni.2010.10.008.
49. Parvin, J.D.; Moscona, A.; Pan, W.T.; Leider, J.M.; Palese, P. Measurement of the
mutation rates of animal viruses: influenza A virus and poliovirus type 1. J Virol 1986, 59,
377-383, doi:10.1128/JVI.59.2.377-383.1986.
50. Vieira, S.M.; Pagovich, O.E.; Kriegel, M.A. Diet, microbiota and autoimmune diseases.
Lupus 2014, 23, 518-526, doi:10.1177/0961203313501401.

Abstract (if available)

Abstract The progression of cervical cancer from persistent Human Papillomavirus (HPV) infection remains a global health concern. The standard of care for cervical cancer is surgical resection, chemotherapy, or radiation, which is suitable for patients in the early stages of cancer. However, developing a treatment for recurrent, persistent, and metastatic cervical carcinoma remains challenging. Therefore, the investigating and targeting of the tumor microenvironment with various immunotherapies in the hopes of converting immunologically cold tumors to hot tumors have been at the forefront of research. Newman et al. hypothesized that repurposing a seasonal Influenza vaccine and injecting it into the tumor could elicit a proinflammatory immune response, thus overcoming the highly immunosuppressive tumor microenvironment seen in HPV-induced tumors. However, in this thesis, the data suggests that this conversion from an immunologically cold tumor to a hot tumor does not occur with the intratumoral injection of a repurposed Influenza vaccine. Instead, tumor reduction and complete regression are dependent on the length of time between the priming and boosting (PBIs) of a therapeutic vaccine in tumor models. This study, therefore, also used an HPV16E7E6 Venezuelan equine encephalitis virus replicon particle (VRP) vaccine to determine the effects of six different PBIs in an HPV model. Analysis of the data demonstrates that sufficient HPV 16 E7-specific CD8+ T-cells formation and proliferation was achieved with a minimum of a one-week prime-boost interval. Furthermore, longer PBIs resulted in higher differentiation of memory T cells and increased cytotoxicity. Our studies suggested a novel approach to apply therapeutic vaccines to tumor microenvironment and stimulate effective immunotherapies for cervical cancer.

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Asset Metadata

Creator Martinez, Emma Antoinette (author)

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

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2023-05

Publication Date 04/11/2023

Defense Date 03/30/2023

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

Tag cervical cancer,HPV,immunology,immunotherapy,intratumoral injections,OAI-PMH Harvest,repurposed vaccines,tumor microenvironment,virology

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Kast, Martin (committee chair), Mullen, Peter (committee member), Yuan, Weiming (committee member)

Creator Email em.martinez0287@gmail.com,emmamart@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113010395

Unique identifier UC113010395

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Legacy Identifier etd-MartinezEm-11604

Document Type Dissertation

Format theses (aat)

Rights Martinez, Emma Antoinette

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Type texts

Source 20230412-usctheses-batch-1020 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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