Cancer Letters 471 (2020) 88–102

Contents lists available at ScienceDirect

Cancer Letters
journal homepage: www.elsevier.com/locate/canlet

Mini-review

Human papillomavirus vaccine against cervical cancer: Opportunity and T

challenge
Renjie Wanga,1, Wei Panb,1, Lei Jina,1, Weiming Huanga, Yuehan Lia, Di Wua, Chun Gaoa,
Ding Maa, Shujie Liaoa,∗
a
Department of Obstetrics and Gynecology, Cancer Biology Research Center, Tongji Hospital, Tongji Medical College of HUST, Wuhan, Hubei, 430030, PR China
b
School of Economics and Management, Wuhan University, Wuhan, Hubei, 430072, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: Cervical cancer is one of the most common cancers threatening women's health, and the persistent infection of
Human papillomavirus high-risk human papillomavirus (HPV) is closely related to the pathogenesis of cervical cancer and many other
Cervical cancer cancers. The carcinogenesis is a complex process from precancerous lesion to cancer, which provides an ex-
Prophylactic vaccine cellent window for clinical prevention, diagnosis, and treatment. However, despite the various preventions and
Therapeutic vaccine
treatments such as HPV screening, prophylactic HPV vaccines, surgery, radiotherapy, and chemotherapy, the
disease burden remains heavy worldwide. Currently, three types of prophylactic vaccines, quadrivalent HPV
vaccine, bivalent HPV vaccine, and a new nonavalent HPV vaccine, are commercially available. Although these
vaccines are effective in protecting against 90% of HPV infection, they provide limited benefits to eliminate pre-
existing infections. Therefore, new progress has been made in the development of therapeutic vaccines.
Therapeutic vaccines differ from prophylactic vaccines in that they aim to stimulate cell-mediated immunity and
kill the infected cells rather than neutralizing antibodies. This review aims at systematically covering the pro-
gress, current status and future prospects of various vaccines in development for the prevention and treatment of
HPV-associated lesions and cancers and laying foundations for the development of the new original vaccine.

1. Introduction
Cervical cancer is one of the most common cancers threatening
women's health and is the fourth most common cancer in women
worldwide. According to the World Health Organization (WHO), cer-
vical cancer is the second most common cancer in women living in less
developed regions with an estimated 570000 cases and 311000 deaths
in 2018, and more than 85% of these deaths occurring in less developed
regions [1]. Currently, the study clarifies that cervical cancer is caused
by sexually transmitted infection with human papillomavirus (HPV),
and 84% of HPV-related cancer lesions are cervical cancer [2]. HPV
infection is the most prevalent sexually transmitted disease, which re-
sults in over 14000000 individuals annually and 80% of sexually active
individuals in their lifetime to be infected from HPV [3].
HPV is a double-stranded circular DNA virus with a genome of ap-
proximately 8000 base pairs. The genome encodes six early regulatory
proteins(E1, E2, E4, E5, E6, and E7) and two late structural proteins(L1
and L2) [4]. There are more than 100 types of HPV, of which at least 14
high-risk HPV types have been defined as carcinogenic [1]. HPV
infection is limited to the basal cells of the epithelium. HPV lesions arise
from the unchecked cell proliferation and mutations and lead to cancer
ultimately [5,6]. Due to the rapid immune clearance, most HPV infec-
tions do not cause symptoms and resolve spontaneously within 1 or 2
years; while those women who develop cervical cancer can test positive
for a high-risk HPV genotype 3–5 years prior to cancer [6]. Persistent
high-risk HPV infection has been shown to be involved in the increased
risk of high-grade cervical intraepithelial neoplasia (CIN) and cancer
[7]. The two HPV types, HPV16 and HPV18, have been identified as the
most prevalent types associated with cervical cancer and are accoun-
table for 70% of cervical cancers and precancerous cervical lesions [1].
Moreover, high-risk HPV types are responsible for anogenital and or-
opharyngeal cancer [8]. Immunosuppression is also a high-risk factor
for persistent infections with HPV and HPV-associated diseases [9].
However, studies have found that some patients with cervical cancer
are tested HPV-negative regardless of the detection methods. The rea-
sons lie in that testing results include the true negative and false ne-
gative [10]. Patients with HPV-negative cervical cancer are easily
missed diagnosis in routine screening, thus missing the opportunity for

∗
Corresponding author.
E-mail address: sjliao@tjh.tjmu.edu.cn (S. Liao).
1
Renjie Wang, Wei Pan and Lei Jin contributed equally to this work.

https://doi.org/10.1016/j.canlet.2019.11.039
Received 29 September 2019; Received in revised form 28 November 2019; Accepted 30 November 2019
0304-3835/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
R. Wang, et al.
early intervention and treatment. Though the proportion of these pa-
tients is small, more attention needs to be paid.
The comprehensive cervical cancer control is composed of primary
prevention (vaccination against HPV), secondary prevention (screening
and treatment of precancerous lesions), tertiary prevention (diagnosis
and treatment of invasive cervical cancer) and palliative care.
Screening of precancerous and cancerous lesions and the primary pre-
vention through vaccination allow reducing the incidence of HPV-re-
lated disease and death effectively [3]. Three types of prophylactic
vaccines, quadrivalent HPV vaccine, bivalent HPV vaccine, and the
nonavalent HPV vaccine have been approved for use in many countries.
These three vaccines prevent 70%–90% of the HPV-related cancers and
reveal safety and effectiveness in randomized trials and post-marketing
surveillance. The quadrivalent and nonavalent HPV vaccines also in-
clude the protection against HPV6 and HPV11, which cause anogenital
warts.
Currently, at least 118 million women have received one dose of
HPV vaccine worldwide [11]. These vaccines are effective at protecting
against HPV infection and neoplastic diseases; however, they are pro-
phylactic vaccines and provide no therapeutic benefit and limited
benefits to eliminating pre-existing infections. Moreover, the impact of
vaccination may not reduce the cancer incidence apparently because of
the long-time required for the development of precancerous lesions
[12]. Therefore, the ability to stimulate cell-mediated immune response
and kill the infected cells rather than neutralizing antibodies (nAbs) has
led to increasing interest in therapeutic vaccines. These vaccines could
have a significant impact on the morbidity and mortality related to
HPV. Different classes of therapeutic vaccines have been developed.
The safety and efficacy of therapeutic vaccines are the main concerns
on their development and utilization.
The extensive studies of virology and epidemiology have provided
an understanding of HPV and carcinogenesis and revealed opportu-
nities to prevent and treat HPV-related diseases. The immunotherapy
for cervical cancer and other diseases related to HPV infection has
become a research hotspot. In this review, we summarized the recent
advances in HPV-related cancer biology and the viral action in carci-
nogenesis. Then we discussed the progress, current status, and pro-
spects of various vaccines in development for the prevention and
treatment. This review focused on the development and safety of cur-
rent HPV prophylactic vaccines and the prospects for the landscape of
therapeutic vaccines.
2. HPV and cervical cancer
2.1. HPV genome and structure
The HPV belongs to the Papillomaviridae family of small, none-
nveloped, double-stranded DNA viruses, able to target epithelial cells of
skin, oral and anogenital mucosa [13]. The viral genome consists of
three regions:(1) early (E) region, containing several open reading
frames (ORFs), which encode the replication proteins (E1, E2, and E4)
and the oncoproteins (E5, E6, and E7); (2) late (L) region, containing
the late genes (L1 and L2), which encode major (L1) and minor (L2)
virus capsid proteins. The capsid of HPV is 50–60 nm in diameter and a
T = 7 icosahedral formation consisting of 72 L1 protein pentamers
(360 copies). The L2 protein is located in the center of the pentamers in
an L2:L1 ratio of 1:5–1:10 [14]; (3) upstream regulatory region (URR),
also known as non-coding region (NCR) or long control region (LCR),
which is located between the L1 and E6 ORFs and contains the reg-
ulatory elements associated with the viral DNA replication and tran-
scription [13].
2.2. HPV classification and oncogenic genotypes
According to the PV study group within the International
Committee on Taxonomy of Viruses [15], there are 53 genera in the
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Cancer Letters 471 (2020) 88–102
family Papillomaviridae, among which 5 genera (alphapapilloma-
viruses, betapapillomaviruses, gammapapillomaviruses, mupapilloma-
viruses, and nupapillomaviruses) are associated with HPVs. The phy-
logenetic classification of HPVs is characterized by genotype and based
on the L1 ORF sequence homology, which is the most conserved ORF. A
novel HPV type is defined as differences over 10% on L1 ORF sequence
from other known genotypes, while a subtype is defined as differences
between 2 and 10%. The HPV types are numbered based on the date of
discovery [16,17]. Currently, more than 200 HPV types have been
identified by the International HPV Reference Center [18], and the
number has been a rapid increase with the application of new mole-
cular tools such as next-generation sequencing during the past decade
[19].
HPVs induces different lesions and they can result in overlapping
outcomes. The betapapillomaviruses and gammapapillomaviruses are
mainly cutaneous HPV types [20]. Based on epidemiological data, the
International Agency for Research on Cancer (IARC) designates a sub-
group of alphapapillomaviruses (HPV16, HPV18, HPV31, HPV33,
HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59)
as group 1 (carcinogenic), which is considered as high-risk HPV types.
The IARC group 2A (probably carcinogenic) and group 2B (possibly
carcinogenic) include several HPV types (such as HPV26, HPV53,
HPV66, HPV67, HPV68, HPV70, HPV73, and HPV82). The high-risk
HPV types are the etiological agents of anogenital and oropharyngeal
cancer, such as cervix, vagina, vulva, penis, anus, and head and neck
cancers [21]. Of new cancer cases in 2012, 15.4% of cancers worldwide
are caused by carcinogenic infections [22]. 96% of cervical cancers are
attributed to the 13 HPV types in groups 1 and 2A (HPV68) [23].
However, little studies are indicating that HPV68 is a high-risk HPV.
Additional alphapapillomaviruses (HPV26, HPV30, HPV34, HPV53,
HPV66, HPV67, HPV69, HPV70, HPV73, HPV82, HPV85, and HPV97)
are associated with infrequent incidences of cervical cancer and are
referred to as group 2B, which is hard to assess the carcinogenicity
[10]. Moreover, the alphapapillomaviruses also includes low-risk HPV
types that have been linked to benign lesions, such as HPV 6 and HPV
11, which are responsible for approximately 90% of anogenital wart
cases [24].
2.3. HPV pathogenesis and cervical carcinogenesis
Located between the vagina and the uterus, the cervix is char-
acterized by the simple columnar secretory epithelium. In contrast, the
vaginal cavity is the stratified non-keratinizing squamous epithelium.
The squamocolumnar junction is particularly vulnerable to transfor-
mation by high-risk HPV and is the area in which over 90% of lower
genital tract malignancies initiate [25,26]. As there are two kinds of
epithelial cells (squamous and glandular cells) in the transition zone,
cervical cancers can occur in two different forms. Mucosa and skin are
the most common areas of infection for HPV [27]. Women, especially
newly sexually active adolescent and young adult women, may acquire
HPV via sexual exposures with an infected partner. HPV infects the
basal epithelial cells through the epithelial abrasion [28] and subse-
quently induces cervical dysplasia and CIN, which typically develops
into cervical cancer due to the persistent infection of high-risk HPV. The
carcinogenic progression to cervical cancer is a slow process and can be
divided into several stages. In the first stage, HPV infection can be
detected in clinical, but the majority is usually transient and becomes
undetectable without intervention within 1–2 years due to the viral
clearance. After infection, the early HPV genes (E1, E2, E4, E5, E6, and
E7) are expressed in the productive lifecycle. In the upper layers of
epithelium, the viral genome is replicated, the late HPV genes (L1 and
L2) and E4 are expressed, and progeny viral particles are assembled.
The shed virions repeat the virus life cycle. Some HPV infections persist
beyond 1 year and thus increasing the risk of progression to high-grade
squamous intraepithelial lesion (CIN2/3) or potentially invasive carci-
noma if untreated [29]. The development of cervical cancer is
R. Wang, et al.
dependent not only on the negative regulation of cell-cycle control and
the accumulation of genetic damage by viral oncoproteins, but also the
immune evasion [30].
2.3.1. HPV infection and immune response
Most natural HPV infections are restricted to the intraepithelial
layer of the mucosa and do not develop into cancer. Approximately
90% of patients with HPV infection present an innate and humoral
immune-mediated viral clearance within a few months after viral in-
fection [31,32]. 10% of the patients have a persistent infection and an
increased risk of cancer in about 1% of all patients [32]. The persistent
HPV infection is an essential event for the development of cervical
cancer. The competition between persistence and clearance is central in
the HPV carcinogenesis. During the progression process of precancerous
lesion, the host immune system reveals infiltration of CD4+, CD8+
lymphocytes and macrophages, an increase of proinflammatory cyto-
kines, and induction of neutralizing antibodies [33]. However, the
immune response against infection is slow and induces low antibody
titers. The nAbs are triggered after viral infection and target only the
viral particles instead of virus-infected cells, which thus cannot cure the
infection. Moreover, the role of macrophages and natural killer (NK)
cells involved in the immune response is also unclear. The main an-
tigen-presenting cell (APC) in the epithelium, Langerhans cells, are
essential in the recognition of HPV infection and the induction of cel-
lular immune response [34].
The combination of innate and adaptive immunity prevents HPV
infection [34]. The effector T cells targeting early viral proteins can
eliminate virus-infected cells. The helper T cells identifying L1 protein
can induce the nAbs, which can prevent virus transmission and re-
infection of the host [35]. However, the immune response able to
protect from reinfection by the same or even other HPV types is dis-
puted [36]. In a phase of HPV persistence during which the host im-
mune system fails to eliminate the virus, the expression of E6 and E7
protein can promote lesion progression, which typically results from the
methylation of the E2 promoter and viral integration, contributing to
immune deviation [4,37]. Despite there are abundant HPV-specific T
cells in neoplastic tissue, the immune system cannot eradicate the
tumor, suggesting the existence of an immunosuppressive tumor mi-
croenvironment [38]. The mechanisms of HPV to evade the immune
system include: (1) the downregulation of antigen presentation ma-
chinery such as MHC class I; (2) resistance to the cytotoxic T lympho-
cyte (CTL)-mediated cytotoxicity; (3) aberrant expression of immune
factors such as the downregulation of interferon, upregulation of in-
terleukin (IL)-10 and transforming growth factor (TGF)-β1; (4) the at-
traction of immune cells that inhibit the immune response such as the
immature dendritic cells (DCs), tolerogenic DCs, T regulatory cells
(Treg), tumor-associated macrophages (TAMs), and myeloid derived
suppressor cells (MDSCs) [38,39]. In addition, the overexpression of E6
and E7 protein compromises cellular DNA repair, leading to genomic
instability and immune escape [40].
2.3.2. The life cycle of HPV
Epithelial abrasion leads to the denudation of the basement mem-
brane (BM) and guarantees the HPV access to the basal keratinocytes.
During the HPV infects the epithelium, the virus sequentially engages
the host proteins and delivers the genetic information into target cells.
This multistep process starts with binding of the major capsid protein
L1 to heparin sulfate proteoglycan (HSPGs) on the BM, inducing con-
formational changes in the viral capsid [41]. Recent studies also reveal
that the L1 protein can also bind to other integrins, such as laminin 5
[42]. The conformational change in the capsid exposes a conserved site
on the amino terminus of minor capsid protein L2, which is then
cleaved by extracellular furin [43]. The cleavage reveals several con-
served protective epitopes of L2 protein and is critical to promote a
second conformational change in viral capsid that allows the virus
binding to cell receptors, such as α6β4 integrin [44]. This is followed by
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Cancer Letters 471 (2020) 88–102
virus uptake and internalization via endosomal vesicles into the target
cells [45]. Once HPV has reached the cell, L2 protein is necessary for
infection as the endosomal virus engages the retromer and travels to-
wards the nucleus [46,47]. The L2 protein chaperones the viral genome
to promyelocytic leukemia nuclear bodies for efficient viral transcrip-
tion [48,49].
As the HPV does not express polymerases and is unable to self-re-
plicate, early viral proteins E1 and E2 are expressed first and regulate
the host cell life cycle into the S phase, which supplies DNA poly-
merases for viral replication [27]. Use of the host replication machinery
to perform DNA replication generates 50–100 copies of the genome per
cell [48]. As the cells undergo cell division, viral genomes segregate
between the daughter cells. The late proteins L1 and L2 are regulated in
a cell differentiation-dependent manner and are expressed in mature
squamous cells. As the epithelial differentiation of daughter cells
leaving the BM and moving up, the cells perform the high copy number
HPV replication (> 103 per cell) and the expression of L1 and L2 pro-
tein in the outermost layer of the epithelium, which is recognized as the
terminally differentiated layers of the epithelium. The new virions
which are composed of the newly synthesized viral DNA are deposited
in squames and are shed [50]. Consequently, the virus life cycle is re-
peated. As the immunogenic virions mainly stay in the outer sites of the
epithelium, HPV can escape from the host immune system. Currently,
the prophylactic vaccines targeting the capsid antigens have no ther-
apeutic effect because the basal epithelial cells carrying HPV express
only the early genes and the L1 protein is expressed in the epithelium
before viral shedding. Clearance of infected cells depends on inducing
cell-mediated immune response [51].
Through the expression of the three viral oncoproteins E5, E6, and
E7, HPV obtains the host replication factors and drives the host kera-
tinocyte into S phase [4]. The E6 protein promotes the ubiquitin-de-
pendent proteasomal degradation of apoptosis regulator protein p53
[52] and activates MYC expression and telomerase, which lead to
promoting cell survival and extended cell life. The E7 protein mainly
binds to the hypophosphorylated form of tumor suppressor retino-
blastoma protein (pRb) and promotes the pRb phosphorylation, leading
to the movement of the cell life cycle into the S phase and subsequent
DNA synthesis and cell proliferation. The E6 and E7 oncoprotein modify
the control of the cell cycle and regulate apoptosis, resulting in genomic
instability and eventually cancer [4]. The E5 protein has been identified
as a channel protein that can modulate ion homeostasis, vesicle traf-
ficking, virion production, and viral genome entry [53]. It also stimu-
lates the epidermal growth factor (EGF)-mediated cell proliferation,
inhibits the apoptosis induced by tumor necrosis factor ligand and
CD95 ligand [54], and modulates several cellular pathways involved in
cell adhesion, cell motility and mitogenic signaling [55]. In addition,
the viral genome is able to integrate into the host's chromosomal DNA
at random positions, which is associated with the transition into in-
vasive carcinoma [56]. The integration may disrupt the viral genes
during the linearization of the genome. For example, loss of E2, the
transcriptional repressor of E6 and E7 protein, could cause dysregulated
expression of these oncoproteins and further lead to aberrant pro-
liferation and malignant progression.
2.4. HPV-negative cervical cancer
Invasive cervical cancer (ICC) can be divided into three groups:
squamous cervical cancers (SCC) which account for 75–90% of ICCs,
adenocarcinomas (ADC), and adenosquamous cell carcinomas (ASC)
[57]. Studies have found that almost 100% of the SCC and 86% of the
ADC are HPV positive [58,59]. The prevalence of HPV among ADC
varies between the subtypes. The usual type, intestinal, villoglandular,
signet-ring cell and the endometrioid ADCs have a high prevalence of
HPV positive. While the gastric type, the mesonephric type, and the
endometrioid ADC from the upper part of the endocervix and lower
uterine segment are mainly HPV negative [60]. In clinical, high-risk
R. Wang, et al.
HPV-negative cervical cancer is extremely rare. The reasons for HPV
negative include multiple aspects: the detection methods of HPV, cer-
vical cancers independent of high-risk HPV, cervical cancers that lose
HPV expression, cervical cancers that associated with non-high risk
HPV, and cancers that are misclassified such as uterine endometrioid
adenocarcinoma or metastasis from other primary tumors. The HPV
testing methods and the misclassification of cancers are the common
reasons for false-negative HPV tumors. There are three main options for
cervical cancer screening: cytology, HPV testing, and cytology-HPV co-
testing [61]. Cytology-based screening is the most widely used with a
lower sensitivity that ranges between 50 and 70% compared to HPV
testing with a sensitivity of more than 90% [62]. Screening with HPV
testing (preferably using triage markers) is still superior to cytology
even though the HPV-negative cancers may be missed diagnosis, and
the HPV genotyping cannot definitively differentiate the different steps
in the natural history of cancer such as a transient infection, an early
persistent infection, and a prevalent precancer [60,63]. Moreover, only
a very small proportion of cervical cancers are HPV true negative [40].
HPV vaccination and HPV screening cannot prevent or detect these
cancers. Therefore, the colposcopy and biopsy should be performed in
time for suspected clinical cases, so as to early diagnose and reduce
missed diagnosis. Whether the HPV-negative cervical cancer patients
need an individualized treatment protocol requires further research,
and the primary HPV screening should be continued.
3. HPV vaccine against cervical cancer
3.1. The requirement for vaccine
Inducing nAbs is the main basis for vaccine-induced protection.
When encountering with HPV, the vaccine binds to the virus and pre-
vents it from infecting the epithelium. However, the level of antibodies
produced in natural infections is usually insufficient to prevent sub-
sequent reinfection. The infected cervical site lacks secondary lymphoid
tissue where contains a large number of memory B cells, subsequently
producing antibody and neutralizing the virus before uptake [64]. Be-
sides, to ensure a protective immune response throughout sexually
active life, high and sustained levels of nAbs induced by vaccination are
required. Therefore, an ideal HPV vaccine should provide an improved
protective immune response, protection against all high-risk HPV types
and other probably and possibly carcinogenic types. Currently, the HPV
vaccines are based on virus-like particles (VLPs), which are assembled
from recombinant HPV capsid proteins and are non-infectious due to
the lack of viral DNA. Vaccination with VLPs can induce different types
of type-specific nAbs, which can bind to the native virus particles and
neutralize the virus by preventing uptake by the target cell.
The cellular immune responses also play a crucial role in the re-
gression of precancerous lesions and the clearance of persistent infec-
tion. It has been reported that the increased CD4: CD8 ratios in the
stroma, the CD4+T cell helper responses to E2, and the infiltration of
CD8+T cells specific for the E6 and E7 protein have been detected in
the spontaneous regression of CIN [65–68]. Induction of effective cel-
lular immunity to E6, E7, and other viral antigens, may be critical to the
efficacy of therapeutic HPV vaccines. The ideal HPV therapeutic vac-
cines should target these proteins to induce a robust tumor-specific T-
cell type 1 and cytotoxic lymphocyte responses where CD4+ T-cells
secrete large amounts of cytokines such as IFN-γ and IL2 labeling in-
fected and malignant cells and cytotoxic CD8+ T-cells eliminate in-
fected cells by secreting granzyme B and perforin which lead to cell
death [69,70].
3.2. Prophylactic HPV vaccines
Currently, three licensed prophylactic HPV vaccines are available
(Table 1): Gardasil®, a quadrivalent HPV (4vHPV) vaccine that targets
HPV6, HPV11, HPV16, and HPV18; Cervarix®, a bivalent HPV (2vHPV)
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Cancer Letters 471 (2020) 88–102
vaccine that targets HPV16 and HPV 18; and Gardasil 9®, a nonavalent
HPV (9vHPV) vaccine that targets HPV6, HPV11, HPV16, HPV18,
HPV31, HPV33, HPV45, HPV52, and HPV58 (Fig. 1). The 4vHPV and
9vHPV vaccines were licensed in 2006 and 2014, respectively, and are
produced in the Saccharomyces cerevisiae (S. cerevisiae) expression
system with the amorphous aluminum hydroxyphosphate sulfate
(AAHS) adjuvant. The 4vHPV vaccine is indicated for the prevention of
genital warts, precancerous or dysplastic lesions, and cervical cancer in
females and males aged 9–26 years [71]. The 9vHPV vaccine covers 5
additional HPV types and is approved for the prevention of genital
warts, precancerous or dysplastic lesions, and cervical, vulvar, vaginal,
and anal cancer in females and males aged 9–45 years [72]. The 2vHPV
received first approval in 2007 is produced in the baculovirus expres-
sion vector system with the AS04 adjuvant containing alumi-
niumhydroxide and 3-deacylated monophosphoryl lipid A (MPL) [73].
HPV vaccination has the potential to prevent cervical cancer, CIN and
adenocarcinoma in situ (AIS) [74,75].
These three prophylactic vaccines are the application of VLPs as-
sembled from the recombinant expression of L1. It has been shown that
the recombinant L1 protein can self-assemble into VLPs that are lack of
the oncogenic viral genome and morphologically similar to the native
virions [41,76]. Vaccination with HPV vaccines can protect against
HPV infections and are immune to subsequent viral challenge by the
induction of high and durable titers of nAbs that bind to the native
virion and neutralize the virus. The immune responses induced by the
HPV vaccination are significantly stronger than those seen in natural
HPV infections. However, it has been found that these vaccines provide
limited benefits to eliminate pre-existing infections and elicit type-re-
stricted protection. HPV vaccines need to contain a wider range of VLP
types for broad protection.
3.2.1. Vaccine immunogenicity
Evaluation of the type-specific immune response to HPV following
vaccination includes cellular and humoral assays. The cellular assays
include the evaluation of T cell and memory B cell responses. Currently,
much more emphasis has been put on humoral responses as the nAbs
play a critical role in the protection against infection and subsequent
disease [77]. Serology assays include detecting antibodies (neutralizing
assays and binding assays) and measuring antibody binding strength
(avidity) [78]. Three main types of serological assays have been used to
evaluate the immune responses to HPV vaccines: neutralization assays
[79], competitive immunoassays [80], and VLP enzyme-linked im-
munosorbent assay (ELISA) [81]. The spectrum and potential relevance
of the detected antibodies among these approaches are different. Neu-
tralization assays are considered the most relevant for measuring the
biological activity of the antibodies. Competitive immunoassays, such
as the competitive Luminex immunoassay (cLIA), estimate neutralizing
activity by measuring competition of test serum with neutralizing
monoclonal antibodies. In contrast, ELISAs detect all antibodies, re-
gardless of neutralization ability.
Ongoing research reveals that the antibody titers elicited by vacci-
nation with Gardasil®, Cervarix®, and Gardasil 9® can persist at least
9.9, 10.0, and 5.0 years, respectively, when women aged 9–15 years
receive a 3-dose schedule [82]. The seropositivity rates following vac-
cination with three doses of 4vHPV vaccine for the total im-
munoglobulin (IG)G assay were 94.3, 89.4, 99.5, and 88.8%, respec-
tively [83]. Recent studies have shown that HPV16 and HPV18
antibody levels after vaccination with the 2vHPV vaccine for at least
9.4 years are significantly higher than natural infection levels [84]. The
4vHPV vaccine elicits stable HPV16 antibody levels for at least 9 years
[85]. Besides, the HPV16 and HPV18 antibody responses induced by
the 9vHPV vaccine are comparable to the Gardasil®. Follow vaccination
with 3 doses of Gardasil 9®, the HPV antibodies persisted through 5
years with seropositivity rates ranging from 77.5% to 100% [86].
R. Wang, et al. Cancer Letters 471 (2020) 88–102

Table 1
Characteristics of three commercially available HPV vaccines.
Cervarix Gardasil Gardasil 9

Antigen L1 VLP of HPV-16 and 18 L1 VLP of HPV-6, 11, 16 and 18 L1 VLP of HPV-6, 11, 16, 18, 31, 33, 45, 52 and 58
Expression system Baculovirus expression vector system S. cerevisiae S. cerevisiae
Adjuvant (μg) AS04: AAHS (225) AAHS (500)
MPL (50); Aluminum hydroxide salt (500)
Indications: Females, age between 9 and 25 years Females and males, age between 9 and 26 years Females and males, age between 9 and 45 years
Manufacturer GlaxoSmithKline Biologicals Merck & Co., Inc Merck & Co., Inc

VLP: virus-like particle; S. cerevisiae: Saccharomyces cerevisiae;AS04: Adjuvant System 04; AAHS: amorphous aluminumhydroxyphosphate sulfate.

Fig. 1. A brief history of HPV and HPV

vaccine research
In the 1930s, the carcinogenic potential of
rabbit papillomaviruses in cottontail and
domestic rabbits were demonstrated for the
first time [194,195]. However, research on
the human wart virus remained low. Be-
tween 1974 and 1976, researchers began to
analyze the possible role of HPV in cervical
cancer [196,197]. In 1976, Meisels and
Fortin [198]found the appearance of Koilo-
cytes in cervical smears, which indicated the
presence of papillomavirus infection. In
1983 and 1984, HPV16 and HPV18, the
cervical-cancer-linked HPV type, were iso-
lated from cancer biopsies of the cervix, re-
spectively [199,200]. Between 1985 and
1992, the activities of HPV oncogenes in cervical cancer etiology have been determined [201–205]. In 1987, de Villiers et al. [206] conducted the first epide-
miological study of HPV infections. In 1991, the first VLPs were produced [207]. In 1999, HPV was first proposed as a necessary cause of cervical cancer development
[208]. In 2001, Harro et al. reported the safety and immunogenicity of VLP vaccines in humans [209]. In 2006, the first prophylactic HPV vaccine (Gardasil®) was
licensed. Subsequently, the bivalent HPV vaccine (Cervarix®) and nonavalent HPV vaccine (Gardasil 9®) received approval in 2007 and 2014, respectively.

3.2.2. Vaccine efficacy against cervical cancer and cross-protection
Prophylactic HPV vaccines have been licensed in many countries for
over 10 years. The introduction of HPV vaccines has shown the re-
duction of HPV prevalence and HPV-related diseases, such as genital
warts, CIN and cervical cancer [3,87–89]. The implementation of HPV
vaccination program will result in a dramatical decrease in cervical
cancer rate. Recently, a meta-analysis of 4vHPV vaccines showed that
up to approximately 90% for HPV-6/11/16/18 infection and genital
warts reduced compared to unvaccinated populations. The incidence of
low-grade cytological cervical abnormalities (approximately 45%) and
high-grade histologically proven cervical abnormalities (approximately
85%) continued to decline [88]. The 2vHPV vaccine was evaluated in
phase III randomized, double-blind, controlled Papilloma TRIal against
Cancer In young Adults (PATRICIA). The vaccine showed 92.9% ef-
fective against CIN 2 + associated with HPV-16/18, 54.0% effective
against CIN2+ associated with 12 non-vaccine oncogenic types, and
consistent cross-protective efficacy against HPV-31/33/45/51 in the
woman aged 15–25years [90,91]. The trial also showed high vaccine
efficacy against CIN3+ and AIS irrespective of HPV type in the lesion
[92]. In another phase 3, double-blind, randomized controlled VIVIANE
study, the 2vHPV vaccine showed sustained protection against infec-
tions, cytological abnormalities, and lesions in the woman older than 25
years [93,94]. Efficacy of the 4vHPV and 9vHPV vaccine was evaluated
in a randomized, international, double-blind, phase 2b-3 study in
14215 women aged 16–26 years. Compared to the 4vHPV vaccine, the
9vHPV vaccine represented 97.4%efficacy against high-grade cervical,
vulvar and vaginal disease associated with HPV-31/33/45/52/58 (0.5
and 19·0 cases per 10 000 person-years in the 9vHPV and 4vHPV
groups, respectively) and non-inferior antibody response to HPV-6/11/
16/18 in the 9vHPV and 4vHPV vaccines. The 9vHPV vaccine provided
sustained efficacy up to 6 years and broader coverage and prevention
for approximately 90%of cervical cancers and HPV-related diseases
[95,96]. Overall, all three vaccines have demonstrated sufficiently high
and durable levels of antibody response to protect against infection and
subsequent disease [97]. The vaccine-mediated protection was not
type-specific because of the cross-protection, such as HPV16 with
HPV31 and HPV33, and HPV18 with HPV45. These related types share
considerable amino acid sequence in the major capsid protein L1 [98].
Therefore, 4vHPV and 2vHPV vaccines induced cross-protection against
HPV types which were not included in the vaccines, such as HPV31,
HPV33, HPV45, and HPV51 [91,99]. The 2vHPV vaccine exerted
higher cross-protection for HPV31, HPV33, and HPV45 than the 4vHPV
vaccine. The 2vHPV vaccine showed 93% efficacy against CIN3 irre-
spective of the HPV type, while only 52% of CIN3is related to HPV16
and HPV18 [92,100].
3.2.3. Vaccination schedules and strategies
The prophylactic HPV vaccines were initially licensed using a 3-
dose vaccination schedule. Recent studies have shown that 2 doses or
even 1 dose of 2vHPV vaccine were highly efficacious compared to
the3-dose regimen [101]. An observer-blind study confirmed that su-
perior HPV-16/18 antibody responses were induced in girls aged 9–14
years who received 2 doses of 2vHPV vaccine at months 0 and 6
compared with those who received 2 doses of 4vHPV vaccine at months
0 and 6 or 3 doses of 4vHPV vaccine at months 0, 2, and 6 [102]. The
cost-effectiveness analysis showed that if the vaccines could provide
protection for more than 20 years, the 2-dose vaccination schedule
seemed to be the most cost-effective option, while it was unknown
whether the duration of the 2-dose schedule can last for 20 years [103].
Based on these studies, the World Health Organization (WHO) has now
recommended that people aged between 9 and 15 years should take 2
doses of HPV vaccines at least six months after the first dose (Time
interval between the first and second dose should be 6–15 months).
Individuals who receive their two doses less than five months at in-
tervals and are older than 14 years old or immuno-compromised re-
gardless of age require a third-dose schedule [104]. Additionally, sev-
eral randomized studies of 4vHPV, 2vHPV, and 9vHPV vaccines
demonstrated that women receiving the 2-dose regimen showed a non-

92
R. Wang, et al.
inferior or uncertain immune response compared with the 3-dose re-
gimen [105–107]. Recent studies have shown that a single dose of
4vHPV vaccine [108] or 2vHPV vaccine [109] provide protection
against persistent HPV infection similar to the 3-dose regimen.
3.2.4. Safety and limitations of prophylactic HPV vaccines
To date, more than 200 million doses of prophylactic HPV vaccines
have been administered worldwide [88]. Increased evidence confirms
the vaccine safety. However, a series of safety issues have emerged,
such as pain, redness, or swelling in the arm where the shot was given,
fever, headache or feeling tired, nausea, and muscle or joint pain. The
Centre of Disease Control and Prevention (CDC) and FDA use three
systems to monitor the vaccine safety: The Vaccine Adverse Event Re-
porting System (VAERS), the Vaccine Safety Datalink (VSD), and the
Clinical Immunization Safety Assessment (CISA) Project. In 2014, the
safety of 9vHPV vaccine was evaluated in seven studies prior to ob-
taining the license by the FDA. These pre-licensure studies have in-
dicated that the 9vHPV vaccine has similar safety to 4vHPV vaccine. In
addition, approximately 92% of the 4vHPV vaccine reports were clas-
sified as non-serious [110]. Serious adverse events are very rare after
vaccination with HPV vaccines, and the reasons are unclear. Based on
the VAERS data [111,112] and a series of studies in Southern Italy
[113], Denmark [114], and Indonesia [115], the overall results of these
studies demonstrated that no serious adverse events following im-
munization were medically confirmed to be related to HPV vaccines.
The adverse events related to HPV vaccination, such as lymphadeno-
pathy in 2 clinical cases [116], myasthenia gravis in a 23-year-old
woman [117], were reported on individual cases. Recent evidence has
suggested that HPV infection may affect reproductive health and fer-
tility [118]. The fetal adverse events following HPV vaccination mainly
relate to spontaneous miscarriage, while the vaccines do not seem to
escalate the occurrence of miscarriage. The HPV vaccination during
pregnancy, peripregnancy, or immediately post-conception is not as-
sociated with risks of adverse pregnancy outcomes [119–121]. The HPV
vaccination is not recommended for pregnant women, but breast-
feeding women may get the vaccine and receiving HPV vaccines during
pregnancy should not cause panic [110]. Moreover, the nonavalent
HPV vaccine was evaluated in Sprague-Dawley rats and the researchers
found that there were no effects on fertility or reproductive perfor-
mance and no evidence of developmental toxicity [122], supporting the
safety profile of 9vHPV vaccine. In a word, the HPV vaccine is recently
commercially available, and post-market surveillance must be main-
tained to detect any adverse events following HPV vaccination. Addi-
tional studies are needed to imply the possible cause-effect relationship
between the HPV vaccination and serious advents and to validate the
safety of HPV vaccines.
Currently, these three vaccines have been widely used and effec-
tively prevent HPV infections caused by the targeted types. However,
there is still a considerable population suffering from high-risk HPV
infections and associated diseases. There are some restrictions on this.
Firstly, the main factor limiting vaccination is inadequate population
coverage and global vaccine uptake. Developing countries suffer the
greatest burden of HPV infection and malignancy due to the lack of
resources to implement efficient vaccination and screening programs
[123]. Therefore, many women only detect infections when they have
exceeded CIN 1 or have developed cancer. Additionally, the high prices
of vaccines make them unable to meet the needs of low-income popu-
lations. By mid-2016, only 8% of low-to middle-income countries had
introduced the HPV vaccine, compared with71% in high-income
countries [11]. The refrigeration of the vaccine such as the cold chain
also restricts large-scale deployments in developing countries. This may
be solved by lyophilized formulations or heat-stable capsomer pre-
parations [124]. Secondly, all three vaccines have little benefit for
people already infected with HPV. The therapeutic vaccines that are
capable of inducing a cell-mediated immune response againstE6 and E7
proteins and clearing the infected cells are required. Thirdly, the
93
Cancer Letters 471 (2020) 88–102
limited cross-protection and HPV genotypes in prophylactic HPV vac-
cines. Currently, the 2vHPV and 4vHPV vaccines provide approxi-
mately 100% protection against infection with HPV16 and HPV18
[125]. However, the non-vaccine high-risk HPV types account for ap-
proximately 30% of cervical cancers [126]. Therefore, there is an ur-
gent need for vaccines that broadly target HPV types.
3.2.5. Future prospects of prophylactic HPV vaccines
3.2.5.1. L2-based HPV vaccines. A key feature of L1 VLPs is their high
immunogenicity, even in the absence of adjuvant. Vaccination with
HPV L1 VLPs provides HPV type-specific protection and cross-
protection against certain HPV types. In contrast, the L2 protein
cannot form VLP alone and is poorly immunogenic. The L2 protein is
located inside the HPV capsid and therefore cannot enter the immune
system [127]. Compared with 360 L1 in the capsid, there are only
12–72 L2copies per virion, and thus the interval between L2 is further,
which may result in poor immunogenicity of L2 in the capsid
[128,129]. Despite these differences, vaccination with L2 using
adjuvants such as alum provides long-lasting immunity in animal
models, and passive-transfer studies have shown that low titers of
nAbs with moderate affinity were sufficient for protection [130,131].
Moreover, a variety of strategies have been applied to elicit robust
immune responses, including displaying L2 epitopes on a carrier
protein, the use of peptide multimers, and binding to the fusion
partner [14,132]. Therefore, the L2-based vaccines have the potential
for providing protection against various HPVs at low vaccination prices
due to their simple form [133,134].
3.2.5.2. The second-generation prophylactic HPV vaccines. The three
currently licensed prophylactic HPV vaccines use insect or yeast cell
expression systems and their manufacturing processes are therefore
complex, which is the main reason for the high vaccine price. Numerous
workers have attempted to enhance the production processes for VLP-
based vaccines and have achieved considerable success. HPV L1 VLP-
based vaccines produced in S. cerevisiae, Escherichia coli (E. coli) and
methylotrophic yeast species, such as Hansenulapolymorpha (H.
polymorpha) and Pichia pastoris (P. pastoris) are currently being
studied in preclinical or clinical trials (Table 2).
3.3. Therapeutic HPV vaccines
Prophylactic HPV vaccines aim to induce humoral immunity against
target late proteins L1 or L2, eventually leading to antibody induction
and neutralization of antigens. In contrast to the prophylactic vaccines,
the main aim of therapeutic vaccines is to eliminate the precancerous
lesions and the persistent infection caused by HPV. The early protein E6
and E7 are expressed constantly in HPV infection and most cervical
cancer and precancerous lesions, but not in normal tissues. More im-
portantly, the sustained expression E6 and E7 are essential for the in-
duction and maintenance of the malignant phenotype of cancer cells,
and therefore E6 and E7 are the ideal target proteins for the develop-
ment of antigen-specific immunotherapy of HPV infections and related
diseases. In the past two decades, various therapeutic HPV vaccines
targeting E6, E7, and other viral antigens have been widely studied,
including live vector-based vaccines, nucleic acid/protein/peptide-
based vaccines, and cell-based vaccines. Therapeutic vaccines are di-
verse and the following table describes the characteristics of vaccine
platform technologies (Table 3) and the progress of current therapeutic
HPV vaccines.
3.3.1. Live vector-based vaccines
Live vector-based vaccines can be subdivided into recombinant viral
and bacterial vector-based vaccines that can carry HPV antigens, re-
plicate inside the host cells, and induce immune responses against HPV.
In addition, they can drive antigen presentation through both major
histocompatibility complex (MHC) class I and class II pathways, thus
R. Wang, et al. Cancer Letters 471 (2020) 88–102

providing high levels of immunogenicity. However, the generation of

Clinical trial number
nAbs against pre-existing virus and bacteria may limit the use of these

NCT03935204

NCT01735006

NCT02710851

NCT01548118

NCT03813940

NCT04083196
vectors and restrict repeated therapy. The potential immunodominance
to the vectors instead of the HPV antigen remains another obstacle in
the use of live vector vaccines [135,136].

3.3.1.1. Bacterial vector vaccines. An ideal vaccine vector should trigger

National Vaccine and Serum

both potent innate and adaptive immune responses in the inoculated
Xiamen Innovax Biotech

Xiamen Innovax Biotech

Xiamen Innovax Biotech

host. Attenuated bacteria can stimulate both cellular and humoral

Biotechnology Co.,Ltd
immune responses to protect the host against challenge of pathogens.

Xiamen University
Shanghai Zerun
Conserved molecular patterns, such as lipoteichoic acid in gram-

Institute, China
Manufacturer

positive bacteria, lipopolysaccharides in gram-negative bacteria,

CO., LTD

CO., LTD

Co., Ltd peptidoglycans, and flagellin, are recognized by pattern recognition

receptors (PRRs) and induce innate immune responses [137]. Using a
bacterial vector to deliver heterologous antigens is an effective method
Phase III

for developing new vaccines. Although safety and efficacy issues have
Phase Ⅱ

Phase Ⅱ

Phase I

Phase I

Phase I
Status

always limited the development of bacterial vector vaccines, several

bacteria vectors have been tested in clinical trials, such as Listeria
monocytogenes (Lm), Lactobacillus casei (L. casei), Lactobacillus lactis and
Salmonella. Compared to other vaccines, bacterial vector shows several
Aluminum

Aluminum

advantages: the induction of innate and adaptive immune response to

hydroxide

hydroxide
Adjuvant

pathogens could enhance attenuated bacteria to enable specific immune

N/A

N/A

N/A

N/A

response against target antigens [138]; Using bacterial vector to deliver

heterologous antigens could reduce the difficulties of target antigen
purification and thus relatively simple and inexpensive to large-scale
Hanson's Yeast

manufacture and inoculation.

Expression

Lm is a gram-positive intracellular facultative anaerobe and is

system

E. coli

E. coli

E. coli

E. coli
Yeast

commonly used as a bacterial vector for vaccine development. By ex-

pressing membrane-active virulence factors listeriolysin O (LLO) and
phospholipase C, Lm can degrade the phagolysosomal membrane and
Acuminata

• Condylomata Acuminata

Acuminata
Intraepithelial

• Human Papillomavirus
Intraepithelial

Cervical
Intraepithelial

entry into the cytoplasm of the host [139]. Therefore, Lm could access
Infection

the cytoplasm of antigen-presenting cells (APCs), lead to the en-

HPV-related disease

Cancer

Cancer

Cancer

dogenous antigen-processing pathway and elicit both CD4+ and CD8+

Infection
• Condylomata

• Condylomata
• HPV-Related
Carcinoma

T cell-mediated immune responses through MHC class II and MHC I

Neoplasia

Neoplasia

Neoplasia
• Persistent
• Cervical
• Cervical
• Cervical

• Cervical
• Vaginal
• Vulvar

class pathways [140,141]. Currently, attenuated Lm has been used for

• HPV

developing as vaccine vector and treatment and prevention of HPV

infection and related diseases. ADXS11-001(ADXS-HPV) is a live atte-
nuated Lm-based immunotherapy that secretes antigen-adjuvant fusion
HPV-6, 11, 16, 18, 31, 33, 45, 52, and

HPV-6, 11, 16, 18, 31, 33, 45, 52, 58,

protein Lm-LLO-E7 targeting HPV-associated tumors [142]. In 2009,

HPV-6,11,16,18,31,33,45,52, and 58

Lm-LLO-E7 vaccine was tested in a phase I trial and was administrated

in 15 patients with advanced cervical cancer. The vaccine was shown to
be safe with severe (grade 3) adverse events in 6 patients, no grade 4
L1 VLP of HPV-16 and 18

L1 VLP of HPV-6 and 11

adverse events, and a reduction in total tumor size in 4 patients [143].

In 2014, ADXS11-001 was evaluated in 110recurrent cervical cancer
patients with and without cisplatin in a randomized phase II study
HPV-16 and 18

[144]. Patients were randomized to either three doses or four doses of

59 and 68

vaccines with cisplatin chemotherapy. It was encouraging that the final

Antigen

12-month survival was 36%, the 18-month survival was 28%, and the
58

response rate was 11%. The incidence of grade 3 adverse events was 2%
Recombinant human papillomavirus bivalent (types 16 and

and most non-serious adverse events were infusion-related, resolved on

11-valent Recombinant Human Papillomavirus Vaccine

their own, or responded to symptomatic treatment. GLBL101c, an oral

vaccination of HPV16 E7-expressing L. casei bacterial vector vaccine,
was tested in 17 patients with HPV16-related CIN3. The authors found
(6,11,16,18,31,33,45,52,58 Type) (E.Coli)

(6,11,16,18,31,33,45,52,58 Type)(E.Coli)
Recombinant Human Papillomavirus Vaccine

Recombinant Human Papillomavirus Vaccine

that the vaccine could elicit E7-specific mucosal immunity in the

HPV vaccines that are in clinical trial.

uterine cervical lesions, with a pathological down-grade to CIN2 in 70%

of patients who took the optimized doses. No patient was found an
adverse event [145]. Moreover, Komatsu A et al. [146] has optimized a
recombinant HPV16 E7-expressing L. casei (IGMKK16E7) to generate
optimal ratio for the amount of E7 and lactobacilli. The IGMKK16E7
18) vaccine (Yeast)

E. coli: Escherichia coli.

showed a higher induction of E7-specific IFN γ-producing cells com-

pared with the GLBL101c.
Product name

3.3.1.2. Viral vector vaccines. Because of the high infection efficiency

Gecolin
Cecolin

and high antigens expression of viral vector-based vaccines and the

Table 2

natural propensity to transduce their genetic information into the host

for replication of the viruses, viral vectors have been extensively tested,

94
R. Wang, et al.
Table 3
Characteristics of vaccine platform technologies.
Vaccine Platform Advantages
Bacterial vector • Highly immunogenic
vaccines • Inducing both potent innate and adaptive immune
responses
• Low difficulties of target antigen purification
• Simple and inexpensive to large-scale manufacture and
inoculation.
Viral vector vaccines • Highly immunogenic
• Wide range of selection for available vectors
• Can be engineered to include and express multiple genes
such as costimulatory molecules/cytokines
• Inducing
responses
both potent innate and adaptive immune
DNA-based vaccines • Stable, safe, easy to manufacture and purify
• Capable of repeated vaccination for long-term protection
• Can be engineered to include and express multiple genes
such as costimulatory molecules/cytokines
• Multiple delivery methods available
RNA-based vaccines • Easy to produce
• Wide range of host cells
• Safety with no risk of chromosomal integration or
cellular transformation
• Efficient delivery and high expression levels of
recombinant antigens due to high capacity of
autonomous RNA replication
Peptide-based • Stable, safe, easy to produce and store
vaccines • Can include multiple epitopes
• Can be modified for better MHC binding
Protein-based • Stable, safe, easy to produce
vaccines • No limitation of MHC restriction
Dendritic cell-based • Highly immunogenic
vaccines • Serve as natural adjuvants
• Multiple methods of antigen loading
• Inducing
responses
both potent innate and adaptive immune
MHC: major histocompatibility complex; DCs: dendritic cells.
such as adenoviruses (Ad), adeno-associated viruses, alphaviruses, and
vaccinia viruses. Foreign genes that code immunogenic proteins from
pathogens are used to replace non-essential viral genes, and thus the
recombinant viral vectors could transduce the target cell and express
the encoded antigens.
Adenovirus, an80-100 nm, non-enveloped, double-stranded DNA
virus, represents one of the most promising vaccine platforms used for
gene therapy and vaccine vectors. Ad vector vaccines have many ad-
vantages that make them attractive and widely used [147–149]: (1)
Deletion of the E1 region which is critical for the initiation of virus
replication and replacement with the foreign gene results in replication-
defective virions that have low risk of insertional oncogenesis for
human use; (2) Deletion of theE2, E3 and E4 regions increase the ca-
pacity for transgene insertion, and multiple transgenes such as the
target antigen and other genes that enhance the response to vaccination
can be inserted into the recombinant viral vectors; (3) Inducing robust
immune responses that are associated with pro-inflammatory cytokines
and both the innate and adaptive immunity. However, Ad is ubiquitous
in humans and up to 60% of adults in Europe and the USA have high
titers of nAbs to adenovirus type 5 (Ad5). Therefore, animal or rare
human Ad serotypes such as Ad26 and Ad35 are currently evaluated to
overcome type-specific anti-vector immunity [150,151]. In 2017, Khan
et al. [152]developed replication‐deficient Ad26 and Ad35-based
vector vaccines that consisted of fusion proteins of HPV E2, E6, and E7
for HPV16 and HPV18 related disease. They found considerable ther-
apeutic efficacy in the TC‐1 mouse model, indicating that the developed
vaccine vectors form a promising therapeutic vaccine candidate for
active immunotherapy. Subsequently, Çuburu Net al. [153]evaluated
95
Cancer Letters 471 (2020) 88–102
Disadvantages Reference
• Potentially toxic/harmful to patients [135,136,138]
• Potential immunodominance to the vectors
• Generation of neutralizing antibodies restrict the
efficacy of repeated therapy
• Potentially toxic/harmful to patients [12,135,136,147–149,154,155]
• Potential immunodominance to the vectors
• Generation of neutralizing antibodies restrict the
efficacy of repeated therapy
• Low immunogenicity [158,159]
• Potential risk of chromosomal integration
• Lack intrinsic ability to self-amplify and spread to
surrounding cells
• Poor stability [167–169]
• Inability to spread intercellularly
• Preparation/production is labor intensive
• Difficult to prepare in large quantity
• Low immunogenicity [161,170]
• MHC specific that need to match the patient's
human leukocyte antigen (HLA) haplotypes
• Low immunogenicity [175,178]
• Inducing more humoral responses than cell-
mediated responses
• Difficult for large-scale production [182,183]
• Lack of a consensus on the optimal methods of DCs
preparation
• Different culture techniques may result in
inconsistent vaccine quality
• Limited
apoptosis
lifespan due to the T cell-mediated
the immunogenicity of this vaccine in prime-boost immunization regi-
mens via heterologous methods combining intramuscular and in-
travaginal immunization in mice. They found that systemic prime fol-
lowing local intravaginal boost represented the most promising
combination to efficiently induce considerable CD8+ T cell responses.
Vaccinia virus is a double-stranded DNA virus, which belongs to the
Poxviridae family. The poxvirus has a large and stable genome that can
express large amounts of foreign antigen. Modified vaccinia virus
Ankara (MVA) is licensed as the third-generation vaccine against
smallpox and serves as an effective vector platform for the development
of new vaccines. The MVA has several features that make the re-
combinant MVA platform highly suitable as a heterologous viral vector:
(1)large capacity for transgene insertion; (2) ease of inexpensive man-
ufacture; (3)safe administration by the intradermal, intranasal, in-
travaginal and intrarectal routes; (4) thermostability for application in
resource-poor settings without a cold chain [12,154,155]. Recently,
Rosales R et al. evaluated an MVA viral vector targeting HPV16 E2
(MVA-E2)to treat intraepithelial lesions associated with the HPV in-
fection in a phase III trial of a total of 1176 female and 180 male pa-
tients. MVA E2 virus particles were directly injected into the uterus,
urethra, vulva or anus. After treatment with MVA E2, 89.3% of the
female patients and all the male patients observed complete elimination
of lesions, and no apparent side effects were generated. However, there
is no control vaccine in the study, the true vaccine efficacy is uncertain
[156].
3.3.2. Nucleic acid/protein/peptide-based vaccines
3.3.2.1. DNA-based vaccines. DNA-based vaccines have become a safe
R. Wang, et al.
alternative to standard live and inactivated vaccines for human and
animal infections [157]. They exhibit several advantages over
traditional strategies: (1) easy to manufacture and purify; (2) high
purity and stability compared to protein-based vaccines; (3) safe
compared to live vector-based vaccines; (4) capable of inducing both
humoral and cell‐mediated immune responses; (5) unlike the live
vector-based vaccine and protein-based vaccines, they do not produce
nAbs to the vector, allowing for repeated vaccination for long-term
protection [158,159]. However, the main limitation of DNA-based
vaccines is poor immunogenicity. To increase immunogenicity,
strategies such as optimizing delivery approaches with the use of
electroporation, encapsulation, gene gun or laser therapy, fusions of
antigens with T-cells activating molecules, the use of
immunomodulators such as potent adjuvants, priming with DNA
vectors followed by boosting with viral vectors have been
investigated, and modification of the properties of antigen-presenting
cells, especially dendritic cells (DCs) [160–162]. In 2014, Kim et al.
[163] developed the DNA vaccine GX-188E that expressed HPV16/18
E6 and E7 antigens and the Fms-like tyrosine kinase-3 ligand (Flt3L),
which could activate DCs. They used electroporation to enhance
immunization. The vaccine could elicitE6/E7-specific IFN-γ-secreting
T-cell responses in 9 patients with HPV16/18 + CIN 3, with 7 patients
displaying complete lesion regression within 36 weeks of follow up and
no serious vaccine-associated adverse events in all patients. In a
randomized, double-blind, placebo-controlled phase IIb study [164],
the VGX-3100, synthetic plasmids targeting E6 and E7 proteins of
HPV16 and HPV18, delivered by electroporation, was tested in women
with CIN2/3. Histopathological regression was observed in 49.5% of
vaccine recipients compared with 30.6% of placebo recipients. VGX-
3100 is the first and the most successful therapeutic DNA vaccine to
show efficacy against CIN2/3to date. Currently, the efficacy, safety, and
tolerability of VGX-3100 are evaluated in women with histologically
confirmed HPV16/18-positive CIN2/3 in a phase III trial
(NCT03185013). In another phase II study, VGX-3100 alone or in
combination with imiquimod is tested in women with HPV16/18-
positive high-grade lesions of the vulva (NCT03180684).
3.3.2.2. RNA-based vaccines. RNA-based vaccines are based on RNA
replicon systems derived from single-stranded RNA viruses of positive
or negative polarity and the virus vectors are non‐pathogenic [161].
Various RNA viruses such as retroviruses, lentiviruses, alphaviruses,
flaviviruses rhabdoviruses, measles viruses, Newcastle disease viruses,
and picornaviruses have been engineered as vectors for expression of
antigens and treatment of diseases [165]. Currently, numerous studies
have demonstrated that self-replicating RNA viral vectors could provide
efficient delivery and high expression levels of recombinant
heterologous antigens due to high capacity of autonomous RNA
replication, and administration of RNA viral vectors could generate
strong immune responses and provide protection against challenges of
infectious agents and tumor cells. By acting as agonists for TLR7 and
TLR8, RNA viral vectors can also induce the innate immune responses
[166]. Easy to produce virus, wide range of host cells, safety with no
risk of chromosomal integration or cellular transformation as the viral
RNA degrades within 3–5 days, targeting DCs, rapid generation of RNA
copies and high expression levels in the cytoplasm, and the combination
with either polymer- or liposome-based encapsulation strategies make
the RNA viral vectors an attractive approach [167,168]. However, the
limitations of RNA-based vaccines are poor stability and inability to
spread intercellularly [169]. To date, there is few clinical testing on
RNA-based vaccines associated with HPV-related diseases.
3.3.2.3. Peptide-based vaccines. Peptide-based vaccines have several
advantages, particularly concerning stability, safety, and ease
production and storage [161,170]. However, limitations include low
immunogenicity and MHC specific that need to match the patient's
human leukocyte antigen (HLA) haplotypes [171,172]. Therefore,
96
Cancer Letters 471 (2020) 88–102
adjuvants are required to enhance the immune response. Peptide-
based vaccines can be divided into synthetic long peptides (SLPs) and
specific epitope (short) peptides. The short peptides are restricted to the
patient's specific HLA type and need preliminary HLA typing before
administration [160]. Short peptide-based therapeutic HPV vaccines
(CIGB-228) adjuvated with very small size proteoliposomes (VSSP) was
tested in 7 women with HPV16-positive high-grade CIN. The vaccine-
induced IFN-γ-associated T-cell response and 5 patients had lesion
regression and HPV clearance [173].
Compared to short peptides, SLPs do not require patient selection
with specific MHC profile, but they need to be processed and presented
by APCs [174]. In the dose-escalation phase I clinical study, PepCan, a
peptide-based vaccine consisting of 4 HPV16 E6 synthetic peptides and
a novel adjuvant Candin, was carried out in 24 patients with CIN2/3.
The trial showed that no dose-limiting toxicity, histological regression
of disease in 45%of patients, a significant decrease in viral load, and an
increased immune response [175]. Additionally, the phase II trial
evaluating the efficacy and safety of PepCan in cervical high-grade
squamous intraepithelial lesions is ongoing (NCT02481414). Kenter
et al. [176]tested the immunogenicity and the efficacy of a synthetic
long-peptide vaccine in 20 women with high-grade HPV16 related
vulvar intraepithelial neoplasia (VIN). At 12 months of follow-up,
complete responses were found in 47% of the patients and maintained
at 24 months. Patients with a complete response had a stronger IFN-γ-
associated CD4+ andCD8+ T-cell response. In 2013, the HPV16 syn-
thetic long-peptide vaccine consisting of 13 HPV16 E6 and HPV16 E7
overlapping long peptides and combination with Montanide ISA-51
adjuvant was evaluated in 20 women with advanced or recurrent cer-
vical cancer. The HPV16-specific T-cell response associated with IFN-γ,
TNFα, IL-5 and/or IL-10 was found in 9 patients. However, neither
regressions of the tumor nor prevention of the progressive disease were
developed [177]. Moreover, the novel therapeutic synthetic long pep-
tide vaccine targeting HPV16 ISA101/ISA101b is ongoing to assess the
safety, tolerability and the HPV-specific immune responses in women
with advanced or recurrent cervical cancer (NCT02128126).
3.3.2.4. Protein-based vaccines. Protein-based vaccines can avoid the
limitation of MHC restriction due to the numerous CD4+ and CD8+T
epitopes compared to peptide-based vaccines. However, the major
limitations are that they may induce the antibody responses rather
than CTL responses because of the preferential presentation via MHC
class II. Therefore, adjuvants have been used for therapeutic
vaccination to enhance the APC presentation, target the antigen to
DCs, and improve immunogenicity [175,178]. Several protein-based
vaccines have entered clinical trials. TA-CIN, a subunit vaccine
comprising an HPV16 E6E7L2 fusion protein, has been proven safe
and immunogenic in a number of clinical trials [179,180]. Daayana
et al. [181]treated 19 women with VIN grades 2 and 3 using a topical
immunomodulator (imiquimod) followed by TA-CIN. At week 52, it
was shown that 63% of patients observed the complete histological
regression of VIN and increased local infiltration of CD4+ and CD8+ T
cells. Currently, TA-CIN vaccine is evaluating in 14 patients with
HPV16-associated cervical cancer to determine the safety and
feasibility as well as the induction of HPV antigen-specific immune
response (NCT02405221).
3.3.3. Cell-based vaccines
3.3.3.1. Dendritic cell-based vaccines. Dendritic cells (DCs) are ideal
candidates for immunotherapy strategies as they have been recognized
as the most potent APCs in vivo, capable of mediating and inducing
both innate immune response and adaptive immune response. DCs have
a strong ability to acquire and process antigens for presentation to T
cells and express high levels of costimulatory or coinhibitory molecules
[182]. Moreover, DCs can serve as natural adjuvants to increase the
potency of antigen-specific immunotherapy against cancer [183]. DCs
can be generated in vitro through optimal methods. Then the DCs can
R. Wang, et al.
be loaded with specific peptide or antigens to make a vaccine. The DC-
based vaccines can be adoptively transferred into patients and maintain
their capacity to induce specific CTL responses and exert efficient
antiviral or antitumor responses. DC-based vaccines have several
limitations, such as difficult for large-scale production, lack of a
consensus on the optimal methods of DCs preparation, different
culture techniques may result in inconsistent vaccine quality, and
limit lifespan due to the T cell-mediated apoptosis [183]. To prolong
DCs survival, short interfering RNA (siRNAs)against pro-apoptotic
molecules have been developed [184,185].
DC-based vaccines can be divided into DCs pulsed with HPV-specific
peptides/protein antigens or DCs transduced with DNA/viral vectors
encoding foreign antigens [186]. DCs can be prepared as mature or
immature cells. Most trials utilized the mature DCs as the immature
types lack the full T cell co-stimulatory activity [178]. However, it is
unclear whether DCs maturation is necessary to stimulate an immune
response in vaccines. The immature DCs has the potential to reduce
vaccine production cost compared to the mature DCs [187]. Therefore,
the pre-immature DCs (PIDCs) have been tested as cancer vaccines.
Rahma et al. [187] used the PIDC pulsed with HPV16 E6 or E7 peptide
to treat 32patients with advanced cervical cancers. The specific im-
mune response was 63% (E6) and 58% (E7) of the patients. Further
investigations are required to evaluate PIDCs for peptide delivery in
vaccines.
3.3.3.2. Adoptive T-cell therapy. Adoptive T-cell therapy (ACT) involves
the identification and expansion of desired specificity and phenotype T
cells in vitro and the reinfusion of antigen-specific T cells. ACT allows
for appropriate in vitro manipulation and selection of the T cells for
immunotherapy, which produces 10-fold greater frequencies of
antigen-specific T cells than those by using current vaccine regimens
alone [188]. Stevanovic et al. [189]evaluated ACT in 9 patients
diagnosed with metastatic cervical cancer. HPV-targeted tumor-
infiltrating T cells (HPV-TILs) were selected and reinfused into
patients. Objective tumor responses were observed in 3 patients and
2 complete responses were observed at 22 and 15 months after
treatment. Recently, Doran et al. [190] developed autologous
genetically engineered T cells expressing a T-cell receptor directed
against HPV16 E6 and treated 12 patients with metastatic HPV-
associated epithelial cancers in Phase I/II Study. Regression of
metastatic epithelial cancer and objective responses were observed
and the subsequent trial of T cell receptor gene therapy for
HPV16 + cancers targeting E7 is conducting (NCT02858310).
However, it is thought that CTLs alone may not be sufficient to
eliminate cancer cells in patients with advanced HPV cancers. The
evasion of the immune system and the treatment of solid tumors remain
a challenge. ACT needs to address issues such as tumor heterogeneity,
antigen escape, immunosuppressive microenvironment, and fragile
blood vessel status [191–193].
4. Conclusion
Ten years after the introduction of HPV vaccines, there has been a
great success in the development of prophylactic HPV vaccines against
certain types of HPV infections, showing efficacy and safety in pre-
venting persistent infections of precancerous lesions by approximately
95% of the vaccinated individuals. A series of limitations such as lim-
ited HPV type for prevention and high cost of manufacture restrict the
widespread use. Various types of second-generation prophylactic HPV
vaccines and HPV L2-based vaccines are currently undergoing pre-
clinical or clinical trials. Moreover, therapeutic HPV vaccines are ur-
gently required to reduce the burden of cervical cancer as the licensed
HPV vaccines cannot help those people who are already infected with
HPV and developing the associated tumors. In contrast to the prophy-
lactic HPV vaccines, progress in the development of therapeutic stra-
tegies has so far been very slow. Though several studies showed
97
Cancer Letters 471 (2020) 88–102
promising clinical responses, there was no clear relationship between
induced immune responses and clinical responders. This should con-
tribute to the lack of knowledge about the mechanisms of immune re-
sponse that control and clear the HPV-infected cells in the host.
Meanwhile, research on the cancer microenvironment and immune
evasion mechanism are still unclear. We believe that when more in-
formation regarding the immune mechanisms against HPV infection is
obtained, strategies to design better vaccines, including the use of ef-
ficient adjuvants will lead to the development of effective in the future.
Funding
This work was supported by the National Natural Science
Foundation of China (81672085; 81372804; 71871169; U1933120;
71373188; U1333115; 30901586); the Chinese medical association of
clinical medicine special funds for scientific research projects
(17020400709), the Hubei Provincial Natural Science Foundation of
China (2019CFA062).
Declaration of competing interest
The authors have no conflicts of interest to report.
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