"219", 28.5.
03/dk köthen GmbH
A. M. Kaufmann
C. Backsch
A. Schneider
M. Dürst
HPV induzierte Karzinogenese der cervix uteri: molekulare Mechanismen und
Impfstoffentwicklung
Zusammenfassung
Prädisponierend für die Entstehung eines Zervixkarzinoms ist
die Infektion mit humanen ¹high-riskª Papillomviren (HPV). Es
gibt nur wenige Zervixkarzinome, bei denen keine HPV DNA
nachweisbar ist. Mechanismen der Immortalisierung von Epi-
thelzellen durch Interaktion mit Tumorsuppressorgenen p53
und pRB durch virale Onkogene E6 und E7 wurden definiert. Die
Progression einer HPV-infizierten Zelle zu einem malignen Phä-
notyp erfordert weitere genetische Veränderungen des Wirts-
zellgenoms. Mit dem Auftreten von chromosomalen Aberratio-
nen kann es zur Mutation oder dem Verlust von Tumorsuppress-
orgenen (TSG) und zur Aktivierung und Amplifikation von Onko-
genen kommen, die im Prozess der Tumorgenese involviert sind.
Die Onkogenamplifikation scheint mit Ausnahme weniger Ber-
ichte allerdings in der Zervixkarzinogenese nicht bedeutsam zu
sein. Demgegenüber zeigen zytogenetische und ¹loss of hetero-
zygosityª LOH-Untersuchungen an CIN-Läsionen und an invasi-
ven Zervixkarzinomen den häufigen Verlust von spezifischen
chromosomalen Regionen, die auf die Lokalisation von TSG hin-
weisen. Genetische Veränderungen der Chromosomen 3p, 6p
und 11q wurden häufig in der frühen Tumorentwicklung gefun-
den. Primäre invasive Karzinome zeigten zusätzliche Allelver-
luste an Chromosomenbereichen 6q, 17p und 18q. Als biolo-
gische Marker für die diagnostische und prognostische Einschät-
zung von ¹high-riskª HPV Infektionen und maligne Progression
sind für bestimmte Fragestellungen p16INK4, p27Kip1, und NET-I/
C4.8 zu nennen. Putative Seneszenzgenloci mit Relevanz für
HPV-induzierte Karzinogenese sind auf Chromosomen 2, 4 und
10 lokalisiert. Gene für Telomerasesuppression werden auf den
Chromosomen 3, 4, und 6 vermutet. Natürliche Immunität gegen
HPV Infektionen existieren. Daher sind Immuntherapien eine at-
Gynäkologische Molekularbiologie, Frauenklinik, Friedrich-Schiller-Universität Jena
Dr. Andreas M. Kaufmann ´ Gynäkologische Molekularbiologie ´ Frauenklinik ´ FSU Jena ´ Bachstrasse 18 ´ 07743
Jena ´ Germany ´ Tel.: +49/36 41/93 42 73 ´ Fax: +49/36 41/93 4216 ´ E mail: andreas.kaufmann@med.uni-je-
Zentralbl Gynakol 2002; 124: 511±524  J. A. Barth Verlag in Georg Thieme Verlag KG ´ ISSN 0044-4197
HPV induced cervical carcinogenesis:
molecular basis and vaccine development
Review
Abstract
Association of infection with papillomavirus and dysplasia of the
cervix uteri has been firmly established. There are only few cer-
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vical cancers where no HPV DNA is detectable. The mechanism of
epithelial cell immortalization by interaction with tumour sup-
pressor genes p53 and pRb by viral oncogenes E6 and E7 is eluci-
dated. Progression of the HPV infected cell to a malignant pheno-
type involves further modification of host gene expression and/
or mutations.
The appearance of chromosomal aberrations can lead to muta-
tional inactivation or loss of tumour suppressor genes (TSG), ac-
tivation and amplification of oncogenes, with importance for the
process of carcinogenesis. Oncogene amplification, with excep-
tion of few reports, seems not to be a major mechanism in cervi- 511
cal carcinogenesis. In contrast, cytogenetic and loss of heterozyg-
osity (LOH) results from CIN and invasive cancer demonstrate al-
terations at specific chromosomal regions, pointing at localisa-
tion of TSG. Genetic alterations at chromosomes 3p, 6p, 11q
were frequently found early in tumour development. Primary in-
vasive carcinoma showed additional allelic losses at chromo-
some arms 6q, 17p and 18q. Useful biological diagnostic and
prognostic markers for high-risk HPV infection and malignant
progression may be p16INK4, p27Kip1, and NET-I/C4.8. Putative se-
nescence genes relevant for HPV-induced carcinogenesis are lo-
calized on chromosomes 2, 4 and 10. Genes for Telomerase sup-
pression are presumably located on chromosomes 3, 4 and 6.
Natural immune responses to HPV infection exist. Therefore, im-
mune therapy is an attractive possibility for prevention and ther-
apy of HPV infection. To date, vaccine development has reached
clinical evaluation. Prophylaxis aims at the induction of virus
neutralizing antibodies to capsid proteins. Virus-like particle
Affiliation
Correspondence
na.de
Bibliography
"219", 28.5.03/dk köthen GmbH
traktive Möglichkeit für die Prävention und Therapie von HPV In-
fektionen. Derzeit hat die Vakzinentwicklung das Stadium der
klinischen Prüfung erreicht. Prophylaxe zielt auf die Induktion
virus-neutralisierender Antikörper gegen Kapsidproteine. Auf
Virus-ähnliche Partikel basierende Impfstoffe werden in klini-
schen Versuchen geprüft. Wegen der langen Verzögerungsper-
iode zwischen Infektion und klinischer Manifestation werden
klinische Untersuchungen einer langen Zeit bedürfen, bis schlüs-
sige Ergebnisse vorliegen. Die grundsätzliche Expression viraler
und vielleicht zellulärer Gene in infizierten Epithelien und Tu-
morzellen bietet Zielstrukturen für therapeutische Vorgehens-
weisen. Dass die meisten Dysplasien spontan regredieren zeigt,
dass die Virusinfektion immunogen ist. In einigen Patientinnen
muss die Immunantwort durch Impfungen verstärkt werden,
Review
um effektiv zu sein. Verschiedene Strategien werden in klini-
schen Versuchen getestet und andere sind in präklinischer Ent-
wicklung. Die Aufgabe wird sein, die Immunsuppression von
HPV-Infizierten zu umgehen.
Schlüsselwörter
Humane Papillomaviren (HPV) ´ Progression ´ Genomverände-
rungen ´ Prophylaxe ´ Immuntherapie ´ Klinische Studien
Introduction
Human Papillomavirus and Cervical Cancer
Yearly approximately 500 000 women worldwide develop cervi-
cal cancer, making this malignancy the second leading cause of
cancer death in women. Despite the availability of PAP smear
screening the incidence of invasive cervical cancer does not
512 seem to be diminishing. A wide variety of epidemiological and
virological data have identified a clear and consistent association
of human papillomavirus (HPV) infection with the development
of cervical cancer over the past 15 years. More than 99 % of cervi-
cal cancers and their precursor lesions, cervical intraepithelial
neoplasia (CIN) contain HPV DNA [1]. Over a hundred HPV geno-
types have been identified so far. About 80 % of cervical cancer is
associated with four ªhigh-riskº types of HPV (types 16, 18, 31,
45) and the remaining 20 % are associated with a dozen other on-
cogenic types [for review, see 2]. HPV16 is present in approxi-
mately 50 % of all cervical cancers [3] and consequently is the fo-
cus of most recent HPV vaccine developments.
Molecular Mechanisms of Progression
Pathogenesis of Papillomavirus Infection
Primary infection by HPV takes place within the basal cells of the
epithelium probably at the transition of columnar to cylinder
epithelium of the cervix uteri. The viral DNA persists within this
compartment. Upon differentiation of the cells, vegetative virus
replication is initiated. The action of the viral proteins E6 and E7
which bring the differentiated cells back into S-phase thus facil-
itating the replication of the viral DNA appears to be a key event.
This also leads to hyperproliferation of cells and thus to the typi-
cal macroscopic features of a papillomavirus infection, i. e. wart
formation. Papillomavirus infection of the anogenital tract indu-
ces either genital warts (mostly related to HPV types 6 or 11) or
cervical intraepithelial neoplasia (ªhigh riskº HPV types). Virus
persistence for many years is required for development of cancer.
Kaufmann AM et al. HPV induced cervical ¼ Zentralbl Gynakol 2002; 124: 511 ± 524
vaccines are currently tested in clinical trials. Due to the long lag
period between infection and clinical manifestation trials will
take a long time until conclusive results are obtained. Mandatory
expression of viral and perhaps certain cellular genes in infected
epithelial and tumour cells offers targets for therapeutic
approaches. Since most dysplasia clears spontaneously the viral
infection is immunogenic to some extent. However, in some indi-
viduals the immune response has to be stimulated by vaccina-
tion in order to be effective. Several strategies are being tested
in clinical trials and others are in preclinical development. The
task will be to circumvent immunosuppressive features of the
HPV infected cells.
Key words
Human Papillomavirus (HPV) ´ progression ´ genetic alteration ´
prophylaxis ´ immunotherapy ´ clinical trials
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Very likely, the continued expression of the viral oncogenes E6
and E7 leads to accumulation of cellular changes, e. g. overex-
pression of cellular oncogenes and loss of tumour suppressor
genes (see below) finally resulting in invasive cell growth. It
was shown that these viral oncoproteins are required not only
in the initial phases of transformation but also for maintaining
the proliferative state of a tumour cell. This feature makes them
unique targets for the induction of a virus-specific anti-tumour
immune response (see below).
Molecular Mechanisms of HPV Induced Immortalization
Acquisition of immortality has been defined as a two step pro-
cess involving the mortality phases M1 and M2 [4]. M1 is over-
come due to expression of the viral oncogenes E6 and E7, that
are consistently expressed in precursor lesions (CIN) and cervical
cancer, giving rise to an extended lifespan [5]. Molecular, bio-
chemical and cellular studies have unequivocally demonstrated
that E6 and E7 lead to malignant transformation of epithelial
cells [for review, see 6]. The mechanism of transformation is not
completely understood but it is clear that the proteins E6 and E7
through their interaction with the cellular tumour suppressor
gene products p53 and pRB are pivotal in this process. E6 binds,
inactivates and promotes the degradation of p53, while E7 binds
and inactivates pRb [7 ± 9]. The affinity of E6 to p53 leads to in-
creased degradation of p53 via the ubiquitination pathway [8].
The deregulation of pRB leads to an increased transcription of cy-
clin E by E2F, which is separated from the pRb/E2F complex by
the blocking of cyclin D1 [10]. E2F itself acts as a transactivator
at the promoters of many genes involved in cell cycle progression
[11]. E6 and E7 have been implicated in mitotic infidelity by their
ability to induce centromere-related mitotic disturbances [12].
While the E7 oncoprotein rapidly drives centrosome duplication
errors in cells that appear phenotypically normal, expression of
the E6 oncoprotein results in an accumulation of supernumerary
centrosomes in multinucleated cells. The E7 has been shown to
"219", 28.5.03/dk köthen GmbH
dysregulate cdk2 activity, a major determinant for the initiation
of centrosome duplication. HPV-induced centrosome abnormal-
ities, multipolar mitoses, and aneuploidy often occur at early
stages during cervical carcinogenesis and increase with malig-
nant conversion [12]. Overexpression of E5 protein can modify
cell growth and results in neoplastic transformation in some ex-
perimental systems [13, 14]. E5 seems to be important in the ear-
ly course of infection. With the integration of viral DNA into the
host genome part of the E5 genome (including the coding se-
quence) is deleted or uncoupled from its promoter, suggesting
that E5 is not necessary to maintain the malignant state [15 ±
17]. One major activity of the HPV 16 E5 protein is the modula-
tion of the ligand-dependent activation of the epidermal growth
factor receptor (EGFR) [18]. It has been postulated that this effect
is mediated by binding of E5 to the 16 K proteolipid located at the
endosomal membranes which may disturb vacuolar-ATPase
function [19]. In contrast Rodriguez et al. [18] found that EGFR
activation by HPV 16 E5 is working through a vacuolar-ATPase in-
dependent route. HPV E5 also enhances signalling via the G pro-
tein-coupled endothelin receptor [20] and may also contribute to
cell transformation by repressing expression of the cyclin-de-
pendent kinase inhibitor, p21 [21].
While M1 represents a critical phase for the expansion of the
natural cellular lifespan, M2 describes a point to overcome mor-
tality for true immortalization for which additional genetic
events are necessary [22]. Immortality is frequently associated
with the reactivation of telomerase activity [23, 24]. Putative
genes that suppress telomerase activity in HeLa cells localize on
chromosomes 3 and/or 4 [25]. Results from Steenbergen et al.
[26] showed that chromosome 6 may harbour a repressor of
hTERT transcription, the loss of which may be involved in HPV-
mediated immortalization. Several investigators have used mi-
crocell-mediated chromosome transfer to identify human chro-
mosomes involved in immortalization [27 ± 29]. They found loci
with putative senescence genes involved in HPV-induced carci-
nogenesis that were localized on chromosomes 2 [27], 4 [28],
and 10 [29]. Bertram et al. [30, 31] identified a gene (mortality
factor 4 ± MORF 4) that reverses the immortal phenotype of a
subset of cells (complementation group B). It is a member of a
novel family of transcription factor-like genes. Since the protein
is localized in the nucleus and has a DNA binding motif, it has the
potential as a transcription factor [30].
Events in the Progression from HPV Infection to
Cervical Cancer
Clinical, pathological and virological studies have defined a pro-
gression of events in the development of cervical cancer [for re-
view, see 32, 33]. The pathogenesis of cervical cancer is initiated
by HPV infection of cervical epithelium during sexual inter-
course. The majority of genital HPV infections are transient.
However, a fraction of infections persist and initiate transforma-
tion events within cervical epithelium. The cervical epithelial
changes associated with this initial set of events, pathologically
classified as low-grade SIL (LSIL or CIN 1), are associated with
continued viral replication and virus shedding [for review, see
32, 33]. The progression from high-grade SIL (CIN II/III) to inva-
sive cancer occurs at high frequency. In the majority of cases,
progression is associated with conversion of the viral genome
from an episomal form to an integrated form, along with deletion
Kaufmann AM et al. HPV induced cervical ¼ Zentralbl Gynakol 2002; 124: 511 ± 524
or inactivation of E2, a negative regulator of E6 and E7 expression
[32].
The use of a novel quantitative real-time PCR technique by Peit-
saro et al. [34] demonstrated that integrated HPV type 16 is al-
ready present in low-grade CIN lesions. Only one of 31 cervical
lesions contained exclusively episomal HPV 16 DNA. Rapid pro-
gression of the CIN lesions was closely associated with heavy
load of integrated HPV 16. Less sensitive techniques, such as
Southern blotting, PCR and two-dimensional electrophoresis
and high-copy number load of episomal forms can mask the
presence of low-level integrated HPV [34]. In contrast Nagao et
al. [35] found, that the physical status of HPV 16 DNA in 50 clini-
cal samples was exclusively episomal in 42 %, concomitant in
Review
28 %, and integrated in 30 %, respectively ± also by a real-time
PCR method. There are two other methods for examination of in-
tegration sites and functional aspects of integration: the ligation-
mediated PCR (DIPS-PCR) [36] and the amplification of papillo-
mavirus oncogene transcript (APOT-assay) [37]. Development of
invasive cancer requires additional genetic events facilitated by
E6- and E7-mediated inactivation of the genome guardians p53
and pRb, suppression of apoptosis and genomic instability [32].
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Molecular Progression to Cervical Cancer
The enhanced genomic instability is expressed by the appear-
ance of chromosomal aberrations that can lead to mutation or
the loss of tumour suppressor genes (TSG), activation and ampli-
fication of oncogenes, with importance for the process of carci-
nogenesis. Oncogene amplification seems not to be an important
event in cervical carcinogenesis except for an amplification of
c-myc and H-ras 1 in some progressive tumour stages [38, 39].
Gene amplification was found in neoplasia and in cervical cancer
only in 7 % (stage I) and in 9 % (stage II) of cervical cancers as com- 513
pared to other aberrations [40].
Loss of heterozygosity (LOH) is one of the most important
mechanisms for inactivation of tumour suppressor genes. In CIN
lesions, frequent LOH was found at chromosome arms 3p [41 ±
44], 4p, 4q, 5p and 6p [42, 44 ± 46] and 11q [42, 44, 47 ± 49]. Re-
gions with frequent allelic losses are listed in Table 1. For exam-
ple Chatterjee et al. [46] identified two minimal regions of dele-
tions at 6p25 and 6p21.3. The high-grade CINs exhibited 91.7 %
LOH, and low-grade CINs had 50 % LOH in these regions, respec-
tively. These findings implicate the presence of at least two tu-
mour suppressor genes on 6p relevant to cervical carcinoma
and suggest that these genetic alterations occur very early in tu-
mour development. In contrast to allelic losses the study of He-
selmeyer et al. [50] confirmed the importance of a gain of chro-
mosome arm 3q at the transition from preinvasive to invasive
cervical carcinoma.
Primary invasive carcinoma showed additional LOH at chromo-
some arms 6q, 17p and 18q (Table 1). In lymph node metastases,
an additional locus on the X chromosome displays LOH [42]. Los-
ses of chromosome 11p and 18q were associated with poor prog-
nosis in cervical carcinomas without lymph node metastasis [51,
52]. Frequent aberrations were found on chromosome 1 with
higher frequencies of LOH in Stage III and IV tumours suggesting
that chromosome 1 changes are late events in cervical carcino-
genesis [53]. The average number postulated for chromosomal
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al. [62] showed that HPV 16 350T variants were significantly

Table 1 Frequently found allelic losses in cervical dysplasia over-represented in p53 arginine homozygous women with cer-
vical cancer. This suggests that in p53 ARGININE/ARGININE
Chromo- Chromosomal Localisation Reference women infection with HPV 16 350T variants may confer a higher
somal
Aberration risk for cervical cancer.

A: severe dysplasia (LOH analyses) Furthermore the products of HLA (human leukocyte antigen)
3p- 3p14.2, 3p14.3±21.1, 3p21, Wistuba et al. 1997 [41] class I and II genes are highly polymorphic and play a major role
3p22±4.2 in regulating T cell responses to foreign antigens, including viral
3p25®pter, 3p21, 3p14 Kersemaekers et al. 1999 [42] antigens. It is evident that the HLA allele DQB1*03 plays an im-
3p14.1±12 Rader et al. 1998 [44]
3p25, 3p14 Chung et al. 1992 [43] portant role in the pathogenesis of cervical cancer. Cuzick et al.
4p- 4p15±16 Kersemaekers et al. 1 999 [42] [63] confirmed that the allele DQB1*0301 alone and in combina-
4q- not mapped Mitra et al. 1995 [45] tion with allele DRB1*0401 is associated with risk for cervical
5p- 5p15.1±15.2 Mitra et al. 1995 [45]
6p- 6p21.3 Kersemaekers et al. 1999 [42] cancer. In contrast, a marginally significant protective effect
Review

6p23 Rader et al. 1998 [44] against cervical cancer was found for DQB1*0501 but no protec-
6p21.3, 6p25 Chatterjee et al. 2001 [46] tive effect could be seen for allele DRB1*1301 [63].
11q- 11q23.3 Rader et al. 1998 [44]
O'Sullivan et al. 2001 [49]
11q23.3±25 Evans et al. 1998 [48] A specific diagnostic marker in HPV infection seems to be over-
11q22±24 Kersemaekers et al. 1999 [42] expression of p16. Expression of the viral oncogenes of high-risk
11q22, 11q23.3 Kersemaekers et al. 1999 [42]
Not mapped Larson et al. 1997 [47] HPV types in dysplastic cervical cells leads to increased expres-
B: cervical cancer (additional to aberrations in A) (LOH analyses) sion of p16INK4. The elevated p16 levels are explained by a nega-
6q- 6q14±16.2, 6q22.3±23.1 Kersemaekers et al. 1999 [42] tive feedback control mechanism through pRB [64]. Indeed Klaes

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10p- 10p14±15 PoigneØ et al. 2001 [29]
17p- 17p13 Kersemaekers et al. 1999 [42]
18q- 18q22-qter Kersemaekers et al. 1999 [42]
aberrations in severe dysplasia is 0.4, in invasive stages (FIGO I)
4.0, and in progressive invasive stages (FIGO II b±IV) 8.2 [50, 54].
514 These analyses confirmed in a great variety of severe dysplasia
and cervical carcinoma the occurrence of chromosomal aberra-
tions, that point at the localization of tumour suppressor genes
and oncogenes. For example, the Fhit gene, which is mapped at
chromosome 3p14.2, is a candidate tumour suppressor gene
that has been implicated in the development of cervical carcino-
ma [55, 56]. Fhit protein was detected in normal cells, whereas
dysplastic cells (independently of HPV infection and HPV sub-
types) showed reduced expression of Fhit. These data suggest
that loss of Fhit expression occurs in the early stages of cervical
carcinogenesis [56]. O'Sullivan et al. [49] found a relationship be-
tween LOH (38.8 %) at chromosome 11q23.3 and the presence of
extensive tumour plugs in lymphovascular spaces (LVS) in
stage I b cervical carcinoma, suggesting that genes at this locus
may regulate vasculoinvasion. This region appears to be impor-
tant in tumour progression.
et al. found that 150 of 152 high-grade dysplastic lesions (CIN II
to invasive cancer) showed diffuse and strong overexpression of
the p16INK4, whereas normal cervical epithelium or inflammatory
or metaplastic lesions were not stained by a p16-specific anti-
body [65]. These data demonstrate that p16 is a specific biomark-
er to identify dysplastic cervical epithelia in sections of cervical
biopsy samples or cervical smears.
Specific prognostic markers for cervical carcinogenesis may be
the cellular proteins Net1/C4.8 and p27. The potential impor-
tance of NET-I/C4.8 expression in cervical carcinogenesis is un-
derlined by an invariably strong expression in all undifferenti-
ated squamous cell carcinoma examined. Notably, expression of
the protein throughout the entire epithelium is only evident for a
subset of CIN III [66]. P27Kip1, a member of the cyclin dependent
kinase inhibitor family, expression is decreased in microinvasive
and invasive carcinomas [67, 68]). The inhibitory effect of this
CDK-inhibitor can be inactivated through binding of E7 (HPV
16) to p27 [69]. Huang et al. [67] could clearly demonstrate that
loss of p27 expression significantly correlates with lymph node
metastasis, but the level of p27 expression was not a significant
predictor of survival in cervical carcinoma. The potential use of
p27 and NET-I/C4.8 as prognostic markers will have to be eval-
uated in further studies, whereas immunocytochemical staining
for p16 has recently been introduced as triage for uncertain PAP
smears.

The PTEN/MMAC1 is often deleted or mutated in several differ-
ent types of human tumours, but for cervical cancer aberrant
function is an uncommon event [57].
In 1998 Storey [58] and colleagues postulated a predisposition to
tumourigenesis for patients who carry homozygously the tu-
mour suppressor protein p53 with an amino acid ªARGININEº at
position 72. However, other studies could not confirm these re-
sults [59 ± 61]. The relationship between p53 polymorphism and
other potential surrogate markers in combination with HPV var-
iants is poorly understood. However, the results from van Duin et
The characterization of key chromosomal changes and expres-
sion of progression markers may in future identify patients at
high risk for progression and with need of enhanced intervention
that possibly will encompass immunotherapy.
Vaccine Development
Natural Immune Response to Infections with HPV
Papillomaviruses are not highly immunogenic during the natural
course of infection. Antibodies arise only in a fraction of patients
and there is no sign of inflammation within a wart [70, 71]. The
reason for this immunologic ignorance may be the inability of

Kaufmann AM et al. HPV induced cervical ¼ Zentralbl Gynakol 2002; 124: 511 ± 524
"219", 28.5.03/dk köthen GmbH

the keratinocytes to function as antigen-presenting cells [72].

Antibodies to early proteins are detected almost exclusively in
patients with invasive HPV-related tumours, suggesting that ex-
posure of the antigens is a consequence of disintegration of indi-
vidual cells [73, 74]. It has been speculated that the particular se-
questering of the virus during its life cycle permits an individual
infection to persist for a long time and guarantees the circulation
of the virus family as a whole. Nevertheless there is mounting
evidence that the immune system does control viral infection
and the development of the diseases associated therewith. The
role of the immune system in controlling papillomavirus infec-
tions is underlined by the increased prevalence of clinical and
subclinical HPV infections in immunosuppressed patients (HIV-
infected patients or organ transplant recipients) [75 ± 77]. Fur-

ther evidence for an immunological control is given by the accu-
mulation of lymphocytes and the presence of proinflammatory
cytokines (e. g. IL-12) in regressing warts [71]. HPV-specific cel-
lular immune responses (T helper and cytotoxic T cells) can be
found both in patients with and without HPV-associated lesions
[78, 79]. A strong argument for a definitive role of cell-mediated
immune responses in controlling of persistent infection came
from a recent observation by Höpfl et al. [80] demonstrating a
Review
Fig. 1 Different mechanisms have to be employed for prophylactic
and therapeutic vaccination. Preventive approaches mainly relay on in-
duction of virus-neutralizing antibodies inhibiting binding of particles
to epithelial cells or uncoating of viral DNA within the cell. Therapeutic
vaccination will need to eliminate infected cells by cytolysis. Effectors
will be HPV antigen-specific T cells induced by immunization protocols.

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positive skin reaction against HPV 16 E7-specific peptides in
most patients with regressing cervical lesions but not in those
cases whose clinical infection persisted.

Taken together, there is good evidence for vaccine efficacy from

the natural history of HPV infections, experimental in vitro stud-
ies, and from animal infection and tumour therapy models. Here
we will concentrate on the translational research and early clin-
ical trials aiming at proofing safety and efficacy in humans.

Development of HPV Vaccines 515

As decribed above, the virologic, genetic and pathological pro-
gression of HPV is well-defined from initial infection to dysplas-
tic lesion and malignant tumour formation. The limited number
of well-defined foreign (viral) antigens provide a unique oppor-
tunity for intervention with antigen specific immune therapy at
various stages of HPV infection and tumourigenesis. Conceptual-
ly, two different types of HPV vaccines can be designed: prophy-
lactic vaccines that prevent HPV infection and therapeutic (cura-
tive) vaccines that would induce regression of established HPV
infection and dysplasia (Fig. 1). Both strategies will have to be ap-
plied at different ages of the vaccinees, before onset of sexual ac-
tivity or after diagnosis of HPV infection and/or lesion manifesta-
tion, respectively (Fig. 2). However, immune therapy of late stage
cancer may be hampered by immune evasion of the heavily im-
mune selected cancer cell.
Prophylactic Vaccines in Clinical Trials
Vaccines that protect the host from infection have to be adminis-
tered before the first potential contact. In case of HPV this means
that effective prophylactic vaccination will have to take place
early enough in advance to sexual contact to allow the acquisi-
tion of protective immunity. It will be necessary to vaccinate
both males and females at young age. Protection from experi-
mental infection in animal models has been demonstrated fol-
lowing passive transfer of IgG from papillomavirus-immunized
animals to naive animals [81, 82]. The results from these protec-
tive passive vaccine studies indicate that humoral responses ef-
Fig. 2 Of 100 infected women 20 will develop a precancerous lesion
(CIN) and 1 or 2 an invasive carcinoma (CA). Prophylactic vaccination
aims at prevention of primary infection and has to be effective before
first exposition to HPV. Therapeutic vaccination will be done when HPV
DNA or cervical dysplasia has been detected. In invasive cancer thera-
peutic vaccination can be envisaged as an adjuvant treatment to con-
trol minimal residual disease.
fect protection from experimental infection. To completely pre-
vent sexual transmission of genital HPV infection, virus neutral-
izing antibodies must act at mucosal surfaces, which are the nat-
ural site of infection. Antibodies both pass from plasma into gen-
ital secretions and are synthesized by local plasma cells [83, 84].
The plasma cell precursors that migrate to the genital tract are
derived primarily from mucosal lymphoid tissues and predomi-
nantly secrete IgA.
The papillomavirus major capsid protein L1, when overexpressed
in mammalian [85, 86], insect [87], yeast [88] or bacterial cells
[89, 90], spontaneously assembles to form virus-like particles
(VLPs) that are devoid of the oncogenic viral genome. Parenteral
injection of these VLPs elicits high titers of serum-neutralizing
antibodies and protection from experimental challenge with in-
fectious virus in several animal papillomavirus models [81, 91,
92]. Recently, oral vaccination with HPV VLPs in mice has been
shown to induce systemic virus-neutralizing antibodies, sug-

Kaufmann AM et al. HPV induced cervical ¼ Zentralbl Gynakol 2002; 124: 511 ± 524
"219", 28.5.03/dk köthen GmbH
gesting that the HPV VLPs may be antigenically stable in the en-
vironment of the gastrointestinal tract. These studies provide the
possibility of vaccinating large populations with HPV VLP with-
out using syringes [93]. Although VLP vaccination provides im-
munity from experimental inoculation, it was unclear up to now
whether this extends to protection against natural transmission
of genital HPV. The promising results generated in preclinical an-
imal studies have led to phase I/II clinical trials using HPV VLP
vaccines delivered intramuscularly. To date three clinical studies
with VLP were reported.
Harro and colleagues have performed a phase I trial at the NIH to
determine safety and efficacy in respect to antibody induction of a
HPV 16 L1 VLP vaccine [94]. Healthy volunteers were vaccinated
Review
three times, with 10 or 50 microgram of HPV 16 L1 VLPs with or
without adjuvants. All persons tolerated the vaccine without any
side effects. The vaccination turned out to be highly immunogenic
even in the absence of adjuvant. All vaccinees seroconverted and
had titers up to 40 times higher than in naturally infected persons.
The positive sera were able to neutralize infectivity of pseudovir-
ions suggesting that such antibodies will be able to protect from
virus infection. Currently, phase III clinical trials using intramus-
cular administration of the HPV 16 L1 VLP vaccine are being per-
formed in Guanacaste, Costa Rica. However, given the long lag be-
tween infection and onset of disease, the benefit of a prophylactic
vaccination program will take many years to become visible in re-
ducing the rate of cervical cancer [95].
The first conclusive evidence that a prophylactic vaccine will
protect women from HPV infection and CIN development comes
from a recent trial performed in college girls in the Seattle/USA
area and sponsored by Merck Research Laboratories [96]. Volun-
516 teers (women, 16±23 years of age) who screened negative for
HPV 16 and dysplastic lesions were randomized into verum or
placebo groups. They received three vaccinations with 40 micro-
gram of HPV 16 L1 VLP or placebo formulated with alumn adju-
vant at day 0, month 2, and month 6, and were followed every
6 months by HPV 16 testing and cytology. Cytologically suspec-
tive women were referred to colposcopy and biopsies were ob-
tained for analysis. Of the VLP-vaccinated women 99.7 % sero-
converted with specific antibody titers 58 times as high as in nat-
ural infection. While in the placebo group of 765 women 41 cases
of new persistent HPV16 infection and 9 cases of HPV 16 related
CIN occurred, none of the 768 women receiving VLP vaccine ac-
quired infection or dysplasia over the median follow-up time of
17.4 months. This is giving a 100 % vaccine efficacy and points to-
ward induction of sterilizing immunity. These encouraging re-
sults will have to be repeated in a larger study to show that clin-
ical disease is prevented. In addition, multivalent vaccines to
protect against other HPV types will be evaluated.
VLP vaccination for low-risk HPV types like HPV 6 and HPV 11
may reduce the incidence of anogenital warts. A HPV 11 L1 VLP
candidate vaccine was tested in healthy volunteers in a phase I
dose finding trial. It was well tolerated and induced high levels
of neutralizing antibodies as well as robust proliferative T cell re-
sponses [97].
In another attempt to develop a vaccine for anogenital warts, 33
patients with persisting anogenital condylomata were vaccina-
Kaufmann AM et al. HPV induced cervical ¼ Zentralbl Gynakol 2002; 124: 511 ± 524
ted with HPV 6 b L1 VLPs without any adjuvant three times or
more. In 25 of 33 vaccinees complete and lasting regression was
observed over the 20 week observation period. The vaccine was
well tolerated and induced seroconversion in 22 of 32 patients
with 9 having antibodies prior to vaccination. Six of eight with
preexisting low titers had an increase in antibody titer. Interest-
ingly, in a DTH skin test with the antigen, all patients acquired a
positive reaction, pointing at T cell induction that may be respon-
sible for the therapeutic effect of resolving preexisting warts
[98].
Although this result of a direct therapeutic effect induced by
L1 VLP was unexpected, it shows that in principle therapeutic
vaccines might work and take the burden of infection and dys-
plasia from patients. This is another argument for the investiga-
tion of therapeutic vaccination strategies.
Therapeutic Vaccines in Clinical Trials
Therapeutic vaccines should induce specific cell-mediated im-
munity that prevents the development of lesions and eliminates
infection, preexisting lesions or even malignant tumours. Theo-
retically, the induced specific cellular immunity can directly tar-
Downloaded by: University of Florida. Copyrighted material.
get HPV viral products, HPV-induced cellular products, or both.
However, most of the experimental vaccination systems use car-
cinoma-associated HPV proteins, particularly E6 and E7, as tar-
gets for specific cellular immunity. L1 and L2 capsid proteins are
unlikely to be suitable targets for therapeutic vaccine develop-
ment because these proteins are not detectably expressed in bas-
al epithelial cells of benign lesions or in abnormal proliferative
cells of premalignant and malignant lesions [99, 100]. Since E6
and E7 are consistently expressed in most cervical cancers and
their precursor lesions but absent from normal tissues, these vir-
al oncoproteins represent promising targets for the development
of antigen-specific therapeutic vaccines for HPV-associated cer-
vical malignancies and their precursor lesions. While most tu-
mour-specific antigens are derived from normal or mutated pro-
teins, E6 and E7 are completely foreign viral proteins, and may
therefore harbor more antigenic peptides/epitopes than a mu-
tant cellular protein. Furthermore, since E6 and E7 are required
for the induction and maintenance of the malignant phenotype
of cancer cells [101], cervical cancer cells are unlikely to evade
an immune response through antigen loss. Finally, studies in an-
imal models suggest that vaccination targeting papillomavirus
early proteins such as E7 can generate therapeutic as well as pro-
tective effects [102]. Therefore, E6 and E7 proteins represent
good targets for developing antigen-specific immunotherapies
or vaccines for cervical cancer.
Various forms of HPV vaccines, such as peptide-based [103], pro-
tein-based [104, 105], DNA-based [106 ± 110], chimeric VLP-
based [111 ± 113], vector-based [114 ± 116], and cell-based vac-
cines [117] have been described in experimental systems target-
ing HPV 16 E6 and/or E7 proteins. Researchers mainly focus on
E7 because it is more abundantly expressed and better character-
ized immunologically than E6. Furthermore, its sequence is more
conserved than the E6 gene [118].
The following sections will concentrate on and describe thera-
peutic vaccines that have been tested in clinical trials. These ex-
perimental vaccines are summarized in Table 2.
"219", 28.5.03/dk köthen GmbH
Table 2 HPV vaccines in clinical trials
Class of vaccine Type of study
Peptide-based Phase I, cervical and vaginal cancer
(therapeutic)
Phase I/II, advanced cervical carcinoma
Phase I, high grade CIN/VIN
Protein-based Phase I, healthy volunteers
(therapeutic)
Phase II a, patients w/anogenital warts
Phase I, healthy volunteers
open lable trial, patients w/anogenital warts
VLP based Phase I, patients w/anogenital warts
(prophylactic)
Phase I, heatlhy volunteers
Phase I, healthy volunteers
Phase I, healthy volunteers
Vector based Phase I/II, terminal cervical cancer
(therapeutic)
Phase I/II, early invasive cervical cancer
Cell based Case report, terminal cervical cancer patient
(therapeutic)
Pilot study, individual terminal cervical cancer
patients
Peptide-Based Vaccines
Several HPV 16 E7-specific CTL epitopes have been characterized
for the HLA-A.2 haplotype [119, 120]. The presence of such epi-
topes on cervical cancer cell lines was shown and epitope specif-
ic T cells are able to lyse such cervical cancer cells in vitro [121 ±
123]. In human studies, some HPV-associated cancer patients
vaccinated with peptides derived from HPV 16 E7 showed CTL re-
sponses.
A lipidated HLA A.2-restricted epitope of HPV 16 E7 was given
4 times and induction of epitope specific T cells monitored. Four
of 10 patients had specific CTL already before vaccination, 5 of
7 displayed reactivity after two vaccinations and 2 of 3 after re-
ceiving all four vaccinations. However, there were no clinical re-
sponses or treatment toxicities observed [124].
In one phase I/II trial, a dose escalation study with a mixture of
two HPV 16 E7 epitopes and an unrelated T helper peptide emul-
sified in adjuvant were applied to 19 terminal patients. There was
a low count in overall lymphocytes seen in 11 of 19 patients indi-
cating a status of immunosuppression. No correlation between
dosage and clinical outcome could be seen and induction of
specific T cells to the T helper epitope was seen in 4 of 12 patients.
However, no CTL responses to the HPV 16 E7 peptides were de-
tectable. No adverse side effects of peptide-based HPV vaccines
were observed in these cervical cancer patients [125, 126].
Therefore, a phase I study in women with HPV 16 positive CIN II/
III or vulvar intraepithelial neoplasia (VIN) with HLA-A.2 restrict-
Verum Reference
lipidated HPV 16 E7 epitope Steller et al. 1998 [124]
HLA-A.2 haplotype-restricted HPV 16 E7 peptide van Driel et al. 1999 [125]
HLA-A.2 haplotype-restricted HPV 16 E7 peptide Munderspach et al. 2000 [127]
recombinant HPV 6 L2 E7 fusion protein Thompson et al. 1999 [131]
recombinant HPV 6 L2 E7 fusion protein Lacey et al. 1999 [105]
recombinant HPV 16 L2 E6 E7 fusion protein De Jong et al. 2002 [130]
Review
Hsp65-HPV 16 E7 fusion protein Goldstone et al. 2002 [129]
HPV 6 b L1 VLP Zhang et al. 2000 [98]
HPV 16 L1 VLP Harro et al. 2001 [94]
HPV 11 L1 VLP Evans et al. 2001 [97]
HPV 16 L1 VLP Koutsky et al. 2002 [96]
recombinant vaccinia virus (Wyeth) containing Borysiewicz et al. 1996 [114]
modified HPV 16/18 E6 E7
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same Kaufmann et al. 2002 [151]
autologous DC pulsed with HPV 18 E7 protein, Santin et al. 2002 [145]
T cell adoptive transfer
autologous DC pulsed with HPV 16 or HPV 18 Ferrara and Nonn et al. [146]
E7 protein
ed E7 epitopes was performed. Patients were treated with esca-
lating doses up to 2 mg of peptide emulsified in incomplete 517
FreundÂs adjuvant by four immunizations each three weeks apart.
Toxicity was modest with only grade I or II local reactions and
grade II systemic symptoms at the highest concentration. Only 3
of 18 patients cleared their dysplasia completely and 6 had par-
tial regression measured by colposcopy. Six of 6 patients ana-
lysed showed an immune cell infiltrate (dendritic cells) in the
biopsy after vaccination. Although E7 specific cytokine release
and cytolysis assays were increased in 10 of 16 patients, no posi-
tive DTH skin test was observed in any of the vaccinees. Twelve
of 18 patients cleared the virus DNA from the scrapings but all
biopsy specimens were still positive by in situ RNA hybridization
[127]. Although the virus load seemed to be reduced the clinical
outcome of the therapy was questionable. The potency of HPV 16
E7 peptide-based vaccines will have to be further enhanced by
the use of adjuvants that are immunostimulatory, direct the pre-
sentation of the peptides to professional antigen presenting cells,
and induce a cytolytic and T helper response for sustained im-
munity.
A way to circumvent HLA-restriction by vaccination with pep-
tides that are minimal T cell epitopes is to use longer overlapping
peptides representing the whole sequence of an antigen. Such
peptide ªlibrariesº represent all possible epitopes of a given anti-
gen. When used for vaccination of mice an individual long pep-
tide encompassing the minimal T cell epitope was superior prob-
ably due to delivery of additional T helper cell epitopes. Antigen
presenting cells (APC) were obviously able to process the rele-
Kaufmann AM et al. HPV induced cervical ¼ Zentralbl Gynakol 2002; 124: 511 ± 524
"219", 28.5.03/dk köthen GmbH
vant peptides from the long peptide, as they do from whole pro-
teins (see below) [128]. Therefore synthetic long overlapping
peptides of antigens may replace minimal T cell epitopes or re-
combinantly expressed full length protein in future trials.
Protein-Based Vaccines
The application of peptide-based vaccines is limited by HLA re-
striction and the necessity to define specific CTL epitopes. Most
CTL epitopes of HPV 16 E6 and/or E7 in patients with HLA other
than HLA-A.2 remain undefined, making it difficult to use pep-
tide-based vaccines in such situations. In addition, the prepara-
tion of peptide-based vaccines for use on a large scale is ineffi-
cient and laborious. These limitations can potentially be over-
come by using protein-based vaccines. Protein-based vaccines
Review
can present all possible epitopes of a protein to the immune sys-
tem, thus bypassing the HLA restriction. Furthermore, with a
protein vaccine, dangerous side effects such as insertional gene
activation and transformation, a potential concern with the use
of recombinant virus vaccines (see below), are not an issue. On
the other hand, protein as antigen is more likely to induce sero-
logic than cellular responses, if not directed to a T helper 1/cyto-
toxic T cell response. In animal models, a growing number of
modified exogenous protein antigens, including HPV 16 E7, have
been shown to be capable of generating MHC class I-restricted
CTL responses.
One possibility to enhance the immunogenicity of HPV proteins
is fusion to immunostimulatory proteins. Candidates are heat
shock proteins that represent danger signals for the immune sys-
tem and carry antigenic peptides [129]. A fusion protein consist-
ing of hsp65 from bacille Calmette-Guerin and HPV 16 E7 was
constructed and tested in patients with high-grade squamous in-
518 traepithelial lesions by Stressgene Biotechnologies (Collegeville,
PA, USA). Monthly 3 times 500 microgram were given to 22 pa-
tients with anogenital warts. After 24 weeks of treatment 3 of
14 patients with warts at baseline had complete resolution and
10 had size reduction between 70 to 95 % diminishing the abla-
tion procedure. The remaining patient's warts increased in size.
No serious or severe adverse events were observed. The effect
seemed not to be strictly HPV type specific.
Another type of fusion protein under investigation by Xenova
Ltd. (Cambridge, UK) consists of the HPV 16 L2 minor capsid pro-
tein fused to the oncoproteins E6 and E7 of the same HPV type
termed TA-CIN. In a phase I study 40 healthy volunteers were
vaccinated in a dose escalating study without adjuvant by intra-
muscular injection three times. Both IgG antibodies and prolif-
erative T cell responses were elicited at all doses tested. At the
highest dose 8 of 11 subjects had developed T cell immunity to
the oncoproteins E6 and E7 measured by IFN-gamma ELISpot as-
says [130]. No serious adverse events were seen in the trial while
proving immunogenicity of the vaccine. Therefore, clinical trials
in patients with persisting CIN or VIN lesions are under way.
A similar construct consisting of HPV 6 L2 E7 termed TA-GW was
tested in phase I and II trials in healthy volunteers or patients
with genital warts, respectively. While humoral and cellular im-
munogenicity and safety was demonstrated in the phase I trial,
the phase II showed complete regression of warts in 5 of 25 pa-
tients and lasting clearance in all 13 patients that had wart re-
Kaufmann AM et al. HPV induced cervical ¼ Zentralbl Gynakol 2002; 124: 511 ± 524
gression after vaccine alone, or after having additional conven-
tional therapy [105, 131].
Taken together, approaches relying on fusion proteins seem to
enhance the immunogenicity of the target antigen considerably.
Chimeric HPV VLP-Based Vaccines
Although immunization with HPV VLPs can protect animals from
experimental papillomavirus infections, induces high titer neu-
tralizing antibodies in the serum, and could lead to anogenital
wart regression [98], immunization with L1 VLPs is not expected
to generally generate therapeutic effects for established HPV in-
fections. Preexisting HPV infection is highly prevalent and the in-
fected population represents an important target for the elimi-
nation of HPV-related disease, a task that likely requires the gen-
eration of protective humoral immunity as well as therapeutic T
cell-mediated immunity against HPV antigens. To create a pre-
ventive and therapeutic VLP-based vaccine, several E7 chimeric
VLPs consisting of the L1 major capsid protein plus the E7 protein
or peptide have been created. These E7 chimeric VLPs have been
shown to generate significant E7-specific CTL activities and E7-
specific antitumour effects in animal models [111 ± 113] and by
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in vitro vaccination in humans [132]. Furthermore, E7 chimeric
VLPs are indistinguishable from parental VLPs in their ability to
elicit high titers of neutralizing antibodies [111]. These findings
suggest that E7 chimeric VLPs may potentially generate L1-
specific protective humoral immune responses and E7-specific
therapeutic cellular immune responses in vaccinated individ-
uals. Currently, clinical grade HPV 16 L1/E7 chimeric VLPs, are
under investigation in a multicenter phase I/II clinical trial in
Germany. Safety, immune responses, and clinical outcome are
endpoints of the study. Results are expected in early 2003 [Medi-
Gene homepage:
(http://www.medigene.com/englisch/index_e.php)].
Cell-Based Vaccines
Cell-based vaccines for cancer immune therapy can be concep-
tually divided into two broad categories: dendritic cell (DC)-
based vaccines and cytokine-transduced tumour cell-based vac-
cines. DCs are the most potent professional APCs, specialized to
prime helper and killer T cells in vivo. Ex vivo preparation and
modification of DCs, therefore, represents an attractive vaccine
strategy that is capable of enhancing T cell-mediated immunity
against tumours. The understanding that DCs can be generated
from hematopoietic progenitors in the setting of various cyto-
kines, mainly GM-CSF and Interleukin-4 or Flt3 ligand, has cre-
ated the opportunity to use a tumour cell-based vaccine trans-
duced with GM-CSF or Flt3-ligand cytokines to expand and
prime DCs in vivo [for review, see 133].
DC-Based Vaccines
Recent advances have revealed the origin of DCs, their antigen
uptake mechanisms, and the signals that stimulate their migra-
tion and maturation into immune stimulatory APCs [for review,
see 134, 135]. Several strategies for the generation of large num-
bers of active DCs ex vivo focus on the use of cytokine factors to
induce the differentiation of primitive hematopoietic precursors
into DCs [136 ± 139]. DCs derived from cultured hematopoietic
progenitors appear to have an APC function similar to purified
mature DCs. Ex vivo generation of DCs, therefore, provides a
"219", 28.5.03/dk köthen GmbH
source of professional APCs for use as potent cellular adjuvants in
experimental immune therapy. There are several vaccine strate-
gies using DCs loaded with HPV antigens by pulsing with pep-
tides, recombinant proteins, cloned or tumour extracted RNA/
DNA, or tumour lysate.
DC Pulsed with Peptides/Proteins
Presentation of peptides derived from HPV E6 and/or E7 to the
immune system by DCs is a promising method of circumventing
tumour-mediated immune suppression. Treatment of tumours
with peptide-pulsed DCs has resulted in sustained tumour re-
gression in several different tumour models [for review, see 140].
Several in vitro studies in the human system have shown convin-
cingly that DC derived from healthy individuals and pulsed with
peptides or full length proteins induce antigen specific T cell re-
sponses [141 ± 143]. Another study demonstrated that DCs de-
rived from patients can be pulsed with fusion proteins such as
E6/E7 and used to generate E6/E7-specific CTLs in vitro [144].
This approach has been taken into early clinical trials or pilot
studies. Santin et al. have reported on a case study of a HPV 18-
positive patient that was treated 14 times with HPV 18 E7 pulsed
autologous DC (3±5 million per injection) together with IL-2 and
adoptive transfer of in vitro restimulated autologous T cells. The
patient had an uncommon stabilization of the disease over a
period of 20 months, when finally metastatic disease reoccurred
and she died. Although no permanent remission was achieved,
disease progression was inhibited, the patients performance
and quality-of-life improved without any significant adverse ef-
fects [145].
A similar approach was undertaken by Ferrara and Nonn et al.
[146]. In a series of compassionate therapies in 15 individual
late stage cervical cancer patients monocyte-derived autologous
DCs loaded ex vivo with recombinant HPV 16 or 18 E7 protein
were applied. Although no objective clinical response was seen
in any patient, the vaccination induced antibodies in 3 of 11 and
specific T cell responses in 4 of 11 patients [146].
DC-based therapy is at an early stage and improvements in DC
differentiation and numbers, antigen delivery, application route,
and adjuvants may be necessary to see therapeutic effects in
such patients. Also it may well be that late stage tumours are re-
sistant to immune therapy as outlined below and therapeutic
success will only be seen in patients suffering from earlier stage
disease.
Tumour Cell-Based Vaccines
The use of tumour cell-based vaccines may not be suitable for
the treatment of early-stage, precancerous HPV-associated le-
sions because of the risks and controversy associated with ad-
ministering modified tumour cells to these patients. Therefore,
tumour cell-based vaccination is likely reserved for patients
with advanced HPV-associated cancer. Transduction of tumour
cells with genes encoding costimulatory molecules or cytokines
may enhance immunogenicity leading to T cell activation and
antitumour effects after vaccination [for review, see 133]. To
date no clinical studies have been reported in cervical cancer.
Several HPV-related tumour cell-based vaccines have been re-
ported in preclinical model systems. For example, vaccines em-
Kaufmann AM et al. HPV induced cervical ¼ Zentralbl Gynakol 2002; 124: 511 ± 524
ploying HPV-transformed tumour cells transduced with cytokine
genes such as IL-12 [147] and IL-2 [148] have been demonstrated
to generate strong antitumour effects in mice. Recently, an E7-
expressing GM-CSF gene-transduced allogeneic tumour cell-
based vaccine has been shown to generate E7-specific CTL activ-
ities and protective antitumour immunity in immunized mice
[149]. These preclinical data indicate that tumour cell-based vac-
cines may be useful for the control of minimal residual diseases
in patients with advanced HPV-associated cervical cancers.
Viral-Vector Vaccines Based on Vaccinia
Vaccinia virus, a member of the poxvirus family, has been used
successfully in the eradication program of the small pox epi-
demic. Its safety profiles and clinical application are well estab-
Review
lished. Nowadays it can be used to mediate the transfer of genes
into host APCs. This strategy offers several appealing features in-
cluding high efficiency of infection and high levels of recombi-
nant gene expression [150]. Infection with recombinant vaccinia
and expression of the desired gene product occurs quickly and
the genome can accommodate large recombinant gene inser-
tions. The availability of highly attenuated strains like MVA or of
replication-deficient recombinant poxvirus, such as canarypox
Downloaded by: University of Florida. Copyrighted material.
virus, has provided the opportunity to use this recombinant virus
as a vector for gene transfer into human APCs. Furthermore, vac-
cinia is a lytic virus and thus the chance of integration into the
host genome is extremely small. These characteristics make vac-
cinia a suitable vector for tumour vaccines.
A recombinant vaccinia termed TA-HPV encoding HPV 16 and
HPV 18 E6/E7 has been developed and has been used for a phase
I clinical trial in late stage cervical cancer patients [114]. No sig-
nificant complications or environmental spread of the virus was
noted in this trial. The induction of specific CTL and prolonged 519
survival of 1 of 3 patients paved the way for a phase I/II trial in
patients with early invasive cancer (stage I b±II a). Twentynine
patients were vaccinated twice, once before and once after radi-
cal hysterectomy. Side effects of the vaccination were mild and
predominantly local. A de novo induction of HPV specific anti-
bodies was observed in 8 of 17 evaluable patients and of specific
CTL in 4 of 29 patients. This multicenter study conducted by the
European Organization for Research and Treatment of Cancer
(EORTC) proved the safety and potential immunogenicity of TA-
HPV [151]. Therefore, the vaccine is being tested in combination
with other approaches and for additional indications. Patients
with recurrent high grade VIN were treated once with TA-HPV.
In 9 of 14 vaccinees an improvement of symptoms was noted,
with 8 of 18 partial regressions that could be confirmed histolo-
gically in 5 of 18 patients. Two patients cleared HPV infection
completely [152].
Specific Problem: Immune Evasion
To ensure propagation HPV have evolved as well adapted ªmole-
cular parasitesº and have developed strategies to evade the im-
mune system [153]. Therefore, HPV infections are non virulent,
do not induce inflammation, and persist for prolonged periods
of time. However, eventually the immune system reacts leading
to regression of dysplasia and clearance of HPV from the tissue.
This happens in about > 95 % of infections [154]. Given the high
prevalence of infection, the progression to invasive cancer is a re-
latively rare event involving host factors as outlined above [155].
"219", 28.5.03/dk köthen GmbH
Investigation of the antigen processing/presentation machinery
in cervical cancer cell lines and tumours showed defects on every
level [151, 156, 157]. For example, loss of HLA-A, B, C expression is
a frequent event in a high number (up to 70 %) of late stage cervi-
cal cancers [146]. This might reflect that basically the viral anti-
gens, that are presented by the tumour cell in a HLA-restricted
manner, are good antigens inducing an effective immune re-
sponse. However, upon immune selection of the genetically un-
stable and heterogeneous tumour cell population variants grow
out that are resistant against immune attack. In addition to HLA
loss, cervical cancer cells can secrete immune suppressive factors
like TGF-beta [158] and can acquire mechanisms to inactivate
granzymes and perforins [159]. This might compromise the suc-
cess and explain the failure of immune therapies in late stage pa-
Review
tients as observed in the early clinical trials described. However,
after proving safety of vaccines in late stage patients, vaccination
in dysplasia or early invasive cervical cancer (FIGO stage I b±II a),
or of patients with advanced disease but selected for full HLA ex-
pression by tumour cells, the effectivity of vaccinations may
come apparent.
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