Extract
Note: Please read the complete
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Introduction
Cervical cancer is one of most common cancers in women. Human papillomaviruses (HPV), particularly HPV-16, are
associated with most cervical
cancers[1,2]. HPV oncogenic proteins, E6 and E7, are expressed in most HPV-containing cervical
cancers and are constitutively expressed in cervical carcinoma cells. They are the only viral proteins whose
expression is required for the maintenance of the transformed
state[3_5]. Compared with E6, E7 has stronger
immunogenicity[6], therefore, E7 is an attractive target for the specific
immunotherapy of cervical carcinoma.
Cytotoxic T lymphocyte (CTL) response is an important
defense mechanism against tumors[7,8]. CTL recognize
immunogenic peptides derived from cellular proteins presented
to the cell surface in the content of MHC class I molecules.
Many studies demonstrate that immunization with antigenic
peptides alone is not sufficient to elicit an efficient CTL
response[9]. Studies have shown that helper T lymphocyte
(Th) immunity is also considered vital for the CTL induc-
tion[10_13]. Lipopeptide formulations have been successfully
used to induce CTL responses and have recently gained
considerable interest and are a promising vaccine
candid-ate[14_16]. Moreover, the peptide of the antigen HPV
E786_93 is restricted to human leukocyte antigen (HLA)-A*2010, the
most common HLA class I molecule, which makes it a good
candidate for vaccination[17]. The
TT830_843 peptide, derived from tetanus toxoid, was chosen as a source of T cell help
because of its promiscuous binding to human MHC class II
antigens and its promiscuous recognition by T
cells[18]. A dipalmitic lipopeptide could induce epitope-specific CTL in
phase I clinical studies[19,20], but it is difficult to be
synthesized and purified. So we constructed a monopalmitic
lipopeptide (Table 1).
We evaluated the ability of the lipopeptide to provoke
in vivo- and in vitro-specific CTL responses in
HLA-A*0201Kb transgenic (Tg) mice and peripheral blood mononuclear cells
(PBMC) preparations from MHC-matched healthy donors.
We also studied the presentation of peptides in T2 cells and
dendritic cell (DC) maturation induced by peptides.
Our findings show that the lipopeptide can induce a
strong CTL response, the mechanisms of which are
associated with an increase in the stability and persistence of the
antigenic complex formed with the lipopeptide and in the
production of interleukin (IL)-12 by DC. These findings
suggest that the lipopeptide against cervical cancer can be
considered as a more effective vaccine type for a
human.
Materials and methods
Peptide synthesis and characterization The
HLA-A*0201-restricted the CTL epitope derived from the HPV
oncogenic protein, E7, referred to here as the P3 peptide,
was used in this study (Table 1)[21]. The lipopeptide
construct is referred to here as the P1 lipopeptide. An
unlipid-ated version denoted P2 was used as the control peptide
(Table 1).
All peptides were synthesized on an ABI 431A
instrument using standard Fmoc technology (PE Applied
Biosystems, Foster city, CA, USA)[22]. The solid support
was Wang resin (Sigma, St Louis, MO, USA). The Fmoc
protecting the group was removed by 20% peperidine in
N-methylpyrrolidone (NMP). To synthesize the lipopeptide, a
Boc-Lys-(Fmoc)-OH group was incorporated at the
N-terminal extremity, which allowed the selective removal of the
Fmoc group by 20% peperidine in NMP. Then the cold
peptide-resin was placed into the cold cleavage mixture (0.25 mL
ethanedithiol, 0.25 mL water, 9.5 mL trifluoroacetic acid). The
reaction mixture was stirred at room temperature for 1.5 h.
Then these peptides were removed from the resin support
and precipitated by adding cold diethyl ether and lyophilized.
These crude peptides were purified by RP-HPLC. All
peptides were checked for homogeneity by analytical RP-HPLC
and for identity by an electrospray mass spectrometer. The
purity was >85% for all peptides. The peptides were
dissolved in DMSO at a concentration of 20 g/L and stored at
-20 °C.
Cells lines Human transporter associated with antigen
processing (TAP)-deficient T2, cervical carcinoma Caski cell
lines were obtained from the American Type Culture
Collection (ATCC, Manassas, VA, USA) and maintained in culture
according to the supplier's specifications. A stable
trans-fectant of the jurkat human cell line that expresses the
chimeric class I molecule, A*0201Kb, was kindly provided by Dr
Jehad CHARO (Max Delbruck Center for Molecular
Medi-cine, Berlin, Germany). The cell lines were cultured in
RPMI-1640 (Gibco, Grand Island, NY, USA) with glutamax
containing 10% fetal calf serum (FCS), antibiotics,
N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid
(HEPES), and 0.4 g/L G418 (Gibco, USA).
Animal immunization procedures and generation of CTL
HLA-A*0201Kb Tg mice were purchased from The Jackson
Laboratory (Bar Harbor, ME, USA). The mice were bred and
maintained in specific, pathogen-free facilities. Groups of
8-12 week-old HLA-A*0201Kb female Tg mice (5 mice per group)
were immunized with synthetic peptide +/_ incomplete
Freund's adjuvant (IFA). Synthetic peptides were injected
with 50 nmol per mice at the base of the tail for subcutaneous
route. Transgenic mice were boosted 2 weeks later with the
same synthetic peptide. After 14 d, the mice were killed and
the spleens were aseptically removed. Splenic single cell
suspensions were produced by teasing the organs through
a sterile nylon mesh as previously
described[21]. The splenic suspensions
(3×106) from the immunized mice were cocultured
with 1 µg/mL P3 peptide in 5% CO2 at 37 °C. Cytotoxic
effector populations were harvested after 5 d of
in vitro culture.
Generation of CTL in healthy donors The CTL
induction in vitro was performed in accordance with the
previously described procedures[23,24]. In brief, the PBMC of 3
HLA-A*0201 healthy donors from the blood bank of the
Third Military Medical University (Chongqing, China) were
first obtained with centrifugation at a Ficoll-Paque density
gradient and then cultured in RPMI-1640 medium
supplemented with 10% FCS, 100 U/mL penicillin, and 0.1 g/L
streptomycin. These cells were pulsed by the synthetic
peptides, respectively, at a final concentration of 10 µmol/L.
The PBMC were restimulated every 7 d with fresh medium
containing these peptides. Recombinant human interleukin
(IL)-2 (Sigma, USA) at a concentration of 30 IU/mL was added
to the culture medium on d 1 after every stimulation. On d 6
after the third round of restimulation, the cells were harvested
and tested by cytotoxicity assay.
Cytotoxicity assay The cytotoxic activity of the cell
culture was determined by a standard 4 h chromium release
assay[25]. To measure the peptide-specific responses, T2
cells and jurkat A*0201Kb cells were loaded with 10
μmol/L peptide at 37 °C for 1 h. Peptide-plused T2 cells, jurkat
A*0201Kb cells, and the Caski cell line were labeled with 3.7
MBq Na251CrO4 for 1.5 h at 37 °C. Target cells
(104/ well) were transferred into a round-bottomed, 96-well plate. Varying
numbers of CTL were added to give a final volume of 200 µL
and incubated for 4 h at 37 °C. At the end of the assay, the
supernatant was harvested and counted. The percentage of
specific lysis was calculated as follow: (experimental release
-spontaneous release)/(maximal release-spontaneous
release)×100. Spontaneous and maximal release were determined in
the presence of medium or 1% Triton X-100, respectively.
Cell isolation and generation of DC from
PBMC The generation of DC from PBMC was performed as described
previously[26]. In brief, peripheral blood was collected from
health volunteers (n=3) and fractionated over Ficoll-Paque
by a standard procedure. To derive DC, PBMC were seeded
(2×106/mL) into 6-well plates in RPMI-1640 medium
supplemented with 100 units/mL penicillin and 0.1 g/L streptomycin.
After 2 h of incubation at 37 °C, nonadherent cells were
removed and the adherent cells were cultured in RPMI-1640
medium supplemented with 10% FCS, 50 ng/mL
granulocyte-macrophage colony-stimulating factor (GM-CSF) (Sigma,
USA), 500 U/mL IL-4 (Sigma, USA), 100 U/mL penicillin, and
0.1 g/L streptomycin for 4 d at 37 °C. Immature DC were
incubated in vitro for 48 h with P1, P2, or P3 peptides.
Medium-treated, immature DC and tumor necrosis factor
(TNF)-α-stimulated DC were included as negative and positive
controls, respectively. The phenotype of DC was analyzed
by flow cytometry.
Interferon (IFN)-g and IL-12 detection
IFN-g secretion in stimulated in vitro (IVS) culture supernatants and IL-12 in
immature DC cultures were measured by a commercially
available, two-site sandwich ELISA (Diaclone, Besancon,
France) according to the manufacturer's instructions.
Collection of the supernatant was performed at 72 h after IVS.
DC were pulsed with P1, P2, or P3 peptides and incubated
with autologous PBMC in RPMI 1640 medium. After 48 h of
culture, IL-12 concentrations in the supernatant were
determined. Recombinant IFN-g and IL-12 were used for the
preparation of a standard curve. Each sample was tested in
duplicate.
Peptide-binding assay To determine whether synthetic
peptides could bind to HLA-A*0201 molecules,
peptide-induced HLA-A*0201 upregulation in T2 cells was examined
according to a protocol described
previously[27]. Briefly, T2 cells were incubated with 10
µmol/L peptides and 5 µg/mL human
β2-microglobulin (Sigma, USA) in serum-free Iscove's
Modified Dulbecco's medium for 18 h at 37 °C. The
expression of HLA-A*0201 on T2 cells was then determined using
anti-HLA-A*0201 mAb B B7.2 followed by a fluorescein
isothiocyanate (FITC)-conjugated goat anti-mouse antibody
(Becton Dickinson, Mountain View, CA, USA). The samples
were analyzed on a FACScan Calibur (Becton Dickinson, USA).
Stability of HLA-A*0201-peptide complexes on cell
surface[22] The T2 cells were incubated with 10 µmol/L peptides
as described for the peptide-binding assay. After 18 h
incubation at 37 °C, the T2 cells were washed. At the indicated
times, they were stained with mAb B B7.2 and analyzed by
flow cytometry.
Flow cytometry Standard flow cytometric analysis was
used to assess surface expression of various markers using
the following mAb directly conjugated with either
phycoerythrin (PE) or FITC: FITC-CD40, FITC-CD80, FITC-CD83,
and PE-HLA-DR (Pharmingen, San Diego, CA, USA). IgG
isotype-matched control antibodies were used in all of the
experiments. After staining, the cells were washed and fixed
in 1% paraformaldehyde before analysis on a FACScan
Calibur (Becton Dickinson, USA).
Statistical analysis The Student's
t-test was performed to evaluate the significance of the results.
P values less than or equal to 0.05 were considered significant.
Results
Immunological effect in transgenic mice after
vaccination with peptides To assess their in
vivo immunogenicity, HLA-A*0201Kb Tg mice were immunized with each of the
peptide in IFA. Splenocytes from immunized mice were
restimulated in vitro and tested for IFN-g production and
CTL reactivity. CTL from P1-immunized mice showed strong
IFN-g production, an indicator of CTL reactivity. CTL from
P1-immunized mice efficiently lysed jurkat
A*0201Kb cells added with the
E786_93 peptide, but did not lyse jurkat
A*0201Kb cells alone. These results indicated that the
cytotoxicity of induced CTL was peptide specific. CTL from
P2-immunized mice also showed modest IFN-g production and
cytotoxicity. In contrast, immunization with the P3 peptide
in IFA was shown to have no specific reactivity (Figure 1).
Induction of CTL by the lipopeptide without an
adjuvant To further explore the potential of the lipopeptide for human
vaccination, we investigated whether the lipid part of the
lipopeptide construct could bypass the requirement for IFA.
Separate groups of Tg mice were immunized either with the
lipopeptide or with an equimolar amount of the unlipidated
version. Peptide-specific CTL responses were examined in
the spleens of Tg mice. CTL from lipopeptide-immunized
mice efficiently lysed jurkat A*0201Kb added with the
E786_93 peptide, but not the unlipidated analogue P2. These data
indicated that the lipopeptide could efficiently induce
specific CTL responses in vivo without the need for an adjuvant
(Figure 2).
In vitro generation of peptide-specific CTL from healthy
human donors To investigate the capacity of peptides to
mobilize a human CTL repertoire, specific CTL were
generated in vitro using healthy HLA-A*0201+donor PBMC with
peptides. As shown in Figure 3, CTL induced with P1, P2,
and P3 peptides lysed T2 cells pulsed with
E786_93 and produced strong IFN-g, but did not lyse T2 cells alone.
Moreover, CTL induced by the P1 peptide elicited the
strongest cytotoxic activity. To analyze the ability of specific
CTL to lyse the endogenously HPV E7-expressing tumor cell,
the HPV E7-positive HLA-A*0201-expressing cervical
cancer cell line, Caski, was used as the target cell in a standard
51Cr-release assay. As shown in Figure 3, CTL were able to
efficiently lyse Caski tumor cells (Figure 3).
Peptide-binding affinity to HLA-A*0201 molecules
Peptide-binding affinity to HLA-A*0201 molecules was analyzed
by a binding assay on the TAP-deficient, human T2 cell line.
The stabilization assay was based on the fact that in T2
cells, a large proportion of MHC class I molecules are devoid
of endogenous peptides and are unstable at 37 °C. This
could be prevented by the addition of exogenous peptides
that bind within the MHC class I Ag-presenting groove,
stabilizing the 3-D structure of the complex. According to our
results, stabilization of MHC class I on T2 cells could be
obtained with the lipopeptide (Figure 4).
Complex stability assay The kinetics of complex
dissociation was evaluated by measuring the time necessary for
the complex to disappear from the cell surface. Our
observations revealed an increase in the persistence of the antigenic
complex formed with the P1 peptide (Figure 5).
DC maturation induced by peptides To check whether
the addition of peptides induce DC maturation, the DC were
incubated further for 48 h with or without peptides.
TNF-α was used as a maturation control. The mean surface
fluorescence intensity of molecules involved in the antigen
presentation and in costimulation was not increased, and CD83
expression was not induced by the peptides (Figure 6).
IL-12 secretion of DC induced by peptides
To check whether the exposure of DC to peptides increases IL-12
secretion, the DC were pulsed with P1, P2, or P3 peptides
and incubated with autologous PBMC in RPMI 1640 medium.
After 48 h of culture, IL-12 concentrations in the
supernatant were determined. There was an upregulation of
production of IL-12 following incubation of DC with P1, P2, or P3
peptides, and the effect of the P1 peptide was the strongest
(Figure 7).
Discussion
Traditional immunization includes vaccines that use live
attenuated organisms, inactivated organisms, whole proteins,
and naked DNA. From a practical and safe standpoint,
however, live, attenuated vaccines raise issues related to
manufacturing and safety that may preclude their widespread
use. Peptide-based approaches offer several potential
advantages over conventional whole proteins or naked DNA
in terms of purity, lot-to-lot consistency, cost of production,
and a high specificity in eliciting immune
response[15,28_31]. This technology has proven to be an extremely useful
strategy to avoid potentially harmful immune responses.
However, immunization with antigenic peptides alone is not
sufficient to elicit an efficient CTL
response[9]. It is important for peptide-based vaccine development to improve the
immunogenicity of peptides.
Our study showed P2 containing Th and CTL epitopes
could induce CTL response, but P3 only containing the CTL
epitope could not do so in Tg mice. The result showed the
necessity for the help effect of Th cells in the induction of
CTL, which was in agreement with recent
studies[12,32]. Our study also showed that P1 containing Th, CTL, and lipid
part had already induced the strongest CTL response, which
showed that linkage of Th, the CTL epitope, and lipid part
could improve the immunogenicity of the CTL epitope. Of
particular interest was the finding that peptide-specific CTL
were able to lyse Caski tumor cells, which increased the
possible clinical use of the lipopeptide as a cancer vaccine.
We investigated whether the lipopeptide could bypass
the need for IFA. Our study confirmed that a lipopeptide
alone could induce CTL response, and demonstrated that
such an immunogen bypassed the requirement for an adjuvant. The toxicity of a lipopeptide was low and a patient
could easily tolerate the toxicity, so the lipopeptide might be
a good vaccine for human use[15,16,33].
Loing and colleagues reported that the lipopeptide could
gain access into the MHC-loading compartment of T2 cells
without the help of the TAP transport system, and the
TAP-deficient T2 cells could specifically present the lipopeptide
to CD8+ T cells[22]. Our data indicated that the lipopeptide
could be processed and presented by T2 cells. Moreover,
studies have shown that the lipopeptide could also be
presented by powerful antigen-presenting cells
(APC)[34,35], which provide an additional advantage to the lipopeptide
construct in terms of immunogenicity potential.
IL-12 forms a link between innate resistance and
adaptive immunity and plays an essential role in antitumor
immune response. IL-12 has been shown to favor a Th1
response and to enhance the generation of
CTL[36]. Our results showed that the peptide could induce the
production of IL-12 of DC, which was one of the mechanisms of the
lipopeptide enhancing the immunogenicity of CTL epitope
peptides.
DC are the only APC capable of activating naive
lymphocytes, resulting in the initiation of protective immune
responses. In the absence of strong DC activation, naive T
cell will differ and die. Only properly matured DC will deliver
the signals required for full memory and/or effector CTL
induction. In other words, immature DC tolerate while
mature DC immunize[37,38]. Our results showed that the
lipopeptide could not directly induce the maturation of DC,
but the lipopeptides could activate Th and enhance the
expression of the CD40 ligand (CD40L) on Th. Then CD40L
on Th interacted with CD40 on DC, by which DC was
activated[39].
In our observation, the binding affinity of peptide
vaccines to the HLA molecules and their stability, as well as
levels of IL-12 production from DC by peptide vaccines
appear to be important for induction of CTL responses. In
our study, however, P3 peptides showed a higher binding
affinity to HLA molecules, as compared to P2 peptides. In
contrast, P2 peptides displayed a higher CTL activity than
P3 peptides. This discrepancy in the importance of the
peptide binding affinity vs CTL induction might be explained by
the possibility that P2 peptides (Th plus CTL epitopes) might
get a help from Th cells for improved CTL induction, as
compared to P3 peptides (CTL epitope alone).
In conclusion, the lipopeptide can induce a strong CTL
response, the mechanisms of which are associated with an
increase in the stability and persistence of the antigenic
complex formed with the P1 peptide and in the production of
IL-12 of DC. These findings suggest that the lipopeptide
against cervical cancer can be considered a more effective
vaccine type for a human. Moreover, the lipopeptide can be
synthesized in pure form, providing the ability to develop
rapidly this type of vaccine at a low cost.
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