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Departments of
* Immunohematology and Blood Transfusion and
Surgery, Leiden University Medical Center, Leiden, The Netherlands;
Corixa Corp., Seattle, WA 92104; and
Coley Pharmaceutical, Langenfeld, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over the years, various vaccines containing exactly fitting MHC class I-binding peptides have been tested for their therapeutic efficacy against both virally and nonvirally induced cancers (11, 12). However, the observed immunological and clinical responses were rather meager. Therefore, ways to develop more powerful cancer vaccines should be thoroughly explored. In a preclinical model we previously achieved considerable success when mice were vaccinated with a H-2Db-restricted HPV16 E7 CTL epitope (E749–57, RAHYNIVTF) emulsified in IFA (13). Immunized mice developed CTL-mediated protection against a subsequent challenge with an otherwise lethal dose of HPV16 E7-expressing tumor cells. Based on these encouraging findings several HPV peptide-based vaccines comprising minimal CTL epitopes were tested in patients with progressive disease (12, 14, 15). Although moderate T cell responses were observed, the clinical benefit was only modest. This may be due to an impaired immune system, which is commonly found in patients diagnosed with cancer, but failure of the vaccines to induce strong sustained immunity is more likely to be the cause. Recent preclinical data demonstrated that tumor-specific CD4+ Th cells critically contribute to the development and efficacy of antitumor responses (16, 17, 18, 19, 20). The effectiveness of these Th cells probably lies in their capacity to deliver essential activation signals to professional APCs, such as dendritic cells (DC), needed for an optimal priming of tumor-specific CTL (21, 22, 23, 24). In addition, Ag-specific CD4+ T cells may provide CTL with essential growth stimuli during the effector phase (25). In line with this notion, several studies show that effective CTL priming can be induced by the inclusion of Th epitopes in peptide vaccines (26, 27, 28, 29). Another strategy to power up vaccines is the use of molecularly defined strong DC-activating adjuvants such as oligodeoxynucleotide (ODN)-CpG (30, 31, 32), monophosphoryl lipid A (MPL) (33, 34), anti-CD40 Ab (35), and GM-CSF (36).
We have recently developed a clinical grade vaccine against HPV16 that consists of 32- to 35-aa-long overlapping peptides, together covering the HPV16 E6 and E7 protein sequences. The design of these long peptides was based on the presence of Th and CTL epitopes (3, 37, 38, 39), but we also speculated that, due to the length of the peptides, direct binding to MHC class I molecules would not take place. As a consequence, endocytosis, processing, and presentation by professional APC would be required to induce HPV16-specific CTL, and this would circumvent the potential hazard of peptide-induced tolerance (40).
In a preclinical mouse tumor model we analyzed the T cell response to
one of these 35-aa-long peptides, containing both a HPV16 E7-derived Th
epitope and the HPV16 E749–57 CTL epitope. As
demonstrated earlier (26), the physical linkage between
these two epitope types may potentiate specific CTL responses.
Vaccination with the long peptide resulted in markedly improved CTL
responses in comparison to the minimal essential CTL epitope vaccine.
In-depth analysis revealed that two distinct mechanisms were
responsible. Moreover, vaccination with the long peptide and the
DC-activating agent ODN-CpG induced strong antitumor immunity that
eradicated established tumors with sizes that otherwise resulted in the
death of mice within 
5 days.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6, H-2b) mice were obtained from IFFA Credo (Paris, France). MHC class II-/- B6 mice were purchased from Taconic Farms (Germantown, NY) and CD40-/- B6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Tumor cell line TC-1 was generated by retroviral transduction of lung fibroblasts of C57BL/6 origin by HPV16 E6/E7 and c-H-ras oncogenes (41). Tumor cell line 13.2 was derived from MEC (B6) transformed with adenovirus type 5-derived E1 protein in which the H-2Db E1A epitope was replaced with the HPV16 E749–57 CTL epitope. Isolation and culture of the HPV16 E7-specific CTL clone 9.5, the p53-specific CTL clone 1H11, and the nonrelevant 9.42 CTL clone have been described elsewhere (42, 43, 44). D1 cells are long-term growth factor-dependent immature splenic DC derived from C57BL/6 mice and were cultured as described (45, 46).
Peptides
Peptides were generated as described before (13). The purity of the peptides was determined by reverse-phase HPLC and was found to be routinely >90% pure. Peptides were dissolved in 0.5% DMSO in PBS and, if not used immediately, stored at -20°C. The nomenclature refers to the numbers of the first and last amino acid. The HPV16-derived peptide E749–57 (RAHYNIVTF) was selected because it was identified as an H-2Db CTL epitope (13). The E743–77 35-residue long peptide (GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR), which represents one of the peptides of a good manufacturing practice-grade vaccine against HPV16-induced tumors currently tested in a clinical trial, contains the E749–57 CTL epitope with its natural flanking sequences. (CTL epitope underlined). HPLC and mass spectrometry demonstrated that no small fragments representing the minimal CTL epitope were present in this peptide lot. The DRAHYNIVTF sequence, which is encompassed in this peptide, represents a Th epitope (47).
Tetramers and Abs
PE-labeled H-2Db epitope
E749–57 (RAHYNIVTF)-containing tetramers were
constructed and used for the analysis of peptide-specific CTL immunity
as described earlier (48). FITC-labeled anti-CD8b.2 Ab
(Ly-3.2; clone 53-5.8), allophycocyanin-labeled anti-CD4 Ab
(L3T4; clone RM4-5), and PE-labeled anti-IFN-
Ab (clone XMG1.2)
(BD PharMingen, San Diego, CA) were used in the various FACS
procedures.
Adjuvants
IFA was obtained from Difco (Detroit, MI). Montanide ISA 51 was purchased from SEPPIC (Paris, France). CpG-ODN 1826, sequence TTCATGACGTTCCTGACGTT, was provided by Coley Pharmaceutical (Langenfeld, Germany) and used at a their suggested optimal working concentration of 50 µg/mouse. GM-CSF was obtained from PeproTech (Rocky Hill, NJ) and used at a concentration of 4 µg/mouse based on the work of Disis et al. (36). The FGK-45 hybridoma cells producing stimulatory anti-CD40 Ab were provided by Dr. A. Rolink (University of Basel, Basel, Switzerland). MPL was provided by Corixa (Seattle, WA) and used at the suggested optimal concentration of 10 µg/mouse.
Immunization strategies
C57BL/6 mice were injected s.c. with either 50 µg E749–57 short peptide or 150 µg E743–77 35-residue long peptide dissolved in PBS to achieve similar molar levels of the E749–57 CTL epitope in both cases. Combinations with various adjuvants were tested. In the case of IFA and Montanide, the dissolved peptides were emulsified in 50% of these respective substances. ODN-CpG, MPL, and GM-CSF were all dissolved in PBS and mixed with the peptides before s.c. vaccination. The total injected volume was 200 µl/mouse. Anti-CD40 Ab was dissolved in PBS and injected separately from the peptides i.v. on days 0, 1, and 2 at an amount of 100 µg per injection (a total volume of 200 µl/mouse) as described before (35). Spleens were harvested after 10 days. In the prime-boost experiments with IFA, mice were boosted with identical vaccines 50 days after priming to allow the formation of memory CTL and Th cells (49). In these experiments spleens were harvested 10 days after the booster immunization. In the therapeutic antitumor experiments, tumor-bearing mice were vaccinated at the time tumors were palpable in all mice and 14 days later, considering the aggressive outgrowth of TC-1 tumors in untreated mice.
T cell cultures
T cells were obtained from immunized mice by culturing spleen cells (5 x 106 cells/well of a 24-well plate) in complete medium in the presence of 0.5 x 106 E749–57-expressing cells (tumor cell line 13.2) or, when indicated, in the presence of D1 cells. Before use, the D1 cells were incubated for 16 h with the E743–77 long peptide and subsequently activated by adding LPS (10 µg/ml) for 6 h and then thoroughly washed. Complete medium consists of IMDM (BioWhittaker, Walkersville, MD) supplemented with 8% FCS, 100 IU/ml penicillin, 2 mM glutamine (ICN, Aurora, OH), and 30 µM 2-ME (Merck, Darmstadt, Germany). Cultures were maintained at 37°C in humidified air containing 5% CO2. No exogenous IL-2 was added. On day 6, dead cells were removed from the culture by centrifugation over a Ficoll density gradient and remaining cells were seeded in 24-well plates at 1 x 106 cells/well. On day 7 tetramer staining or intracellular cytokine staining was performed.
Tetramer staining
Spleen cultures, stimulated with tumor cell line 13.2 for 7 days, were transferred at 40 x 104 cells per well to 96-well V-bottom microtiter plates and washed twice with PBS/BSA 0.5%. Subsequently, PE-labeled E749–57-containing tetramer was added. After 30 min of incubation at room temperature, cells were washed twice with PBS/BSA 0.5% and incubated with FITC-labeled anti-CD8b Ab for 30 min. Subsequently cells were washed twice in PBS/BSA 0.5%, suspended in PBS/BSA 0.5% containing propidium iodide (0.5 µg/ml) and transferred to tubes. Cell samples were analyzed in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences). A total of 20 x 104 events were analyzed for fluorescence intensity. Debris was gated out using the propidium iodide staining and subsequently the CD8+ fraction was gated. Mean background tetramer staining of similarly cultured and stained cells from nonimmunized control mice was found to be <1% of CD8+ cells (0.94%; SD, 0.36%). As a positive control the HPV16 E7-specific CTL clone 9.5 was used.
Intracellular cytokine staining
Spleen cultures were stimulated with D1 cells pulsed with 35-mer
E743–77 for 7 days. Subsequently, the
percentage of CD8+ and CD4+
IFN--producing T cells was measured as described before
(48). LPS-activated nonpulsed D1 cells or D1 cells pulsed
with short peptide or the long peptide (5 µg/ml) were used as
stimulator cells.
Cytokine ELISA
The production of IFN- by the CTL clones 9.5, 1H11, and 9.42
was measured as described elsewhere (50).
Statistics
The survival of mice receiving various treatments was statistically analyzed using the Kaplan-Meier test. Where indicated the survival was significantly prolonged (p < 0.05).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice (B6) were vaccinated once with either the minimal CTL epitope HPV16 E749–57 (short peptide) or the HPV16 E743–77 35-residue long peptide, admixed with IFA. Ten days after vaccination, the spleens were harvested and subjected to an extra round of in vitro stimulation that magnifies but does not alter the hierarchy of in vivo induced CD8+ T cell responses before the percentage of E749–57 peptide-specific CD8+ T cells was determined by H2-Db E749–57 (RAHYNIVTF) tetramer staining (48). Because immunization with short peptide in IFA has been shown to induce E749–57-specific CD8+ T cells that are able to protect mice from a subsequent tumor challenge (13), but which were not demonstrable by tetramer analysis directly ex vivo, we decided to use one round of in vitro stimulation to discriminate nonresponding mice from those with low/high T cell frequencies.
In both groups, three of nine mice responded to the vaccine while
generally 5% of the CD8+ T cells stained with
tetramers (Fig. 1
, A and
B), suggesting that the vaccines perform equally well when
injected once.
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C). In contrast,
mice vaccinated with the long peptide displayed a more robust
E749–57-specific CD8+ T
cell response in 9 of 11 mice, with high numbers of
E749–57-specific CD8+ T
cells (mean, 19%; range, 5–40%) (Fig. 1D). These
experiments demonstrate that vaccination with the long peptide in a
homologous prime-boost regimen is superior to vaccination with the
minimal CTL epitope and suggest that the formation of Th cells, by the
intrinsic Th epitope present in the long peptide (47),
contributes considerably to the level of the CD8+
T cell responses. The high E749–57-specific CD8+ T cell response is dependent on MHC class II-restricted Th type 1 cells and CD40-CD40L interactions
To directly assess whether CD4+ Th cells
were induced after priming, allowing the formation of memory Th cells
before the second vaccination, wild-type B6 mice were vaccinated once
with the long peptide in IFA. Subsequently the percentage of
E743–77-specific IFN--producing
CD4+ T cells was measured by intracellular
cytokine staining. Whereas no responses over background were observed
in naive mice (data not shown), 5% or more of the
CD4+ T cells from vaccinated mice specifically
responded upon stimulation with the long E743–77
peptide (Fig. 2A). To further
demonstrate that the impressive CD8+ T cell
responses, detected following prime-boost vaccinations with the long
E743–77 peptide, were in fact enhanced by MHC
class II-restricted E7-specific CD4+ Th cells,
MHC class II-/- mice were prime-boosted with
the long peptide vaccine. The number of
E749–57-specific CD8+ T
cells detected in the MHC class II-/- mice
(Fig. 1E) was far lower than found in B6 mice after two
vaccinations and comparable to that found after one vaccination with
the long peptide (Fig. 1B). These data not only indicate
that MHC class II-restricted CD4+ Th type 1
responses are induced after one vaccination but also suggest that these
E743–77 peptide-specific Th cells are required
for inducing vigorous CD8+ T cell responses. Most
likely, professional APC that process and present the long peptide upon
the booster immunization are activated through CD40-CD40 ligand (CD40L)
interactions by E743–77 peptide-specific Th
cells (47), and this will lead to enhanced
CD8+ T cell activation.
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F). As expected, the number of
E749–57-specific CD8+ T
cells induced did not exceed that seen after one vaccination with the
long peptide in wild-type B6 mice (Fig. 1B). In both groups
of mice, one of the tested mice displayed a high percentage of
tetramer-positive CD8+ T cells, but this has to
be attributed to individual variation. Single vaccinations with the E743–77 peptide and various DC-activating agents result in vigorous CD4+ and CD8+ T cell responses toward HPV16 E7
If the Th-mediated activation of professional APC is important for
the observed robust CD8+ T cell response, direct
activation of DC by itself should be sufficient to raise adequate
CD8+ T cell responses. To address this point, B6
mice were vaccinated once with the long peptide or the minimal
E749–57 peptide in combination with various
DC-activating agents. IFA and Montanide (a human grade IFA) have been
used extensively by us and others in previous studies. Mice vaccinated
with either the short peptide or the long peptide combined with
anti-CD40 Ab or GM-CSF in PBS displayed no or minimal
CD8+ T cell responses (Fig. 3, A and B).
However, the combination of GM-CSF and IFA resulted in moderate
E749–57-specific CD8+ T
cell responses when the long peptide was administered (Fig. 3C). Mice receiving the minimal peptide mixed with MPL or
ODN-CpG showed clear-cut CD8+ T cell responses in
several mice (Fig. 3, D and E), although the
number of responders (4 of 9 and 4 of 12 mice, respectively) was not
increased compared with mice vaccinated with IFA (Fig. 1A).
In contrast, vaccination with the long peptide admixed with MPL or
ODN-CpG resulted in E749–57-specific
CD8+ T cell responses in all mice. Moreover, the
level of the detected responses was high (up to 40% of stimulated
CD8+ T cells) in the majority of animals.
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-producing cells were measured upon stimulation with the short
and long peptides, respectively. High numbers of IFN-producing
CD8+ T cells were detected, confirming that the
CD8+ T cells detected by the
H2-Db E749–57 tetramers
were functionally active (data not shown). Compared with the use of IFA
as adjuvant (Fig. 2A), the use of the DC-activating agent
(ODN-CpG) raised even higher numbers of
E743–77-specific IFN--producing
CD4+ T cells (15–20% of stimulated
CD4+ T cells; Fig. 2B).
Our prime-boost experiments with the long peptide (Fig. 1, D–F) suggested that the induction of high
numbers of HPV-specific CD8+ T cells was the
result of the activation of APC by HPV-specific
CD4+ T cells. Therefore, direct activation of DC
should be able to bypass a lack of T cell help and as such no
difference in the numbers of HPV-specific CD8+ T
cells among MHC class II-/- mice,
CD40-/- mice, or wild-type mice after
vaccination with the long peptide and a DC-activating agent is
expected. To address this question, mice were vaccinated with the long
peptide and ODN-CpG. As shown in Fig. 3, F and G,
robust CD8+ T cell responses were detected in all
types of mice after one vaccination. Together with the experiments
shown in Fig. 1, these experiments show that proper precursor T cells
are present in all types of mice. Furthermore, it demonstrates that
activation of professional APC is an important aspect in the induction
of high numbers of HPV-specific CD8+ T cells.
Furthermore, the observation that in this Th-independent setting
vaccination with the long peptide is superior to vaccination with the
short peptide suggests that, compared with the minimal CTL epitope that
may bind to MHC class I molecules present on all nucleated cells, the
long peptide is preferentially processed and presented by
professional APCs.
Vaccinations with the long E743–77 peptide and the DC-activating adjuvant ODN-CpG can effectively eradicate HPV16-expressing tumors
Numerous studies show that protection of C57BL/6 mice against
HPV16 E7-expressing tumors is entirely dependent on
E749–57-specific CD8+ T
cells (13, 41, 42, 51). These CD8+
CTL are able to kill the HPV16 E7-expressing epithelial tumor cell line
TC-1 (42, 51). Because it was shown that the percentage of
E749–57 tetramer-positive
CD8+ T cells correlated with cytotoxicity
(48), the antitumor efficacy of the short peptide or the
long peptide vaccine admixed with ODN-CpG, which consistently resulted
in high numbers of CD8+ T cells, was tested by
therapeutically vaccinating tumor-bearing mice in the contralateral
flank (Fig. 4). Vaccinations with the
short or the long peptide combined with IFA were taken along for
comparison (Fig. 4). Notably, the vaccinations were given at the time
that tumors were palpable in all mice (day 10). Based on a pilot
experiment in which tumors treated with one single vaccination
occasionally progressed after initial regression (data not shown), a
booster vaccination was administered 14 days later to fully sustain
E7-specific Th and CTL immunity. Compared with nonvaccinated mice (Fig. 4A), administration of the peptides in IFA showed some delay
in tumor growth but all mice had died by day 50 (Fig. 4, D
and E). In both groups treated with either the short or the
long peptides combined with ODN-CpG, inhibition of tumor growth was
seen 8–12 days after treatment (Fig. 4, B and
C). Complete eradication of the TC-1 tumor was observed in
three of nine mice treated with the short peptide and ODN-CpG (Fig. 4B) and in 2 of 10 control mice who, as a control, received
two injections of ODN-CpG peritumoral (Fig. 5). In all of the control mice injected
with ODN-CpG in the contralateral flank, tumor growth was comparable to
nontreated animals (Fig. 5). In contrast, 8 of 10 mice treated with the
long peptide and ODN-CpG completely eradicated their tumors, some of
them sizing up to 250 mm3 (Fig. 4C).
The mice with the two largest tumors (up to 500
mm3) initially succeeded in reducing the tumors
to a size that was not palpable anymore, but eventually the tumors
became detectable again and progressive tumor growth developed. One of
these mice died spontaneously, whereas the other was killed to
investigate the tumor, bearing in mind the possibility that recurrence
may have resulted from escape mechanisms. The recognition of this
excised tumor by two tumor-specific CTL clones was compared with that
of the original in vitro growing tumor cell line (Fig. 6). Clearly, the recognition of the
original tumor cells was far higher in comparison to the excised tumor,
indicating that escape may have occurred through reduced CTL killing.
Additional FACS analysis showed a 2-fold decreased expression of
Kb and Db MHC class I
molecules on the cell surface of the excised tumor (data not shown).
Because TC-1 already expresses low levels of Kb
and Db on its surface, further down-regulation of
the MHC class I molecules may be one of the multiple mechanisms through
which this tumor could have escaped from the immune system.
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). Mice treated with the long peptide
admixed with ODN-CpG survived significantly longer (Kaplan-Meier
analysis, p < 0.05). These in vivo data are consistent
with the in vitro analysis of the CD4+ and
CD8+ T cell responses after vaccination with the
peptides in IFA or mixed with ODN-CpG.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The high efficacy of our long peptide vaccine may partly result from the fact that the Th and CTL epitope are physically linked to each other, although we have not tested this in the current study. The potential advantage of epitope linkage lies in the increased chance for simultaneous presentation of both the MHC class I- and class II-restricted epitopes on the surface of a single APC, thereby facilitating the delivery of cognate T cell help to CTL priming. A direct comparison of vaccines that used a mixture of nonlinked CTL and Th epitopes with vaccines that used identical, but physically linked, CTL and Th epitopes demonstrated that the latter resulted in more vigorous CTL responses (26, 27, 28, 29). Naturally occurring epitope linkage can be found in most protein Ags, including the HIV-1 envelope protein (52), the IDDM-associated GAD65 protein (53), and the Her-2/neu protein (54). Interestingly, recent vaccinations with Her-2 peptides, comprising both CTL and Th epitopes, resulted in enhanced Her-2-specific Th and CTL activity in cancer patients (54). Our experiments in wild-type B6 and MHC class II-/- mice show that simultaneous vaccination with both CTL and Th epitopes is advantageous for the development of a strong E7-specific CD8+ T cell response. In addition, the experiments in CD40-/- mice demonstrate that particularly the interaction of CD40-CD40L between E7-specific Th cells and APC is responsible for this boost of the CD8+ T cell response.
Comparison of the CD8+ T cell responses induced after a single vaccination with the short peptide or the 35-residue long peptide in combination with DC-activating adjuvants revealed another interesting property of the long peptide vaccine. Under these circumstances the contribution of E7-specific Th cells is negligible because at priming their number is too low to exert this function. Consequently, only differences in physical properties or kinetics of the peptides are likely to play a role. Because of its size, which excludes direct binding to MHC class I molecules, the long peptide has to be taken up by professional APC that are able to process exogenously derived Ags for presentation in MHC class I molecules (55). We demonstrate that direct activation of APC with DC-activating agents, bypassing the need for T cell help, only marginally affects the outcome of the E7-specific CD8+ T cell response when the minimal peptide is used, whereas DC activation induces a superior E7-specific CD8+ T cell response when the long peptide is administered. To confirm that T cell help was not responsible for this effect, the long peptide was coinjected with the DC-activating adjuvant ODN-CpG in MHC class II-/- and CD40-/- mice and, as expected, the observed CD8+ T cell responses were comparable to those seen in wild-type mice. The superior CD8+ T cell-inducing capacity of long peptides plus DC-activating agents over short peptides can be explained by the fact that minimal CTL epitopes show no distinction between MHC class I molecules present on professional APC or the MHC class I molecules on other nucleated cells. In the absence of expression of proper costimulatory molecules, such nonprofessional APC have been shown to exert tolerizing influences on CTL precursors (40). Additionally, long peptides need to be taken up and processed by professional APC. The use of specific DC-activating agents affected especially CD8+ T cell induction by the long peptide vaccine, suggesting that the long peptides are indeed taken up and processed by professional APC. This may create an intracellular reservoir for continuous supply of MHC class II- and MHC class I-bound peptides to the cell surface and as such lead to sustained CD8+ T cell activation. Exact MHC class I-binding short peptides have a short half life at the cell surface and DC loaded in vivo with such peptides will not benefit from resupply to the cell surface from internally processed stores of Ag (56). Finally, recent experiments performed in our department indicate that ODN-CpG not only activates DC but can also improve the MHC class I peptide processing pathway, leading to an increased number of available CTL epitopes on the cell surface of the DC (F. Ossendorp M. Camps, A. Sijts, P. Kloetzel, and C. J. M. Melief, unpublished observations).
We and others have completed phase I/II vaccination studies in which HPV16+ patients were vaccinated with CTL epitopes admixed with Montanide (12, 15), a human grade of IFA with similar properties (57), or admixed with Montanide plus a nonlinked universal Th epitope (14). All together, no or marginal HPV16 CTL responses were detected in these patients. Our data show that minimal peptide epitopes mixed with mineral oil based adjuvants, such as Montanide or IFA, trigger only marginal CD8+ T cell responses in prime-boost regimens. As shown here, the inclusion of a Th epitope markedly improves the vaccine. Increasing the size of the peptide, forcing it to be presented by professional APC, is another way to enhance the vaccination efficacy. In addition, the strength of the vaccine can be further improved by the use of DC-activating adjuvants. We demonstrate that the DC-activating agents MPL (a detoxified form of LPS that can be used in humans) (33), ODN-CpG, and the DC-activating chemokine GM-CSF in IFA help increasing the levels of CD8+ T cell responses induced by the long HPV16 E7-derived peptide. Interestingly, this DC activation also results in increased levels of E7-specific CD4+ Th cells, which may additionally enhance and sustain HPV-specific CTL responses. Indeed, the combination of the DC-activating agent ODN-CpG and the long peptide proved to result in complete eradication of established tumors following prime-boost vaccinations. These data are in line with the results of a recent study, which demonstrated that ODN-CpG markedly enhance the number of tumor-specific CTL (58).
Based on our data, we conclude that vaccination with long peptides containing both CTL and Th cell epitopes and DC-activating agents can contribute to the design of stronger HPV vaccines. We have now initiated a phase I/II study in which a clinically grade long (32–35 residues) overlapping peptide vaccine, covering the entire HPV16 E6 and E7 protein, is used to vaccinate patients with HPV16-induced cervical or vulvar lesions.
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Acknowledgments |
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Footnotes |
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2 S.H.v.d.B. and C.J.M.M. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Cornelius J. M. Melief, Department of Immunohematology and Blood Transfusion, Building 1, E3-Q, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail address: cmelief{at}lumc.nl
4 Abbreviations used in this paper: HPV, human papillomavirus; DC, dendritic cell; MPL, monophosphoryl lipid A; ODN, oligodeoxynucleotide; CD40L, CD40 ligand.
Received for publication February 27, 2002. Accepted for publication April 29, 2002.
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G. G. Kenter, M. J.P. Welters, A.R. P.M. Valentijn, M. J.G. Lowik, D. M.A. Berends-van der Meer, A. P.G. Vloon, J. W. Drijfhout, A. R. Wafelman, J. Oostendorp, G. J. Fleuren, et al. Phase I Immunotherapeutic Trial with Long Peptides Spanning the E6 and E7 Sequences of High-Risk Human Papillomavirus 16 in End-Stage Cervical Cancer Patients Shows Low Toxicity and Robust Immunogenicity Clin. Cancer Res., January 1, 2008; 14(1): 169 - 177. [Abstract] [Full Text] [PDF]
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M. J.P. Welters, G. G. Kenter, S. J. Piersma, A. P.G. Vloon, M. J.G. Lowik, D. M.A. Berends-van der Meer, J. W. Drijfhout, A. R. P.M. Valentijn, A. R. Wafelman, J. Oostendorp, et al. Induction of Tumor-Specific CD4+ and CD8+ T-Cell Immunity in Cervical Cancer Patients by a Human Papillomavirus Type 16 E6 and E7 Long Peptides Vaccine Clin. Cancer Res., January 1, 2008; 14(1): 178 - 187. [Abstract] [Full Text] [PDF]
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 R. Bos, S. van Duikeren, T. van Hall, M. M. Lauwen, M. Parrington, N. L. Berinstein, B. McNeil, C. J. M. Melief, J. S. Verbeek, S. H. van der Burg, et al. Characterization of Antigen-Specific Immune Responses Induced by Canarypox Virus Vaccines J. Immunol., November 1, 2007; 179(9): 6115 - 6122. [Abstract] [Full Text] [PDF]
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M. S. Bijker, S. J. F. van den Eeden, K. L. Franken, C. J. M. Melief, R. Offringa, and S. H. van der Burg CD8+ CTL Priming by Exact Peptide Epitopes in Incomplete Freund's Adjuvant Induces a Vanishing CTL Response, whereas Long Peptides Induce Sustained CTL Reactivity J. Immunol., October 15, 2007; 179(8): 5033 - 5040. [Abstract] [Full Text] [PDF]
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R. J. May, T. Dao, J. Pinilla-Ibarz, T. Korontsvit, V. Zakhaleva, R. H. Zhang, P. Maslak, and D. A. Scheinberg Peptide Epitopes from the Wilms' Tumor 1 Oncoprotein Stimulate CD4+ and CD8+ T Cells That Recognize and Kill Human Malignant Mesothelioma Tumor Cells Clin. Cancer Res., August 1, 2007; 13(15): 4547 - 4555. [Abstract] [Full Text] [PDF]
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 S. Loeser, K. Loser, M. S. Bijker, M. Rangachari, S. H. van der Burg, T. Wada, S. Beissert, C. J.M. Melief, and J. M. Penninger Spontaneous tumor rejection by cbl-b-deficient CD8+ T cells J. Exp. Med., April 16, 2007; 204(4): 879 - 891. [Abstract] [Full Text] [PDF]
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S. H. Bae, Y.-J. Park, J.-B. Park, Y. S. Choi, M. S. Kim, and J.-I. Sin Therapeutic Synergy of Human Papillomavirus E7 Subunit Vaccines plus Cisplatin in an Animal Tumor Model: Causal Involvement of Increased Sensitivity of Cisplatin-Treated Tumors to CTL-Mediated Killing in Therapeutic Synergy Clin. Cancer Res., January 1, 2007; 13(1): 341 - 349. [Abstract] [Full Text] [PDF]
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J. N. Kochenderfer, C. D. Chien, J. L. Simpson, and R. E. Gress Synergism between CpG-Containing Oligodeoxynucleotides and IL-2 Causes Dramatic Enhancement of Vaccine-Elicited CD8+ T Cell Responses J. Immunol., December 15, 2006; 177(12): 8860 - 8873. [Abstract] [Full Text] [PDF]
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G. Bioley, C. Jandus, S. Tuyaerts, D. Rimoldi, W. W. Kwok, D. E. Speiser, J.-M. Tiercy, K. Thielemans, J.-C. Cerottini, and P. Romero Melan-A/MART-1-Specific CD4 T Cells in Melanoma Patients: Identification of New Epitopes and Ex Vivo Visualization of Specific T Cells by MHC Class II Tetramers J. Immunol., November 15, 2006; 177(10): 6769 - 6779. [Abstract] [Full Text] [PDF]
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 K. B. Gendron, A. Rodriguez, and D. A. Sewell Vaccination with human papillomavirus type 16 e7 Peptide with CpG oligonucleotides for prevention of tumor growth in mice. Arch Otolaryngol Head Neck Surg, March 1, 2006; 132(3): 327 - 332. [Abstract] [Full Text] [PDF]
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 D. Wakita, K. Chamoto, Y. Zhang, Y. Narita, D. Noguchi, H. Ohnishi, T. Iguchi, T. Sakai, H. Ikeda, and T. Nishimura An indispensable role of type-1 IFNs for inducing CTL-mediated complete eradication of established tumor tissue by CpG-liposome co-encapsulated with model tumor antigen Int. Immunol., March 1, 2006; 18(3): 425 - 434. [Abstract] [Full Text] [PDF]
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Y. Motomura, S. Senju, T. Nakatsura, H. Matsuyoshi, S. Hirata, M. Monji, H. Komori, D. Fukuma, H. Baba, and Y. Nishimura Embryonic Stem Cell-Derived Dendritic Cells Expressing Glypican-3, a Recently Identified Oncofetal Antigen, Induce Protective Immunity against Highly Metastatic Mouse Melanoma, B16-F10 Cancer Res., February 15, 2006; 66(4): 2414 - 2422. [Abstract] [Full Text] [PDF]
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 A. Baez-Astua, E. Herraez-Hernandez, N. Garbi, H. A. Pasolli, V. Juarez, H. zur Hausen, and A. Cid-Arregui Low-Dose Adenovirus Vaccine Encoding Chimeric Hepatitis B Virus Surface Antigen-Human Papillomavirus Type 16 E7 Proteins Induces Enhanced E7-Specific Antibody and Cytotoxic T-Cell Responses J. Virol., October 15, 2005; 79(20): 12807 - 12817. [Abstract] [Full Text] [PDF]
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K. L. Smith, A. Tristram, K. M. Gallagher, A. N. Fiander, and S. Man Epitope specificity and longevity of a vaccine-induced human T cell response against HPV18 Int. Immunol., February 1, 2005; 17(2): 167 - 176. [Abstract] [Full Text] [PDF]
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X. Preville, D. Ladant, B. Timmerman, and C. Leclerc Eradication of Established Tumors by Vaccination With Recombinant Bordetella pertussis Adenylate Cyclase Carrying the Human Papillomavirus 16 E7 Oncoprotein Cancer Res., January 15, 2005; 65(2): 641 - 649. [Abstract] [Full Text] [PDF]
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H. Shams, P. Klucar, S. E. Weis, A. Lalvani, P. K. Moonan, H. Safi, B. Wizel, K. Ewer, G. T. Nepom, D. M. Lewinsohn, et al. Characterization of a Mycobacterium tuberculosis Peptide That Is Recognized by Human CD4+ and CD8+ T Cells in the Context of Multiple HLA Alleles J. Immunol., August 1, 2004; 173(3): 1966 - 1977. [Abstract] [Full Text] [PDF]
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T. F. Rowley and A. Al-Shamkhani Stimulation by Soluble CD70 Promotes Strong Primary and Secondary CD8+ Cytotoxic T Cell Responses In Vivo J. Immunol., May 15, 2004; 172(10): 6039 - 6046. [Abstract] [Full Text] [PDF]
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E. H. Slager, C. E. van der Minne, M. Kruse, D. D. Krueger, M. Griffioen, and S. Osanto Identification of Multiple HLA-DR-Restricted Epitopes of the Tumor-Associated Antigen CAMEL by CD4+ Th1/Th2 Lymphocytes J. Immunol., April 15, 2004; 172(8): 5095 - 5102. [Abstract] [Full Text] [PDF]
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H. L. Hanson, S. S. Kang, L. A. Norian, K. Matsui, L. A. O'Mara, and P. M. Allen CD4-Directed Peptide Vaccination Augments an Antitumor Response, but Efficacy Is Limited by the Number of CD8+ T Cell Precursors J. Immunol., April 1, 2004; 172(7): 4215 - 4224. [Abstract] [Full Text] [PDF]
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Y.-F. Chen, C.-W. Lin, Y.-P. Tsao, and S.-L. Chen Cytotoxic-T-Lymphocyte Human Papillomavirus Type 16 E5 Peptide with CpG-Oligodeoxynucleotide Can Eliminate Tumor Growth in C57BL/6 Mice J. Virol., February 1, 2004; 78(3): 1333 - 1343. [Abstract] [Full Text] [PDF]
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 B. de Rijke, H. Fredrix, A. Zoetbrood, F. Scherpen, H. Witteveen, T. de Witte, E. van de Wiel-van Kemenade, and H. Dolstra Generation of autologous cytotoxic and helper T-cell responses against the B-cell leukemia-associated antigen HB-1: relevance for precursor B-ALL-specific immunotherapy Blood, October 15, 2003; 102(8): 2885 - 2891. [Abstract] [Full Text] [PDF]
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L. Rivoltini, C. Castelli, M. Carrabba, V. Mazzaferro, L. Pilla, V. Huber, J. Coppa, G. Gallino, C. Scheibenbogen, P. Squarcina, et al. Human Tumor-Derived Heat Shock Protein 96 Mediates In Vitro Activation and In Vivo Expansion of Melanoma- and Colon Carcinoma-Specific T Cells J. Immunol., October 1, 2003; 171(7): 3467 - 3474. [Abstract] [Full Text] [PDF]
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 J. D. Marshall, K. Fearon, C. Abbate, S. Subramanian, P. Yee, J. Gregorio, R. L. Coffman, and G. Van Nest Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions J. Leukoc. Biol., June 1, 2003; 73(6): 781 - 792. [Abstract] [Full Text] [PDF]
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S. Gnjatic, D. Atanackovic, M. Matsuo, E. Jager, S. Y. Lee, D. Valmori, Y.-T. Chen, G. Ritter, A. Knuth, and L. J. Old Cross-Presentation of HLA Class I Epitopes from Exogenous NY-ESO-1 Polypeptides by Nonprofessional APCs J. Immunol., February 1, 2003; 170(3): 1191 - 1196. [Abstract] [Full Text] [PDF]
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J. Li, Y. Sun, and A. Garen Immunization and immunotherapy for cancers involving infection by a human papillomavirus in a mouse model PNAS, December 10, 2002; 99(25): 16232 - 16236. [Abstract] [Full Text] [PDF]
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