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* Institute of Molecular Medicine, John Radcliffe Hospital, and
Wellcome Trust Center for Human Genetics, Oxford, United Kingdom;
Ludwig Institute for Cancer Research, Universite Catholique de Louvain, Brussels, Belgium;
Department of Medicine, University of Wales College of Medicine, Cardiff, United Kingdom; and
¶ Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden
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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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It has become clear that some of the most successful vaccination protocols for inducing epitope-specific CTL are the heterologous prime-boost protocols, involving sequential injections of different vectors encoding the same recombinant Ag. These protocols focus the CTL response toward peptides within the recombinant Ag, which are the only CTL epitopes shared by the different agents (5). Recent results have demonstrated that priming with plasmid DNA and boosting with recombinant modified vaccinia Ankara (MVA)4 generate high levels of specific immunity (6, 7, 8, 9).
To minimize the generation of virus or tumor Ag loss variants (10, 11, 12), it would be desirable to induce CTL responses against a broad range of different epitopes, preferably encoded by distinct proteins. This rationale has led to the generation of polyvalent vaccines encoding strings of CTL epitopes (polyepitope vaccines) and/or proteins. Due to the large number of well-characterized HLA-A2 binding tumor- and virus-derived peptides, polyepitope vaccines are often designed to include peptides that can be presented by A2 to responding CTL. The availability of mice transgenic for A2 has allowed some preclinical testing of the efficacy of these vaccines. Polyepitope vaccine constructs are capable of priming multiple CTL specificities in A2 transgenic mice (13, 14, 15, 16) and also of expanding single CTL specificities to high numbers in nonhuman primates (8, 17). However, evidence is lacking that polyepitope constructs are capable of expanding CTL of multiple different specificities to effective levels. Systematic comparison of the efficacy of different polyvalent vaccination strategies has also been hampered by the technical limitations of assays for directly monitoring CTL responses in A2 transgenic mice (18).
We reasoned that the use of polyepitope vaccines in a prime-boost strategy might lead to dominant expansion of CTL with a narrow CTL repertoire, as a result of the preferential expansion of immunodominant CTL responses. The identity of the mechanisms responsible for the expansion of immunodominant CTL responses is the subject of ongoing debate. Recent results have demonstrated that high-affinity CTL are capable of down-modulating peptide-MHC complexes on APCs (19), providing a mechanism for the preferential expansion of high-affinity CTL during a secondary T cell response.
To address our hypothesis, we developed a novel technique, based on the use of chimeric A2/H-2Kb (Kb) class I tetramers, for directly monitoring A2-restricted CTL responses in the blood of A2 transgenic mice. This technique has allowed us to accurately and rapidly monitor the frequency of CTL induced by prime-boost regimens using a polyepitope construct designed for clinical trial in melanoma patients. We compared protocols using a number of different vectors and correlated the frequency of CTL induced with their cytotoxic activity in vivo. The results suggest a new strategy for optimizing polyvalent CTL responses in human vaccine trials.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mel3 sequence encodes the following CTL epitopes in sequence: tyrosinase1–9 (MLLAVLYCL), melan-A26–35 (ELAGIGILTV) encoding an analog peptide with a substitution at position 2 (A to L) (20), tyrosinase369–377 (YMDGTMSQV), linker (GS), Mage-3167–175 (EVDPIGHLY), Mage-3271–279 (FLWGPRALV), Mage-1161–169 (EADPTGHSY), linker (GS), NY-ESO-1155–167 (QLSLLMWITQCFL), and influenza virus (flu) nucleoprotein (NP)366–374 (ASNENMDAM).
The DNA vector pSG2, used throughout the study, was derived from pRc/CMV (Invitrogen, Paisley, U.K.).
Generation of recombinant vaccinia virus and MVA
Recombinant MVA was made as described by cloning the mel3 polyepitope string into the vaccinia shuttle vector pSC11 (21). Vaccinia viruses (WR strain) expressing mel3, full-length NY-ESO-1 (kindly provided by D. L. Panicali, Therion Biologics, Cambridge, MA), NY-ESO-1157–165 minigene encoding the NY-ESO-1 peptide SLLMWITQC, or tyrosinase were made by cloning each insert into the thymidine kinase gene using the vector pSC11 as previously described (22).
Generation of recombinant SFV
The mel3 polyepitope string was cloned into the transfer vector pSFV4.2-mel3 (23). RNA produced from this vector was used to construct recombinant SFV · mel3 particles.
Recombinant Semliki Forest virus (SFV) stocks were made and purified as described previously (23).
Tetramer synthesis
Tetrameric A2/peptide complexes were synthesized as previously
described (1). A2/Kb/peptide
tetramers were synthesized using chimeric H chain with 
1 and 2
domain from the A2 molecule and the 3 domain from the
Kb molecule. The Kb 3
domain was amplified by PCR using forward primer
5'-AAGGAGACGCTGCAGCGCACGGATTCCCCAAAGGCCCATGTGACC-3' and reverse
primer 5'-CGGATCCCGGCTCCCATCTCAGGGTGAGGGGCTC-3'. This PCR product
was then used as reverse primer together with the forward primer
5'-GGGGGCCATGGGTTCTCATTCTATGAGATATTTCTTCACATCCGTG-3' to generate
the full-length A2/Kb hybrid H chain.
Inhibition of A2/Kb tetramer staining by anti-CD8 Abs and A2/Kb monomers
NY-ESO-1157–165-specific CTL were
incubated with 12 ng/µl A2/Kb tetramers at
22°C for 3 h in the presence of different monomer
concentrations. Cells were then incubated for 15 min on ice with CD8
Ab (clone CT; Caltag Laboratories, Silverstone, U.K.). Cells were
washed twice and resuspended in PBS for FACS analysis. For CD8 blocking
experiments, CT-CD8-specific Abs were added either before or after
20 min of tetramer staining at 37°C.
Isolation of PBL and tetramer staining
Fresh PBL were isolated from blood taken from the tail vein
using red cell lysis buffer (Invitrogen). For tetramer staining, 3
x 105 cells were resuspended in 20 µl RPMI
1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FCS. Cells
were incubated with tetramer for 15 min at 37°C. PBL were then
incubated with rat anti-mouse CD8 (BD PharMingen, San Diego, CA)
for 15 min at 4°C. Cells were washed twice in PBS and resuspended in
PBS for FACScan (BD Biosciences, Mountain View, CA) analysis.
A2 transgenic mice
A2-Kb mice express chimeric A2 1 and
2 domains fused with the Kb 3 domain. They
express the endogenous H-2b class I molecules
(24). HHD mice express a transgenic chimeric
monochain class I molecule in which the C terminus of the human
2-microglobulin (2-m)
is covalently linked to the N terminus of chimeric A2 1 and 2
domains fused with the Db 3 domain
(14). HHD H-2Db and mouse
2-m genes were disrupted
by homologous recombination, resulting in complete lack of
serologically detectable cell surface expression of mouse endogenous
H-2b class I molecules.
Immunization protocols
Plasmid DNA for injection was purified using anion-exchange
chromatography (Qiagen, Hilden, Germany) and diluted in PBS at 1 mg/ml
DNA. DNA (25–50 µg) was injected into each musculus tibialis under
general anesthesia. Ten days after DNA injection, mice were boosted
with 106 PFU of recombinant MVA or vaccinia
viruses, which were diluted in PBS and injected i.v. into the lateral
tail vein. For priming or boosting with mel3·SFV,
108 virus particles were diluted in sterile PBS
and injected into the lateral tail vein. For the polyvirus boosting
protocol, shown in Fig. 7
, mel3·DNA mice were injected with a mixture
of 108 mel3·SFV, 106
NY-ESO-1 vaccinia, and 106 tyrosinase vaccinia in
a total volume of 300 µl.
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, freshly isolated splenocytes were
infected at a multiplicity of infection of 5 with mel3·VAC for 90 min
in RPMI 1640 supplemented with 0.1% BSA at 37°C. To ensure that the
virus was unable to replicate in vivo, mel3·VAC was UV
inactivated as described previously (25). The inability of
UV-inactivated virus particles to replicate was routinely confirmed
using a plaque assay. Cells were then washed three times, resuspended
in sterile PBS, and injected into lateral tail vein. Mice received a
total of 5 x 105, 5 x
106, or 5 x 107
splenocytes.
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, mice primed with flu virus A (H17)
were infected by intranasal injection (20 U of hemagglutinin per
mouse). One month later mice were injected with mel3·DNA followed by
mel3·MVA, as described above.
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In vivo killing assay
Pools of freshly isolated splenocytes from HHD mice were separately incubated in RPMI 1640 medium with different peptides at a concentration of 10-6 M for 2 h. Each cell pool was then labeled with a different concentration of CFSE (Molecular Probes, Eugene, OR) to allow simultaneous tracking of the different populations in vivo (Ref. 26 and I. F. Hermans, J. Yang, and F. Ronchese, unpublished results). Labeled cells were pooled and injected at 107 cells/mouse into the tail vein. A control population without peptide that had been labeled with 5-(and-6-)(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CellTracker Orange; Molecular Probes) was coinjected to assess killing of peptide-pulsed targets relative to unpulsed cells. Mice were bled at the time of injection of fluorochrome-labeled targets to determine their CTL frequencies to different epitopes. Disappearance of peptide/fluorochrome-labeled cells was tracked using FACS analysis of freshly isolated PBL 5 h after the injection. The level of specific cytotoxicity was calculated relative to the unpulsed population labeled with Cell Tracker Orange using the following calculation: 100 - (100 x (percentage pulsed/percentage unpulsed)). WinMDI 2.8 software (J. Trotter, http://facs.scripps.edu) and CellQuest 3.3 software (BD Biosciences) were used to analyze the FACS data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A string of five HLA-A2 and two HLA-A1 melanoma epitopes (mel3) was cloned into four distinct vectors: 1) naked plasmid DNA (mel3·DNA), 2) vaccinia virus (mel3·VAC), 3) MVA virus (mel3·MVA), and 4) SFV (mel3·SFV). To allow monitoring of CTL responses restricted by human as well as mouse class I molecules, we introduced an additional epitope from the flu NP restricted by H2-Db (Db) class I molecules (NP366–374; see Materials and Methods for list of epitopes in mel3).
We and others have previously demonstrated that optimal flanking
residues are important to ensure presentation of class I-restricted
epitopes (22, 27). To assess whether mel3 peptide epitopes
were properly processed, and whether competition for binding to MHC
class I molecules impaired CTL recognition of lower-affinity epitopes,
we infected target cells with mel3·MVA. Each of the seven epitopes
contained within the polyepitope mel3 cassette was simultaneously
presented to specific CTL as measured by specific lysis (Fig. 1).
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). We monitored the CTL response ex vivo
by direct staining of PBL with Db tetramers to
enumerate Ag-specific CTL. Frequencies of tetramer-positive cells were
calculated as a percentage of total CD8-positive cells. Heterologous
prime-boost vaccination strategies (Fig. 2B) were capable of
inducing vaccine-driven CTL responses, with frequencies up to 100 times
greater than frequencies obtained by strategies based on a homologous
Ag delivery system (Fig. 2A). Repeated injections of the
same immunogen into B6 mice led to the expansion of flu NP-specific CTL
ranging from 0.5 to 3% of CD8+ cells, while the
injection of heterologous Ag delivery systems led to much higher
frequencies ranging from 40 to 70% of CD8-positive CTL. All
tetramer+ cells were
CD69high and CD62Llow (data
not shown). Similar results were obtained in the spleen and blood,
although we observed that the percentage of
tetramer+CD8+ cells was
higher in the blood than in the spleen (data not shown), consistent
with recently published results (28).
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To test the ability of the mel3 polyepitope constructs to prime
A2-restricted CTL responses in vivo, A2 transgenic mice were primed by
mel3·DNA and boosted by mel3·MVA or mel3·VAC. Initial experiments
were conducted using transgenic mice, which express chimeric A2
molecules containing the H-2Kb
(Kb) 3 domain (A2-Kb),
and endogenous Db and Kb
molecules (24) (A2/Kb mice).
A2/Kb mice can respond to peptides presented by
Db and Kb as well as
A2.
We routinely use A2 tetramers to characterize A2-restricted CTL
responses in humans (2, 3, 11, 29). Previous results
showed that A2 tetramers were inefficient at detecting A2-restricted
CTL in A2/Kb mice (28). Because
these findings could be accounted for by the lack of mouse CD8 binding
to human A2 molecules (30), we engineered a chimeric
A2/Kb class I molecule containing the mouse
Kb 3 domain and compared
A2/Kb and A2 tetramers for their ability to stain
PBL of A2/Kb transgenic mice immunized with a
combination of DNA and vaccinia encoding the
NY-ESO-1157–165 epitope (Fig. 3A). While wild-type A2
tetramers failed to detect Ag-specific CTL in seven of eight immunized
mice, A2/Kb tetramers were capable of detecting
Ag-specific CTL in all responding mice (Fig. 3A and data not
shown). These results were consistent with the possibility that
A2/Kb tetramers have a higher binding affinity
for mouse A2-restricted CTL due to the interaction of murine CD8 with
the 3 domain of the A2/Kb tetramer. To test
this hypothesis, we generated a CTL line from A2 transgenic mice
specific for the NY-ESO-1157–165 epitope and
demonstrated that binding of A2/Kb tetramers to
the NY-ESO-1157–165-specific CTL line was CD8
dependent, as shown by the lack of tetramer staining in the presence of
anti-CD8 Abs (Fig. 3B). Furthermore, we showed that
monomeric A2/Kb molecules were capable of
inhibiting staining of the
NY-ESO-1157–165-specific CTL line by
A2/Kb NY-ESO-1157–165
tetramers (Fig. 3B). In contrast, A2
NY-ESO-1157–165 monomers failed to inhibit
binding of A2/Kb tetramers to the
NY-ESO-1157–165-specific CTL line (Fig. 3C).
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The creation of the A2/Kb tetramer enabled us to accurately measure ex vivo the frequency of A2-restricted CTL in comparison to the frequency of Db-restricted flu NP366–374 epitope-specific CTL.
Priming of A2/Kb mice with mel3·DNA,
followed by mel3·MVA, induced a dominant
Db-restricted flu
NP366–374-specific response and a much weaker
A2-restricted CTL response to melan-A26–35 (Fig. 4
).
The ability to detect these simultaneous CTL responses allowed us to
study whether previous exposure to flu virus would compromise the
ability of prime-boost protocols to expand
melan-A26–35-specific CTL in
A2/Kb mice. To preimmunize against the
NP366–374 epitope, A2 transgenic mice received a
single injection of flu virus H17. They subsequently received
mel3·DNA followed by mel3·MVA (Fig. 4). Expansion of
NP366–374-specific CTL, before vaccination with
mel3 polyepitope constructs, reduced the expansion of
melan-A26–35-specific CTL, because 7 of 10
immunized mice failed to have
melan-A26–35-specific CTL detectable by tetramer
staining (Fig. 4). These results were extended to other CTL responses
by demonstrating that the presence of preexisting
NY-ESO-1155–167-specific CTL was capable of
inhibiting the expansion of
melan-A26–35-specific CTL in mice immunized with
mel3·VAC (data not shown).
Prime-boost vaccination of HHD mice induces the expansion of a narrow repertoire of melanoma-specific CTL
Because a preexisting flu NP-specific CTL response inhibited the
induction of a melan-A-specific CTL response (Fig. 4), we speculated
that T cell interference might compromise the induction of a broad
immune response in polyvalent prime-boost regimens. Studying the
interplay between A2-restricted responses proved difficult in
A2/Kb mice, because we could only rarely detect
priming to multiple A2-restricted epitopes in these mice (data not
shown). This observation is consistent with previous reports suggesting
that the presence of the endogenous mouse class I molecules
significantly narrows the A2-restricted repertoire (14).
Furthermore, a Db-restricted
NP366–374-specific CTL response induced by our
polyepitope construct in these mice might interfere with the generation
of A2-restricted CTL. This reasoning led us to vaccinate the HHD strain
of A2 transgenic mice, which lack endogenous class I molecules. HHD
mice have a much larger A2-restricted T cell repertoire than
A2/Kb transgenic mice (14) because,
unlike A2/Kb transgenic mice, they express
chimeric human/mouse class I molecules linked to human
2-m in a Db-/- and
mouse 2-m-/-
background.
Priming of HHD mice with mel3·DNA led to the expansion of
melan-A26–35-specific CTL to frequencies
detectable by ex vivo tetramer staining in all vaccinated mice (Fig. 5 and data not shown). In contrast,
expansion of NY-ESO-1157–165- and
tyrosinase369–377-specific CTL was detectable
only occasionally, while responses to the
tyrosinase1–9 and
MAGE-3271–279 were not detected ex vivo in blood
or spleen by tetramer staining (data not shown).
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and data not shown).
The observation that melan-A26–35-specific CTL
comprised the dominant CTL response after a single DNA vaccination
presented an opportunity to study the interplay between CTL specific to
different vaccine encoded determinants in polyvalent prime-boost
vaccination protocols. We observed that boosting of
mel3·DNA-primed HHD mice with mel3·MVA (Fig. 7A), SFV·mel3 (Fig. 7B), or mel3·VAC (Fig. 7C) led to the expansion
of melan-A26–35-specific CTL, up to
80% of total CD8+ T cells. Responses specific to
NY-ESO-1157–165 and
tyrosinase369–377 epitopes were significantly
lower than the melan-A26–35-specific responses
confirming that polyepitope prime-boost vaccination regimens result in
a narrowing of the immune response to a single immunodominant
determinant.
Competition of vaccine-driven CTL for mel3-expressing APCs
Because a preexisting flu memory CTL response can inhibit
expansion of melan-A26–35-specific CTL in
A2/Kb mice (Fig. 4), we investigated whether the
higher numbers of melan-A26–35-specific CTL,
dominating the immune response after DNA priming (Fig. 5), were capable
of interfering with the expansion of
NY-ESO-1157–165- and
tyrosinase369–377-specific CTL during virus
boosting in HHD mice.
Competition for Ag recognition on the surface of APCs may lead to the immunodominance of higher-frequency CTL populations (31, 32, 33), and we reasoned that large numbers of melan-A26–35-specific CTL after mel3·DNA priming could result in either rapid killing (34) or shielding of mel3 expressing APC during boosting in vivo. This may hamper stimulation of CTL specific to NY-ESO-1157–165 and tyrosinase369–377 epitopes expressed by the same APC population during the boosting phase.
If CTL are competing for access to APC, then the injection of
increasing numbers of mel3-expressing APC should reduce CTL competition
and induce stronger NY-ESO-1157–165- and
tyrosinase369–377-specific CTL responses. To
address this hypothesis, mel3·DNA-primed mice were injected with
increasing numbers of mel3·VAC-infected splenocytes. To ensure that
mel3·VAC-infected splenocytes were unable to release infectious virus
particles, mel3·VAC was UV inactivated prior the infection of
splenocytes (see Materials and Methods). The results of
these experiments demonstrated that, while the injection of 5 x
105 mel3·VAC-infected splenocytes resulted in
almost complete dominance of
melan-A26–35-specific CTL (Fig. 8A), the injection of 5
x 106 and 5 x 107
cells was capable of boosting a strong CTL response to both
NY-ESO-1157–165 and
tyrosinase369–377 epitopes (Fig. 8, B
and C). These results demonstrate that competition for Ag
recognition at the surface of APC may significantly skew the immune
response, and strongly suggest that this competition needs to be taken
into account for the development of vaccination strategies to optimally
induce polyvalent A2-restricted CTL responses.
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, A and C). In
addition, this novel boosting regimen resulted in significantly
enhanced cytotoxic activity against target cells presenting the
subdominant epitopes in vivo (Fig. 9, B and D).
Importantly, this enhanced activity was achieved without compromising
activity against the dominant melan-A epitope.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 domain to make tetramers, because
wild-type A2 tetramers could not be used to reliably monitor
A2-restricted CTL in these mice (Fig. 3). As predicted, the binding of
murine CD8 to the Kb 3 domain was a critical
factor in the improved affinity of the A2/Kb
tetramers for mouse A2-restricted CTL (Fig. 3). Polyepitope constructs can induce narrow CTL responses in prime-boost protocols
Comparing several vaccination strategies using ex vivo tetramer
staining, we confirmed that heterologous prime-boost protocols induce
higher levels of vaccine-specific immune responses than immunizations
based on the injections of homologous vectors (Fig. 2). After priming,
any of the viral vectors we used (i.e., vaccinia, MVA, flu, or SFV) was
capable of boosting CTL frequencies to very high levels (Fig. 2
and data not shown).
When the responses to different epitopes within the polyepitope
construct were compared, it was clear that prime-boost protocols skewed
the immune response heavily toward a single epitope within the epitope
string. Initially this immunodominance was observed in
A2/Kb mice where prime-boost vaccination with the
polyepitope resulted in a dominant response to the
Db-restricted flu
NP366–374 epitope (Fig. 4A). When HHD
mice were vaccinated, to eliminate presentation of the flu
NP366–374 epitope through
Db, heterologous prime-boost vaccination resulted
in a predominant expansion of
melan-A26–35-specific CTL, up to 80% of total
CD8+ T cells (Fig. 7, A and
B).
Competition for APC is responsible for the narrow immune response to polyepitope vaccination
By dissecting the interplay between CTL responding to different epitopes in the polyepitope construct, we were able to identify a mechanism controlling the higher frequency of melan-A26–35 response during prime-boost regimens.
Several factors may contribute to the immunodominance of
melan-A26–35-specific CTL during the priming
phase (35). The use of the
melan-A26–35 peptide analog with an A
L
substitution at position 2 was shown to increase its binding affinity
to A2 molecules and its immunogenicity in vivo (20). We
have confirmed these results by showing that the modified
melan-A26–35 peptide analog has a higher binding
affinity than the other mel3-encoded peptides to A2 molecules, as
defined by its ability to compete for recognition of flu matrix peptide
58–66 by flu matrix-specific CTL (data not shown). Skewing of the
primary response (see Fig. 5) may have allowed
melan-A26–35-specific CTL to prevent CTL with
subdominant specificities from making contact with mel3-expressing APC
during the boost. This possibility is consistent with previously
published papers showing the interplay between CTL and APC in different
models (31, 32, 33).
This rationale led to the hypothesis that weakly primed CTL responses
(e.g., NY-ESO-1157–165 and
tyrosinase369–377) should be successfully
expanded by minimizing the competition with dominant
melan-A26–35-specific CTL for APC during
boosting. Consistent with this hypothesis, we showed that
immunodominance of melan-A26–35-specific CTL
could be broken upon injection of either increasing numbers of
mel3·VAC-infected splenocytes (Fig. 8) or by injecting a mixture of
recombinant viruses, each encoding different antigenic determinants
(Fig. 9).
These results demonstrate that polyepitope prime-boost vaccination strategies result in the expansion of a narrow CTL repertoire, due to competition of CTL for APC. The results also define an alternative boosting strategy to the polyepitope prime-boost protocol, which overcomes T cell interference and ensures the expansion of a larger CTL repertoire.
Implications for vaccination strategies in patients
These results are of practical importance, because several clinical trials are currently using heterologous prime-boost vaccination protocols with polyepitope and/or polyprotein constructs (8, 36). To minimize the emergence of tumor and virus Ag escape variants, it is important to ensure that polyvalent vaccine protocols are capable of expanding CTL specific for multiple epitopes.
Our results reveal several important parameters that, if confirmed in clinical trials using polyepitope constructs, would need to be taken into account in designing optimal polyvalent vaccine strategies.
First, we demonstrated that preexisting memory CTL responses
significantly reduce CTL responses specific to other epitopes contained
within the same construct (Fig. 4). Several groups have included
immunodominant flu CTL epitopes within polyvalent vaccines for use as
positive controls during immuno-monitoring (37, 38). Our
results suggest that DNA- or virus-based vaccines should not encode
epitopes expressed by recurrent viruses, as preexisting memory CTL
response may compromise the inductionof CTL responses specific to
other vaccine-encoded CTL determinants.
Second, we have demonstrated that the alphavirus SFV can be used both
as a priming vector in combination with MVA (Fig. 2) and for boosting
in combination with DNA (Fig. 7). Alphaviruses are currently being
studied extensively as vectors in vaccination protocols
(39, 40, 41, 42, 43, 44), and our results confirm that recombinant SFV is
very attractive in prime-boost protocols, particularly because
recombinant SFV expresses few viral structural proteins and the chances
of generating an immune response limited to recombinant proteins may be
higher than with larger viral vectors.
Finally, we demonstrated that T cell competition is likely to play a
role in modifying T cell responses in prime-boost vaccination
strategies. Our results strongly suggest that simultaneous presentation
of different epitopes to a skewed repertoire of primed CTL leads to
dominant expansion of a single CTL specificity. However, boosting
the primed response with APC separately presenting the epitopes results
in comparable expansion of CTL of multiple specificities to effective
levels in vivo (Figs. 4, 8, and 9).
In conclusion, we have developed a novel system for dissecting the ability of different vaccination protocols to optimally induce polyvalent A2-restricted CTL responses. Future clinical trials aimed at inducing a broad-based CTL response should consider restricting the use of polyvalent constructs to the priming phase of the protocol and using separate vectors encoding individual epitopes or proteins for the boosting phase.
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Acknowledgments |
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Footnotes |
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2 M.J.P. and E.M.-L.C. contributed equally to this paper.
3 Address correspondence and reprint requests to Dr. Vincenzo Cerundolo, Nuffield Department of Medicine, Institute of Molecular Medicine, John Radcliffe Hospital, OX3 9DS Oxford, U.K. E-mail address: vincenzo.cerundolo{at}imm.ox.ac.uk
4 Abbreviations used in this paper: MVA, modified vaccinia Ankara; flu, influenza virus; 2-m, 2-microglobulin; NP, nucleoprotein; SFV, Semliki Forest virus.
Received for publication September 21, 2001. Accepted for publication February 20, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 domain of class I influence the magnitude of the xenogeneic response. J. Exp. Med. 170:1091.This article has been cited by other articles:
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  C. Chaise, S. L. Buchan, J. Rice, J. Marquet, H. Rouard, M. Kuentz, G. E. Vittes, V. Molinier-Frenkel, J.-P. Farcet, H. J. Stauss, et al. DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance Blood, October 1, 2008; 112(7): 2956 - 2964. [Abstract] [Full Text] [PDF]
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 P. Mooij, S. S. Balla-Jhagjhoorsingh, G. Koopman, N. Beenhakker, P. van Haaften, I. Baak, I. G. Nieuwenhuis, I. Kondova, R. Wagner, H. Wolf, et al. Differential CD4+ versus CD8+ T-Cell Responses Elicited by Different Poxvirus-Based Human Immunodeficiency Virus Type 1 Vaccine Candidates Provide Comparable Efficacies in Primates J. Virol., March 15, 2008; 82(6): 2975 - 2988. [Abstract] [Full Text] [PDF]
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 P. J. Holst, M. R. Sorensen, C. M. Mandrup Jensen, C. Orskov, A. R. Thomsen, and J. P. Christensen MHC Class II-Associated Invariant Chain Linkage of Antigen Dramatically Improves Cell-Mediated Immunity Induced by Adenovirus Vaccines J. Immunol., March 1, 2008; 180(5): 3339 - 3346. [Abstract] [Full Text] [PDF]
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I. A. Cockburn, S. Chakravarty, M. G. Overstreet, A. Garcia-Sastre, and F. Zavala Memory CD8+ T Cell Responses Expand When Antigen Presentation Overcomes T Cell Self-Regulation J. Immunol., January 1, 2008; 180(1): 64 - 71. [Abstract] [Full Text] [PDF]
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 A. Scardino, M. Alimandi, P. Correale, S. G. Smith, R. Bei, H. Firat, M. G. Cusi, O. Faure, S. Graf-Dubois, G. Cencioni, et al. A Polyepitope DNA Vaccine Targeted to Her-2/ErbB-2 Elicits a Broad Range of Human and Murine CTL Effectors to Protect against Tumor Challenge Cancer Res., July 15, 2007; 67(14): 7028 - 7036. [Abstract] [Full Text] [PDF]
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T. I. Naslund, C. Uyttenhove, E. K. L. Nordstrom, D. Colau, G. Warnier, M. Jondal, B. J. Van den Eynde, and P. Liljestrom Comparative Prime-Boost Vaccinations Using Semliki Forest Virus, Adenovirus, and ALVAC Vectors Demonstrate Differences in the Generation of a Protective Central Memory CTL Response against the P815 Tumor J. Immunol., June 1, 2007; 178(11): 6761 - 6769. [Abstract] [Full Text] [PDF]
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 G. T. Belz, L. Zhang, M. D. H. Lay, F. Kupresanin, and M. P. Davenport Killer T cells regulate antigen presentation for early expansion of memory, but not naive, CD8+ T cell PNAS, April 10, 2007; 104(15): 6341 - 6346. [Abstract] [Full Text] [PDF]
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I. F. Hermans, J. D. Silk, U. Gileadi, S. H. Masri, D. Shepherd, K. J. Farrand, M. Salio, and V. Cerundolo Dendritic Cell Function Can Be Modulated through Cooperative Actions of TLR Ligands and Invariant NKT Cells J. Immunol., March 1, 2007; 178(5): 2721 - 2729. [Abstract] [Full Text] [PDF]
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R. A. Willis, J. W. Kappler, and P. C. Marrack CD8 T cell competition for dendritic cells in vivo is an early event in activation PNAS, August 8, 2006; 103(32): 12063 - 12068. [Abstract] [Full Text] [PDF]
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M. J. Palmowski, U. Gileadi, M. Salio, A. Gallimore, M. Millrain, E. James, C. Addey, D. Scott, J. Dyson, E. Simpson, et al. Role of Immunoproteasomes in Cross-Presentation J. Immunol., July 15, 2006; 177(2): 983 - 990. [Abstract] [Full Text] [PDF]
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J. Rice, S. Dunn, K. Piper, S. L. Buchan, P. A. Moss, and F. K. Stevenson DNA Fusion Vaccines Induce Epitope-Specific Cytotoxic CD8+ T Cells against Human Leukemia-Associated Minor Histocompatibility Antigens. Cancer Res., May 15, 2006; 66(10): 5436 - 5442. [Abstract] [Full Text] [PDF]
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 S. M. Barratt-Boyes, A. C. Soloff, W. Gao, E. Nwanegbo, X. Liu, P. A. Rajakumar, K. N. Brown, P. D. Robbins, M. Murphey-Corb, R. D. Day, et al. Broad cellular immunity with robust memory responses to simian immunodeficiency virus following serial vaccination with adenovirus 5- and 35-based vectors J. Gen. Virol., January 1, 2006; 87(1): 139 - 149. [Abstract] [Full Text] [PDF]
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C. L. Smith, F. Mirza, V. Pasquetto, D. C. Tscharke, M. J. Palmowski, P. R. Dunbar, A. Sette, A. L. Harris, and V. Cerundolo Immunodominance of Poxviral-Specific CTL in a Human Trial of Recombinant-Modified Vaccinia Ankara J. Immunol., December 15, 2005; 175(12): 8431 - 8437. [Abstract] [Full Text] [PDF]
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P. Otahal, S. C. Hutchinson, L. M. Mylin, M. J. Tevethia, S. S. Tevethia, and T. D. Schell Inefficient Cross-Presentation Limits the CD8+ T Cell Response to a Subdominant Tumor Antigen Epitope J. Immunol., July 15, 2005; 175(2): 700 - 712. [Abstract] [Full Text] [PDF]
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 P. M. Lavoie, A. R. Dumont, H. McGrath, A.-E. Kernaleguen, and R.-P. Sekaly Delayed expansion of a restricted T cell repertoire by low-density TCR ligands Int. Immunol., July 1, 2005; 17(7): 931 - 941. [Abstract] [Full Text] [PDF]
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S. Buchan, E. Gronevik, I. Mathiesen, C. A. King, F. K. Stevenson, and J. Rice Electroporation as a "Prime/Boost" Strategy for Naked DNA Vaccination against a Tumor Antigen J. Immunol., May 15, 2005; 174(10): 6292 - 6298. [Abstract] [Full Text] [PDF]
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D. Dieckmann, E. S. Schultz, B. Ring, P. Chames, G. Held, H. R. Hoogenboom, and G. Schuler Optimizing the exogenous antigen loading of monocyte-derived dendritic cells Int. Immunol., May 1, 2005; 17(5): 621 - 635. [Abstract] [Full Text] [PDF]
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N. Schaft, J. Dorrie, P. Thumann, V. E. Beck, I. Muller, E. S. Schultz, E. Kampgen, D. Dieckmann, and G. Schuler Generation of an Optimized Polyvalent Monocyte-Derived Dendritic Cell Vaccine by Transfecting Defined RNAs after Rather Than before Maturation J. Immunol., March 1, 2005; 174(5): 3087 - 3097. [Abstract] [Full Text] [PDF]
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F. K. Stevenson, C. H. Ottensmeier, P. Johnson, D. Zhu, S. L. Buchan, K. J. McCann, J. S. Roddick, A. T. King, F. McNicholl, N. Savelyeva, et al. DNA vaccines to attack cancer PNAS, October 5, 2004; 101(suppl_2): 14646 - 14652. [Abstract] [Full Text] [PDF]
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P. Boudinot, D. Bernard, S. Boubekeur, M.-I. Thoulouze, M. Bremont, and A. Benmansour The glycoprotein of a fish rhabdovirus profiles the virus-specific T-cell repertoire in rainbow trout J. Gen. Virol., October 1, 2004; 85(10): 3099 - 3108. [Abstract] [Full Text] [PDF]
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 A. Nencioni and P. Brossart Cellular Immunotherapy with Dendritic Cells in Cancer: Current Status Stem Cells, July 1, 2004; 22(4): 501 - 513. [Abstract] [Full Text] [PDF]
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 M. Salio, M. J. Palmowski, A. Atzberger, I. F. Hermans, and V. Cerundolo CpG-matured Murine Plasmacytoid Dendritic Cells Are Capable of In Vivo Priming of Functional CD8 T Cell Responses to Endogenous but Not Exogenous Antigens J. Exp. Med., February 17, 2004; 199(4): 567 - 579. [Abstract] [Full Text] [PDF]
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M. J. Palmowski, L. Lopes, Y. Ikeda, M. Salio, V. Cerundolo, and M. K. Collins Intravenous Injection of a Lentiviral Vector Encoding NY-ESO-1 Induces an Effective CTL Response J. Immunol., February 1, 2004; 172(3): 1582 - 1587. [Abstract] [Full Text] [PDF]
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L. Wooldridge, S. L. Hutchinson, E. M. Choi, A. Lissina, E. Jones, F. Mirza, P. R. Dunbar, D. A. Price, V. Cerundolo, and A. K. Sewell Anti-CD8 Antibodies Can Inhibit or Enhance Peptide-MHC Class I (pMHCI) Multimer Binding: This Is Paralleled by Their Effects on CTL Activation and Occurs in the Absence of an Interaction between pMHCI and CD8 on the Cell Surface J. Immunol., December 15, 2003; 171(12): 6650 - 6660. [Abstract] [Full Text] [PDF]
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I. F. Hermans, T. W. Chong, M. J. Palmowski, A. L. Harris, and V. Cerundolo Synergistic Effect of Metronomic Dosing of Cyclophosphamide Combined with Specific Antitumor Immunotherapy in a Murine Melanoma Model Cancer Res., December 1, 2003; 63(23): 8408 - 8413. [Abstract] [Full Text] [PDF]
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E. M.-L. Choi, J.-L. Chen, L. Wooldridge, M. Salio, A. Lissina, N. Lissin, I. F. Hermans, J. D. Silk, F. Mirza, M. J. Palmowski, et al. High Avidity Antigen-Specific CTL Identified by CD8-Independent Tetramer Staining J. Immunol., November 15, 2003; 171(10): 5116 - 5123. [Abstract] [Full Text] [PDF]
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I. F. Hermans, J. D. Silk, U. Gileadi, M. Salio, B. Mathew, G. Ritter, R. Schmidt, A. L. Harris, L. Old, and V. Cerundolo NKT Cells Enhance CD4+ and CD8+ T Cell Responses to Soluble Antigen In Vivo through Direct Interaction with Dendritic Cells J. Immunol., November 15, 2003; 171(10): 5140 - 5147. [Abstract] [Full Text] [PDF]
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C. C. Wilson, D. McKinney, M. Anders, S. MaWhinney, J. Forster, C. Crimi, S. Southwood, A. Sette, R. Chesnut, M. J. Newman, et al. Development of a DNA Vaccine Designed to Induce Cytotoxic T Lymphocyte Responses to Multiple Conserved Epitopes in HIV-1 J. Immunol., November 15, 2003; 171(10): 5611 - 5623. [Abstract] [Full Text] [PDF]
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