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J Virol, March 1998, p. 2246-2252, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Targeting a Polyepitope Protein Incorporating
Multiple Class II-Restricted Viral Epitopes to the
Secretory/Endocytic Pathway Facilitates Immune Recognition by
CD4+ Cytotoxic T Lymphocytes: a Novel Approach to
Vaccine Design
Scott A.
Thomson,1
Scott R.
Burrows,1
Ihor S.
Misko,1
Denis J.
Moss,1
Barbara E. H.
Coupar,2 and
Rajiv
Khanna1,3,*
CRC for Vaccine
Technology1 and
Tumour Immunology
Laboratory, EBV Unit,3 Queensland Institute of
Medical Research, The Bancroft Centre, Brisbane 4006, and
CSIRO, Australian Animal Health Laboratory, Geelong,
Victoria,2 Australia
Received 11 August 1997/Accepted 20 November 1997
 |
ABSTRACT |
The role of CD4+ and CD8+ cells in the
generation of an effective immune response against viral infections is
well established. Moreover, there is an increasing realization that
subunit vaccines which include both CD4+- and
CD8+-T-cell epitopes are highly effective in controlling
viral infections, as opposed to those which are designed to activate a
CD8+- or CD4+-T-cell response alone. One of the
major limitations of epitope-based vaccines designed to stimulate
virus-specific CD4+ T cells is that endogenously expressed
class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are
poorly recognized by CD4+ CTLs. In the present study we
attempted to enhance the efficiency of class II-restricted endogenous
presentation of minimal class II-restricted CTL epitopes by
specifically targeting a polyepitope protein to class II processing
compartments through the endosomal and/or lysosomal pathway. A
significantly enhanced stimulation of virus-specific
CD4+-T-cell clones by antigen-presenting cells (APC)
expressing the recombinant polyepitope protein targeted to the
endocytic/secretory pathway was readily demonstrated in cytotoxicity
assays. In addition, in vitro activation of Epstein-Barr virus- and
influenza virus-specific CD4+ memory CTLs by the
recombinant constructs encoding the polyepitope protein, specifically
targeted to the lysosomal compartment, was also demonstrated. The
enhanced stimulatory capacity of APC expressing a lysosome-targeted
polyepitope protein has important implications for vaccine design.
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INTRODUCTION |
There is now increasing evidence to
suggest that both CD4+ and CD8+ T cells are
critical for the generation of an effective immune response against
intracellular pathogens. Although both CD4+ and
CD8+ T cells recognize nonnative forms of the antigen in
association with major histocompatibility complex (MHC) molecules, the
presentation of antigen to these two types of T lymphocytes occurs
through distinct pathways (24). In fact, the disparity in
antigen presentation to these T cells is not due to processing
differences but rather reflects the differences in the capacities of
class I and class II molecules to bind antigenic determinants in an
intracellular compartment. Indeed, earlier studies have shown that for
processing and interaction with MHC class II molecules, antigen
expressed de novo needs to be targeted to an endosomal or lysosomal
compartment (5). There are two major pathways by which
antigens are targeted to these compartments. The traditional pathway
involves the phagocytosis or endocytosis of exogenous antigens,
followed by degradation by acid proteases in the endosomal or lysosomal
compartments (3, 8, 26, 41). On the other hand, class
II-restricted presentation of endogenously synthesized proteins mainly
involves membrane antigens which are thought to enter the endosomal or
lysosomal pathway by internalization from the cell surface
(11). Although, in certain experimental systems, cytoplasmic
and nuclear proteins may also enter this endogenous pathway, generally
these proteins are targets for the class I processing pathway (9,
14, 20, 27).
One of the major limitations of the epitope-based vaccines designed to
stimulate virus-specific CD4+ T cells is that endogenously
expressed class II-restricted minimal cytotoxic T-lymphocyte (CTL)
epitopes are poorly recognized by CD4+ CTLs (2, 35,
38). Based on these observations, we reasoned that a molecular
approach that directly routes these epitopes into the MHC class II
pathway, such as the endocytic or lysosomal compartments, might
facilitate endogenous presentation to CD4+ T cells. The
lysosome-associated membrane protein (LAMP-1) and the invariant chain
(Ii) are transmembrane proteins which are localized predominantly in
the lysosomes and endosomes, respectively. The cytoplasmic domains of
these proteins contain specific targeting signals that mediate their
translocation to the specific compartments. We therefore designed a
chimeric polyepitope construct capable of encoding multiple class
II-restricted CTL epitopes from Epstein-Barr virus (EBV) and influenza
virus linked to the cytoplasmic and/or transmembrane domains of LAMP-1
and the Ii protein, with the aim of targeting the epitopes to the
endosomal and lysosomal compartments. The data presented in this study
clearly demonstrate that if the endogenously synthesized polyepitope
protein is targeted to the endocytic/secretory pathway, processing and
presentation of all the epitopes are dramatically enhanced. More
importantly, minimal epitope sequences, without any natural flanking
sequences, were adequate for efficient stimulation of the
virus-specific memory CTL response, a result that has important
implications for epitope-based vaccine design.
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MATERIALS AND METHODS |
Establishment and maintenance of cell lines.
EBV-transformed
lymphoblastoid cell lines (LCLs) were established from seropositive
donors by exogenous virus transformation of peripheral B cells by using
the B95.8 or Ag876 virus isolate (25). All cell lines were
routinely maintained in RPMI 1640 containing 2 mM glutamine, 100 IU of
penicillin per ml, and 100 µg of streptomycin per ml plus 10% fetal
calf serum (FCS) (growth medium).
Generation of recombinant vaccinia virus constructs.
The
generation of recombinant vaccinia virus constructs encoding either
multiple class II-restricted epitopes as a polyepitope protein or the
polyepitope protein fused to an endoplasmic reticulum (ER) (adenovirus
E1A ER signal), endosomal (invariant-chain signal), or lysosomal
(LAMP-1 signal) signal sequence is summarized in Fig.
1. The minigene construct which expresses
multiple class II-restricted epitopes as a polyepitope protein (Fig.
1a) was designed by splicing six oligonucleotides together as described previously (37). A total of six different MHC class
II-restricted CTL epitopes from EBV and influenza virus were included
in this construct (Table 1). This
minigene was cloned into pBCB07 (1) by using
BamHI and SalI restriction enzymes to generate
the plasmid pPOLY.
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FIG. 1.
Construction of the class II polyepitope plasmids. (a) A
class II polyepitope minigene was designed and constructed by splicing
six overlapping oligonucleotides together by PCR and splicing by
overlap extension (SOEing). The minigene was then cloned into pBCB07 to
generate pPOLY. The plasmid pPOLY was then modified with DNA which
coded for three different transport signal sequences. A DNA fragment
flanked with appropriate restriction sites which codes for the
adenovirus E1A ER signal sequence was generated by annealing two
oligonucleotides together. This DNA fragment was then cloned into pPOLY
at the 5' end of the minigene to generate pER-POLY. A DNA fragment
which codes for the invariant-chain signal sequence (amino acids 1 to
71) was removed from the invariant-chain cDNA in p33-143 and cloned
into pPOLY at the 5' end of the minigene to generate pINV-POLY. A DNA
fragment which codes for the human LAMP-1 lysosomal signal sequence
(amino acids 354 to 389) was constructed by extending two overlapping
oligonucleotides. This DNA fragment was cloned into pER-POLY at the 3'
end of the minigene to generate pER-POLY-LAMP. These plasmids were
subsequently used to generate four recombinant vaccinia viruses,
Vacc.POLY, Vacc.ER-POLY, Vacc.INV-POLY, and Vacc.ER-POLY-LAMP,
respectively, by marker rescue recombination. TK, thymidine kinase.
wtGFP cDNA from the jellyfish A. victoria was cloned into
the coding sequence of the class II polyepitope protein. Briefly, a DNA
fragment containing the complete wtGFP cDNA except for the stop codon
was removed from pGFP-1N (Clonetech) and cloned in frame at both ends
into the class II polyepitope coding sequence in the plasmids pER-POLY,
pINV-POLY, and pER-POLY-LAMP to generate the plasmids pER-POLY-GFP,
pINV-POLY-GFP, and pER-POLY-GFP-LAMP, respectively. These plasmids were
subsequently used to generate three recombinant vaccinia viruses,
Vacc.ER-POLY-GFP, Vacc.INV-POLY-GFP, and Vacc.ER-POLY-GFP-LAMP,
respectively, by marker rescue recombination.
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To target the polyepitope protein to the endocytic/secretory pathway,
the polyepitope gene in pPOLY was fused to the DNA sequences encoding
either the ER, endosomal, or lysosomal signal sequence (Fig. 1a).
Briefly, a synthetic DNA sequence coding for the adenovirus E1A ER
signal sequence (28) was cloned into pPOLY to generate pER-POLY. A DNA fragment coding for the endosomal signal sequence (amino acids 1 to 71) was cleaved from the invariant-chain cDNA (34) and subcloned into pPOLY to generate pINV-POLY. A
synthetic DNA sequence coding for the lysosomal signal sequence (amino
acids 354 to 389) from human LAMP-1 (13) was constructed and
cloned into the plasmid pER-POLY to generate the plasmid pER-POLY-LAMP. These plasmids were then used to generate the recombinant vaccinia viruses Vacc.POLY, Vacc.ER-POLY, Vacc.INV-POLY, and Vacc.ER-POLY-LAMP by marker rescue recombination as described previously (4).
To examine the localization and expression of the class II polyepitope
protein targeted to the endocytic/secretory pathway,
the wild-type
green fluorescent protein (wtGFP) cDNA from the
jellyfish
Aequorea victoria (
6,
30) was cloned into the
coding
sequence of the polyepitope (Fig.
1b). Briefly, wtGFP cDNA was
excised from the plasmid pGFP-1N (Clonetech) and subcloned into
pER-POLY, pINV-POLY, and pER-POLY-LAMP to create pER-POLY-GFP,
pINV-POLY-GFP, and pER-POLY-GFP-LAMP, respectively. These plasmids
were
then used to generate the recombinant vaccinia viruses
Vacc.ER-POLY-GFP,
Vacc.INV-POLY-GFP, and Vacc.ER-POLY-GFP-LAMP as
described above.
The recombinant vaccinia virus constructs encoding the EBV nuclear
antigen 1 (EBNA1), EBNA2, and BHRF1 antigens of EBV and
a vaccinia
virus construct made by insertion of the pSC11 vector
alone and
negative for thymidine kinase (Vacc.TK
) have been
previously described (
16,
21,
39). In addition,
a
recombinant vaccinia virus construct expressing influenza virus
hemagglutinin (Vacc.HA) was also used in the study (
7).
CTL clones and peptide epitopes.
The MHC class
II-restricted, EBV-specific CTL clones used in this study were LC27
(EBNA2 specific; HLA DQ2/DQ7 restricted), DM2 (EBNA1 specific; HLA DR1
restricted), and SBAg1 (BHRF1 specific; HLA DR4Dw10 restricted). The
specificities of these clones have been defined at the peptide epitope
level: LC27 recognizes the minimal epitope TVFYNIPPMPL (residues 280 to
290) (20), DM2 recognizes the minimal epitope TSLYNLRRGTALA
(residues 515 to 527) (17), and SBAg1 recognizes the minimal
epitope TVVLRYHVLLEEI (residues 45 to 57) (33). These CTL
clones were propagated in growth medium supplemented with recombinant
interleukin-2 and MLA supernatant.
Cytotoxicity assay with recombinant vaccinia virus-infected
targets.
Target cells were infected with recombinant vaccinia
virus at a multiplicity of infection (MOI) of 10:1 for 1 h at
37°C. After overnight infection, cells were washed with growth
medium, incubated with 51Cr for 90 min, and used as targets
in a standard 5-h 51Cr-release assay (19, 25).
In some experiments, monoclonal antibodies (MAb) specific for the
nonpolymorphic determinants on MHC class II (IVA12) or class I (W6/32)
antigens were added in the CTL assays to confirm the MHC restriction
for the CTL clones.
To identify the processing pathway utilized by the class II-restricted
epitopes, recombinant vaccinia virus-infected target
cells were
pretreated with chloroquine (
20). Briefly, target
cells were
initially incubated in growth medium supplemented with
chloroquine (80 µmol/ml) for 4 h at 37°C. Following incubation,
these cells
were washed and resuspended in growth medium supplemented
with
chloroquine (20 µmol/ml) and used as targets in a standard
5-h
51Cr release assay (
25).
To verify the endogenous presentation of the epitopes through the class
II pathway, two different sets of experiments were
carried out. First,
vaccinia virus constructs were inactivated
by UV irradiation before
infecting target cells (
18). Briefly,
these vaccinia virus
constructs were treated with UV light in
an open petri dish on ice with
500 mJ of short-wave UV light in
a UV cross-linker apparatus
(Bio-Rad, Richmond, Calif.). In the
second set of experiments,
unlabelled Vacc.EBNA2-infected LCL
cells were mixed with
51Cr-labelled LCL cells at a ratio of 1:1 and then exposed
to specific
CTLs in a standard CTL assay.
Activation of memory CTL responses with recombinant vaccinia
virus.
Unfractionated mononuclear cells from a donor (HLA A3, A23,
B35, B44, DR1 DRW11 DQW1 DQW3 DRW52) were infected in vitro with Vacc.ER-POLY-LAMP at an MOI of 0.01:1 as described previously (21,
22, 36). After 10 days of culture in growth medium, these cells
were used as polyclonal effectors in a standard 51Cr
release assay against peptide-sensitized or vaccinia virus-infected autologous target cells.
Immunofluorescence assays.
To examine the localization and
expression of the class II polyepitope protein targeted to different
compartments of the cell, HeLa cells were infected with various
recombinant vaccinia viruses at an MOI of 4:1 for 1 h at 37°C.
Following incubation at 37°C for 6 h, these cells were processed
for immunofluorescence as described previously (21).
Briefly, vaccinia virus-infected cells were fixed, permeabilized, and
then incubated with rabbit anti-GFP polyclonal serum (diluted 1/400 in
1% FCS-phosphate-buffered saline [PBS]) for 1 h at room
temperature. The cells were extensively washed with 1% FCS-PBS and
then incubated for 1 h at room temperature with fluorescein
isothiocyanate-conjugated sheep anti-rabbit immunoglobulin G (Silenus,
Melbourne, Australia). The cells were washed extensively with 1%
FCS-PBS and then examined under a fluorescence microscope in the
presence of n-propyl gallate.
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RESULTS AND DISCUSSION |
Endogenously synthesized polyepitope protein targeted to the
endocytic/lysosomal compartment facilitates presentation of multiple
epitopes to CD4+ T cells.
To determine the effect of
endosomal or lysosomal targeting of a polyepitope protein on the
processing efficiency of class II-restricted epitopes, a panel of
well-characterized CD4+ CTL clones were used as effector
cells in a standard 51Cr release assay. LCLs were infected
with recombinant vaccinia virus vectors encoding either the polyepitope
protein (Vacc.POLY) or the polyepitope protein fused to an endosomal
(Vacc.INV-POLY) or lysosomal (Vacc.ER-POLY-LAMP) targeting signal
sequence. It is important to mention here that LAMP signal
sequence-mediated transport of the polyepitope protein to the lysosomal
compartment is dependent on efficient translocation to the secretory
pathway by an ER translocation signal sequence. To ensure that the
polyepitope construct is targeted to the lysosomal compartment, an
N-terminal ER signal sequence was included in the construct. Target
cells infected with recombinant vaccinia virus constructs encoding the polyepitope protein fused to an ER signal sequence (Vacc.ER-POLY) alone
and full-length viral antigens (Vacc.EBNA2, Vacc.EBNA1, or
Vacc.BHRF1) were used as controls in the assay. Targeting of the
polyepitope construct to the endosomal or lysosomal pathway was based
on the earlier observations that endogenous processing of antigens
through the class II pathway requires translocation of these antigens
to an endosomal or lysosomal compartment.
The data presented in Fig.
2 clearly
demonstrate that target cells infected with Vacc.POLY were poorly
recognized by all three
CTL clones (LC27, SBAg1, and DM2). However,
translocation of this
polyepitope construct to the endosomal or
lysosomal compartment
significantly increased the levels of CTL lysis.
The level of
CTL lysis of target cells infected with Vacc.ER-POLY-LAMP
was
consistently higher than that of Vacc.INV-POLY-infected targets
(Fig.
2). This CTL lysis was inhibited in the presence of MAb
IVA12
(anti-HLA DR, DP, and DQ) (Fig.
3).
Interestingly, enhanced
presentation of CTL epitopes was also seen in
target cells infected
with Vacc.ER-POLY (Fig.
2), suggesting that
translocation of minimal
epitopes into the ER compartment also
facilitates endogenous presentation
to CD4
+ T cells.
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FIG. 2.
Endogenously synthesized polyepitope protein targeted to
the endocytic/lysosomal compartment facilitates presentation of
multiple epitopes to CD4+ T cells. Autologous LCL cells
infected with different vaccinia virus recombinants (Vacc.POLY,
Vacc.ER-POLY, Vacc.INV-POLY, Vacc.ER-POLY-LAMP, Vacc.EBNA2, Vacc.BHRF1,
Vacc.EBNA1, and Vacc.TK) were used as targets in standard
CTL assays. CTL clones used in these assays were LC27 (EBNA2-specific),
SBAg1 (BHRF1-specific), and DM2 (EBNA1-specific). LC/Ag876, SB29f, and
DM/B95.8 LCLs were used as host cells for vaccinia virus constructs for
LC27, SBAg1, and DM2 CTL clones, respectively. The LC/Ag876 LCL is not
recognized by LC27, since this cell line is infected with a type 2 EBV
which expresses a variant EBNA2 epitope sequence. SB29f LCL cells are
negative for BHRF1 antigen, while the EBNA1 epitope is not endogenously
processed by DM/B95.8 LCL cells, and thus these cells are not
recognized by SBAg1 and DM2 CTL clones, respectively.
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FIG. 3.
Effect of chloroquine and UV treatment of vaccinia virus
constructs on CTL recognition of target cells. Recombinant vaccinia
virus-infected LC/Ag876 LCL cells were pretreated with chloroquine (for
details, see Materials and Methods) and then exposed to LC27 CTL. To
determine whether presentation of the EBNA2 epitope requires de novo
endogenous synthesis of the viral protein and is not exogenously
processed, LC/Ag876 LCL cells infected with UV-inactivated Vacc.EBNA2,
Vacc.ER-POLY, Vacc.INV.POLY, and Vacc.ER-POLY-LAMP were exposed to the
LC27 clone. In addition, vaccinia virus-infected LC/Ag876 LCL cells
were also exposed to LC27 CTLs with or without the addition of MAb
IVA12 (anti-HLA DR, DP, and DQ) (1:20-diluted ascites). An
effector-to-target ratio of 4:1 was used in the assay.
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One of the surprising results from these experiments relates to the
endogenous presentation of the EBNA1 epitope. Earlier
studies have
shown that although EBNA1 includes potential CTL
epitopes which can be
presented by class II molecules, these epitopes
are not endogenously
processed and presented by virus-infected
cells. The data presented in
Fig.
2C show that the translocation
of the EBNA1 epitope to the
endosomal/lysosomal compartment significantly
improved the endogenous
presentation of this epitope. Consistent
with our earlier observations,
this epitope is not endogenously
processed by target cells infected
with either Vacc.EBNA1 or Vacc.POLY.
Earlier studies have shown that conventional class II antigen
presentation requires assembly of the peptide epitope with class
II
molecules in an acidified vacuolar compartment and is therefore
sensitive to lysosomotropic agents that disrupt acidification
of
endosomes (
5,
12,
24). We therefore tested whether the
presentation of the CTL epitopes, targeted to the endocytic/secretory
compartments, was dependent on this pathway by preventing acidification
of the endosomal compartment with chloroquine treatment. Treatment
of
Vacc.ER-POLY-, Vacc.INV-POLY-, and Vacc.ER-POLY-LAMP-infected
target
cells with chloroquine significantly reduced the level
of CTL lysis by
the clone LC27 (Fig.
3). A similar effect on the
presentation of other
CTL epitopes was also seen following treatment
of target cells with
chloroquine (data not shown).
To exclude the possibility that the observed CTL recognition is due to
peptides present in the vaccinia virus stocks, target
cells were
infected with vaccinia virus constructs that had been
inactivated by
irradiation with UV light. As shown in Fig.
3,
target cells infected
with UV-treated vaccinia virus were not
lysed by the CTLs. Moreover,
premixing of
51Cr-labelled target cells with recombinant
vaccinia virus-infected
cells showed no lysis of target cells (data not
shown). Thus,
de novo synthesis of the CTL epitopes from the
polyepitope transcripts
was required for processing and presentation of
this epitope.
Activation of virus-specific memory CTL response by
Vacc.ER-POLY-LAMP.
The results presented above clearly
demonstrate that processing of endogenously synthesized class
II-restricted epitopes can be significantly enhanced by targeting a
polyepitope protein to the lysosomal compartment. A series of
experiments were carried out to determine whether this polyepitope
construct could be used to stimulate a memory CTL response in vitro
from peripheral blood lymphocytes. Unfractionated mononuclear cells
from an HLA DR1-positive donor were infected with Vacc.ER-POLY-LAMP at
an MOI of 0.01:1 for 10 days, and the resulting polyclonal CTLs were
then used as effectors against peptide-sensitized or vaccinia
virus-infected autologous LCLs in a standard CTL assay. The data
presented in Fig. 4 clearly demonstrate
that Vacc.ER-POLY-LAMP was capable of activating CTL responses to both
EBV and influenza virus epitopes. Consistent with the data presented in
Fig. 2C, a strong EBNA1-specific CTL response was noticed following in
vitro stimulation with Vacc.ER-POLY-LAMP (Fig. 4). It is important to
mention here that in vitro stimulation with Vacc.EBNA1 or Vacc.POLY
failed to stimulate an EBNA1-specific CTL response from the HLA
DR1-positive donor (data not shown). These results suggest that direct
LAMP-1-mediated lysosomal targeting of the EBNA1 epitopes could be used
for stimulating CD4+-CTL responses in a vaccine designed to
control EBV-associated diseases.
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FIG. 4.
Activation of virus-specific memory CTL response by the
polyepitope protein targeted to the lysosomal compartment. Polyclonal
effectors from an HLA DR1-positive donor were generated by
restimulating peripheral blood lymphocytes with Vacc.ER-POLY-LAMP.
Target cells were autologous LCLs either infected with recombinant
vaccinia virus constructs or sensitized with the indicated peptide (10 µmol/ml). Effector/target ratios of 15:1 and 30:1 were used in the
assay.
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Detection of polyepitope protein targeted to the
endocytic/secretory pathway.
To examine the localization and
expression of the class II polyepitope protein targeted to the
endocytic/secretory pathway, wtGFP was incorporated into different
polyepitope constructs. A standard immunofluorescence assay was used to
detect these chimeric molecules in recombinant vaccinia virus-infected
HeLa cells by using a GFP-specific antibody (Fig.
5). It is important to mention here that
our earlier attempts to detect the wtGFP by direct fluorescence were
unsuccessful due to a rapid loss of fluorescence at 37°C (10,
29). A distinct pattern of staining, restricted primarily to the
ER, was seen in cells infected with Vacc.ER-POLY (Fig. 5B), while cells
infected with Vac.ER-POLY-LAMP and Vacc.INV-POLY showed fluorescence in
vesicles which resemble lysosomes in size and perinuclear localization
(Fig. 5C and D). These results are consistent with the previously
described endosomal/lysosomal localization of Ii protein and LAMP-1.
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FIG. 5.
Localization of polyepitope protein targeted to the
endocytic/secretory pathway by immunofluorescence. HeLa cells were
infected with Vacc.TK (A), Vacc.ER-POLY-GFP (B),
Vacc.INV-POLY-GFP (C), or Vacc.ER-POLY-GFP-LAMP (D) at an MOI of 4:1
for 6 h at 37°C. Following incubation, these cells were
processed for immunofluorescence as described in Materials and Methods.
Cells were initially labelled with wtGFP-specific antibody and then
incubated with fluorescein isothiocyanate-conjugated sheep anti-rabbit
immunoglobulin G. The cells were examined under a fluorescence
microscope in the presence of n-propyl gallate.
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This study clearly illustrates that targeting signal sequences can be
utilized to direct multiple class II-restricted CTL
epitopes into the
endosomal and lysosomal compartments. This approach
not only
preferentially translocates the polyepitope protein to
these
compartments but also enhances endogenous presentation of
CTL epitopes.
Furthermore, this strategy was successfully used
to activate a
virus-specific memory CTL response from peripheral
blood lymphocytes.
These observations have a number of important
implications for the
endogenous presentation of MHC class II-restricted
epitopes and for
vaccine design in general. First, the use of
a polyepitope protein
targeted to the lysosomal compartment overcomes
the problems associated
with the use of oncogenic viral antigens
as vaccines. Second, multiple
epitopes from different viruses
can be included in these constructs to
specifically stimulate
CD4
+ T cells, avoiding any
requirement for multiple recombinant whole-protein
sequences (
31,
32). This might be important in settings where
an antibody
response results in enhanced infection. For example,
dengue virus
infection is augmented when nonneutralizing antibodies
complex with
virus (
15). Another important implication of these
results
relates to the ability of the LAMP-1-targeted polyepitope
protein to
activate a CD4
+-CTL response to cytoplasmic or nuclear
antigens that are not
normally targeted to the class II pathway.
Indeed, the data presented
in this study showed that although the EBNA1
antigen is poorly
immunogenic, LAMP-1-mediated targeting of the EBNA1
CTL epitope
significantly enhanced the activation of the EBNA1-specific
CTL
response in vitro. These results indicate that the latter approach
might be used to enhance the EBNA1-specific response in vivo to
control
EBV-associated malignancies where EBV latent gene expression
is
restricted to this antigen. Recent studies have shown that
immunization
with the papillomavirus E7 antigen fused to LAMP-1
induced potent
E7-specific antitumor immunity (
23,
40).
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ACKNOWLEDGMENTS |
This work was supported by grant from the Co-operative Research
center for Vaccine Technology and The National Health and Medical
Research Council (NHMRC). R.K. is supported by a R. Douglas Wright
Fellowship from NHMRC.
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FOOTNOTES |
*
Corresponding author. Mailing address: Queensland
Institute of Medical Research, Bancroft Centre, 300 Herston Rd.,
Brisbane, Australia 4006. Phone: 61-7-3362 0346. Fax: 61-7-3362 0106. E-mail: rajivK{at}qimr.edu.au.
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Abstract
Introduction
