Protective cytotoxic T lymphocyte responses induced by DNA immunization against immunodominant and subdominant epitopes of Listeria monocytogenes are noncompetitive.

Abstract

Taking advantage of the fact that plasmid DNA encoding a single cytotoxic T lymphocyte (CTL) epitope can induce CTLs, we examined the influence of T-cell responses to dominant epitopes on those to a subdominant epitope derived from Listeria monocytogenes. Our data suggest that interaction between T cells against dominant and subdominant epitopes does not operate in the generation of the hierarchy. Furthermore, we found that a single dominant epitope is sufficient for the induction of protective immunity.

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INFECTION AND IMMUNITY,
0019-9567/01/$04.00?0
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
DOI: 10.1128/IAI.69.5.3427–3430.2001
May 2001, p. 3427–3430Vol. 69, No. 5
Protective Cytotoxic T Lymphocyte Responses Induced by DNA
Immunization against Immunodominant and Subdominant Epitopes of
Listeria monocytogenes Are Noncompetitive
TAKASHI YAMADA,1HIROSHI UCHIYAMA,1TOSHI NAGATA,2MASATO UCHIJIMA,2
TAKAFUMI SUDA,1KINGO CHIDA,1HIROTOSHI NAKAMURA,1AND YUKIO KOIDE2*
Second Department of Internal Medicine1and Department of Microbiology and Immunology,2Hamamatsu Univeristy
School of Medicine, Hamamatsu 431-3192, Japan
Received 6 August 2000/Returned for modification 25 October 2000/Accepted 2 February 2001
Taking advantage of the fact that plasmid DNA encoding a single cytotoxic T lymphocyte (CTL) epitope can
induce CTLs, we examined the influence of T-cell responses to dominant epitopes on those to a subdominant
epitope derived from Listeria monocytogenes. Our data suggest that interaction between T cells against domi-
nant and subdominant epitopes does not operate in the generation of the hierarchy. Furthermore, we found
that a single dominant epitope is sufficient for the induction of protective immunity.
Effector CD8?T-cell responses against infection are re-
stricted to a few epitopes compared to the total number of
epitopes potentially available (1, 2, 12, 23). These epitopes are
termed immunodominant, and cytotoxic T lymphocyte (CTL)
responses to subdominant epitopes can be demonstrated in
vivo by the removal of dominant epitopes or the separation of
dominant and subdominant epitopes so that they are presented
on different cells (9, 18, 21). Various factors have been impli-
cated to determine the position of an epitope in the immuno-
dominant hierarchy: (i) antigen processing efficiency (10, 11,
20), (ii) transporter associated with antigen processing (TAP)-
dependent peptide transport (15), (iii) the affinity of the pep-
tide for the major histocompatibility complex (MHC) molecule
(6, 24), (iv) the rate of dissociation of the MHC molecule (26,
28), (v) the transport of MHC-peptide complexes to the cell
surface (17), and (vi) the response to the T-cell repertoire (5,
7, 8).
Listeria monocytogenes is a gram-positive bacterium that
causes life-threatening infections during pregnancy and in im-
munocompromised individuals (14). L. monocytogenes enters
macrophages and hepatocytes and accesses the cytoplasm by
secreting listeriolysin O (LLO), resulting in the induction of a
vigorous MHC class I-restricted CTL response that enables the
rapid clearance of live bacteria (3, 16). Four different L. mono-
cytogenes epitopes are known to be presented to CTLs by the
MHC class I H-2Kdmolecules (4, 22). These epitopes are
derived from bacterial virulent factors, LLO, murein hydrolase
p60, and metalloprotease (mpl). Infection of BALB/c mice
with a sublethal dose of L. monocytogenes induces dominant
CTL responses against LLO 91-99 and p60 217-225 and sub-
dominant responses against p60 449-457 and mpl 84-92. There
is no correlation between the amount of epitope that is pre-
sented by infected cells and the magnitude of the CTL re-
sponse (29).
We have previously shown that intramuscular or gene gun
immunizations with plasmid DNA encoding a single epitope of
L. monocytogenes, LLO 91-99, are able to induce specific CTLs
in a CD4?T-cell-independent manner (19, 27, 31). In the
current study, taking advantage of the fact that a single
epitope-expressing DNA vaccine can induce CTLs without the
influence of the other epitopes, we investigated the influence
of dominant T-cell responses on subdominant responses.
We constructed three plasmids, p91m, p60 217m, and p60
449m, which encode LLO 91-99, p60 217-225, and p60 449-457
of L. monocytogenes, respectively, flanked by ATG start codon
and TAA stop codon, under the control of the cytomegalovirus
immediate-early enhancer-promoter. The DNA sequences in-
serted were adapted to the most frequently used codons in
murine genes (19, 27). DNA immunization was performed into
the shaved abdominal skin of BALB/c mice using the Helios
gene gun system (Bio-Rad Laboratories, Hercules, Calif.).
Then, 2 ?g of plasmid DNA was coated onto 0.5 mg of 1.0-?m
gold particles, and the injection was carried out with 0.5 mg of
gold/shot. Mice were injected three times with 2 ?g of the
plasmids weekly. At 2 weeks after the last immunization, im-
mune splenocytes were cultured in 12-well plates at a density of
2 ? 107/well for 5 days with 2 ? 107syngeneic spleen cells per
well that had been treated with 100 ?g of mitomycin C per ml
and pulsed with 5 ?M concentrations of the each of synthetic
peptides representing LLO 91-99, p60 217-225, and p60 449-
457 for 2 h at 37°C. Each well also received 10 U of human
recombinant interleukin-2 (Hoffmann-La Roche, Nutley, N.J.)
per ml. Cell-mediated cytotoxicity was assayed against J774
(H-2d) pulsed by incubation with 5 ?M concentrations of each
peptide using a conventional51Cr release assay as described
previously (27). As shown in Fig. 1A, gene gun immunizations
of BALB/c mice with p91m and p60 217m expressing the dom-
inant epitopes LLO 91-99 and p60 217-225, respectively, in-
duced vigorous CTL responses, whereas immunizations with
p60 449m encoding a subdominant epitope, p60 449-457, in-
duced only a marginal CTL response. These CTL responses
appeared to be the same as that observed with the immuniza-
tion with live L. monocytogenes (4, 29). The results obtained
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Hamamatsu University School of Medicine,
3600 Handa-cho, Hamamatsu 431-3192, Japan. Phone: 81-53-435-
2334. Fax: 81-53-435-2335. E-mail: koidelb@hama-med.ac.jp.
3427

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imply that competitive interaction between CTLs against dom-
inant and subdominant epitopes does not operate in the liste-
rial antigens. It seems unlikely that the relative magnitudes of
the CTL responses is attributable to quantitative difference of
the three peptides expressed by the plasmids, since a subdomi-
nant epitope, p60 449-457, has been reported to be associated
with H-2Kdmolecules approximately 10 times greater than the
dominant epitope p60 217-225 in L. monocytogenes-infected
cells (25). Actually, we observed only small differences in the
translational efficiencies of these DNA vaccines when we used
our readthrough analysis (27), in which the luciferase cDNA
was fused to downstream of the adapted LLO 91-99, p60 217-
225, and p60 449-457 sequences, resulting in p91m-Luc, p60
217m-Luc, and p60 449m-Luc, respectively, and the conven-
tional luciferase assay was performed with these expression
plasmids using BALB/3T3 murine cells (Fig. 1B). However,
CTL activities do not always represent the in vivo level of
T-cell activities since in vitro restimulation of immunized
splenocytes with a relevant peptide is essential to assess CTL
activities. It has been demonstrated that T cells with dominant
specificities expand optimally to low peptide concentrations,
while those specific for subdominant epitopes respond maxi-
mally to high peptide concentrations (4). We, therefore, exam-
ined the frequencies of T cells specific for LLO 91-99, p60
217-225, and p60 449-457 following gene gun immunization
with p91m, p60 217m, and p60 449m, respectively. Figure 2A
shows the results of ELISPOT assays of peptide-induced
gamma interferon (IFN-?) production to quantify T-cell fre-
quencies induced by each plasmid DNA; these results indicate
that p91m appeared to induce the most dominant frequency,
p60 217m induced an intermediate frequency, and p60 449m
induced the lowest frequency of specific T cells. In vivo deple-
tion of T-cell subsets with monoclonal antibodies against CD4
or CD8 (27) indicated that the IFN-?-secreting cells are CD8?
T cells (data not shown). Fensterle et al. (13) reported that
primary immunization with a plasmid encoding LLO but not
that encoding p60 induced significant specific T-cell responses,
whereas booster immunization of the both plasmids after 45
days induced similar specific T-cell frequencies. Their results
conflict with ours since, although our three immunizations with
an interval of 1 week resulted in a pronounced booster effect
(with a 21-fold increase in the LLO 91-99-specific T-cell fre-
quency), we observed the hierarchy of immunodominance in
the listerial epitopes. This discrepancy is puzzling at present
but may be due to the different DNA vaccines used, i.e., plas-
mids encoding epitopes versus plasmids encoding whole pro-
tein.
To investigate the effect of the in vivo interactions of T
cells induced by the three different plasmid DNAs, p91m,
p60 217m, and p60 449m, we immunized mice with the three
different plasmid DNAs simultaneously at nonoverlapping
sites of the abdominal skin and then evaluated the frequen-
cies of T cells specific for LLO 91-99, p60 217-225, and p60
449-457 using ELISPOT (Fig. 2B). This method was de-
signed to have different antigen-presenting cells present dif-
ferent epitopes to T cells in order to avoid the possibility of
epitope competitions for binding to the same H-2Kdmole-
cules. The data obtained are essentially the same the results
we observed with immunization with a single plasmid DNA
(Fig. 2A), suggesting that competitive interaction between
CTLs against dominant and subdominant epitopes does not
operate in the listerial epitopes. As shown in Fig. 2C, es-
sentially the same results were obtained when mice were
inoculated with gold particles coated with the mixed three
plasmids (1 ?g each). These data imply that the hierarchy of
immunodominace in the listerial epitopes is readily deter-
mined by interaction between responder T cells and CTL
epitopes presented by MHC class I molecules and that com-
FIG. 1. Immunization with DNA vaccine encoding a single domi-
nant or subdominant epitope induces different levels of CTL activity.
(A) BALB/c mice were immunized by gene gun with p91m (I, ?), p60
217m (Œ, ‚), or p60 449m (}, {) three times. Spleen cells from
immunized animals were harvested 2 weeks after the last immuniza-
tion and stimulated in vitro with LLO 91-99-, p60 217-225-, or p60
449-457-peptide pulsed spleen cells for 5 days. The percentage of
specific lysis was determined using J774 cells (H-2d) pulsed with LLO
91-99-peptide (solid symbols) or control medium (open symbols) as
target cells. Results are expressed as the mean ? the standard devia-
tion (SD) for six mice. (B) Translational efficiencies of the three
plasmid DNA vaccines. Relative luciferase activities were assayed by
transient transfections of p91m-Luc, p60 217m-Luc, and p60 449m-Luc
in BALB/3T3 mouse fibroblast cells. The relative luciferase activities
are normalized to Renilla remiformis luciferase activities by the co-
transfected pRL-CMV (Promega, Madison, Wis.). The data are ex-
pressed as the mean ? the SD for six samples.
3428 NOTESINFECT. IMMUN.

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petitive interaction between responder T cells may not op-
erate in the generation of the hierarchy of the listerial
epitopes. An attempt has been performed to delete the two
dominant epitopes, LLO 91-99 and p60 217-225, from L.
monocytogenes by anchor residue mutagenesis to study the
influence in vivo of dominant T-cell response on the sub-
dominant response during the listerial infection (30). Con-
sistent with our data, the experiment demonstrated that the
loss of these two dominant T-cell responses does not en-
hance T-cell responses to the subdominant epitopes (p60
449-457 and mpl 84-92).
Given that the three plasmid DNAs induced different levels
of specific lysis, we wanted to determine the biologic effect of
the plasmids against bacterial challenge. At 72 h after sublethal
FIG. 3. Protective immunity induced by immunization with plasmid DNA encoding a dominant or subdominant epitope and by simultaneous
immunization with the three plasmids. Mice were immunized with p91m, p60 217m, or p60 449m or were simultaneously immunized with these
plasmids three times and, 2 weeks later after the last immunization, were challenged with 2 ? 104CFU of L. monocytogenes intravenously. Bacterial
titers of spleens were determined 72 h after the challenge infection by plating 10-fold dilutions of tissue homogenates on Trypticase soy agar plates.
The results are expressed as the mean ? the SD for six mice. ?, P ? 0.01; ??, P ? 0.02.
FIG. 2. In vivo frequencies of T cells specific for each epitope in mice immunized by gene gun with DNA vaccines encoding a dominant or a
subdominant epitope. (A) BALB/c mice were immunized with p91m, p60 217m, or p60 449, and the numbers of epitope-specific CD8?T cells were
determined using IFN-? ELISPOT assays 2 weeks after the last immunization. Immune splenocytes were incubated in the presence of peptide-
coated or uncoated P815 cells. Peptide-induced IFN-? secretion by a single cell was visualized and quantified in triplicates and is expressed as the
number of IFN-?-secreting cells per 106immune splenocytes. The results are expressed as the mean ? the SD for six mice. The P values are 0.003
for p91m versus p60 217m, 0.002 for p91m versus p60 449m, and 0.0069 for p60 217m versus p60 449m. The Student’s t test was used in all statistical
analyses. (B) In vivo frequencies of T cells specific for each epitope in mice simultaneously immunized with the three plasmid DNA at
non-overlapping sites. BALB/c mice were immunized with the three different plasmid DNAs simultaneously at nonoverlapping sites of the
abdominal skin. At 2 weeks after the last immunization, the number of epitope-specific CD8?T cells was determined by IFN-? ELISPOT assay.
The P values are 0.0071 for LLO 91-99 versus p60 217-225, 0.0023 for LLO 91-99 versus p60 449-457, and 0.0799 for p60 217-225 versus p60
449-457. (C) In vivo frequencies of T cells specific for each epitope in mice immunized with gold particles coated with the mixed three plasmid
DNA. The number of epitope-specific CD8?T cells was determined by IFN-? ELISPOT assay. The results are expressed as the mean ? the SD
for six mice. The P values are 0.0059 for LLO 91-99 versus p60 217-225, 0.0009 for LLO 91-99 versus p60 449-457, and 0.0072 for p60 217-225 versus
p60 449-457.
VOL. 69, 2001NOTES 3429

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listerial challenge (2 ? 104CFU), mice from each group were
sacrificed, and the CFU from the spleens were counted. Con-
sistent with the ELISPOT data, both p91m and p60 217m, but
not p60 449m, induced remarkable protective immunities
against listerial infection (Fig. 3). Of particular interest is that
simultaneous inoculation of these three plasmids failed to sur-
pass a single plasmid expressing a dominant epitope, LLO
91-99 or p60 217-225, in the protection, suggesting that a single
dominant epitope may be sufficient for the induction of CTL-
mediated protective immunity. These data have implications
for the development of vaccines designed to emphasize CTL-
mediated immunity.
This work was supported in part by grants-in Aid from the Ministry
of Education, Science, Sports, and Culture of Japan.
We thank Tetsumichi Matsuo for excellent experimental assistance.
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Editor: S. H. E. Kaufmann
3430NOTESINFECT. IMMUN.