Department of Veterinary Microbiology and
Pathology, Washington State University, Pullman, Washington
99164-7040
Most equine infectious anemia virus (EIAV)-infected horses have
acute clinical disease, but they eventually control the disease and
become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered
an important immune component in the control of infections with
lentiviruses including EIAV, but definitive evidence for CTL in the
control of disease in carrier horses is lacking. By using retroviral
vector-transduced target cells expressing different Gag proteins and
overlapping synthetic peptides of 16 to 25 amino acids, peptides
containing at least 12 Gag CTL epitopes recognized by
virus-stimulated PBMC from six long-term EIAV-infected horses were
identified. All identified peptides were located within Gag matrix
(p15) and capsid (p26) proteins, as no killing of target cells
expressing p11 and p9 occurred. Each of the six horses had CTL
recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two
nonamer peptide epitopes were defined from Gag p26; one (18a) was
likely restricted by class I equine leukocyte alloantigen A5.1
(ELA-A5.1) molecules, and the other (28b-1) was likely restricted by
ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm
killing required 10 nM peptide 18a and 1 nM 28b-1. The results
demonstrated that diverse CTL responses against Gag epitopes were
generated in long-term EIAV-infected horses and indicated that ELA-A
class I molecules were responsible for the diversity of CTL
epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV.
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INTRODUCTION |
Equine infectious anemia
virus (EIAV) belongs to the Lentivirus genus, which includes
human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency
virus (SIV), and several other animal viruses. EIAV causes disease in
horses which is characterized by recurrent febrile episodes associated
with viremia, anemia, and thrombocytopenia (10). Most
infected horses are able to eventually control the disease and become
lifelong EIAV carriers (9). The ability of horses to
restrict EIAV replication to very low levels and to remain free of
clinical disease provides an opportunity to determine the immunologic
mechanisms involved in this lentivirus control.
Immune responses are required for the termination of the acute viremia
during EIAV infection since foals with severe combined immunodeficiency
cannot control the initial viremia following EIAV infection, in
contrast to normal foals (41). Results suggesting that
immune responses are involved in the control of EIAV in carrier horses
include the observation that corticosteroid- and
cyclophosphamide-treated carrier horses have recurrent viremia and
disease (24). Neutralizing antibody can be an important
component of the protective immune response against lentiviral
infections (12). Type-specific neutralizing antibody appears
following the episodes of plasma viremia in EIAV-infected horses
(25); however, there is evidence suggesting that the presence of the neutralizing antibody does not necessarily relate to
the occurrence and control of viremic episodes (8,
25). Detectable neutralizing antibodies to the variant
isolated during a disease episode can appear after the episode is
controlled (8). Neutralizing antibody-escape variants are
isolated from EIAV carrier horses as early as 5 days after
corticosteroid treatment, when the antibody levels have not
significantly changed (24). Further, the viremic episode
induced by corticosteroid treatment can be terminated before the
appearance of neutralizing antibody to the variant causing viremia
(24). Other evidence implicating immune responses other than
neutralizing antibody in EIAV control includes the following: (i) EIAV
carrier horses can resist challenge with a heterologous strain in the
absence of detectable neutralizing antibody to the challenge virus
(23), and (ii) some horses immunized with an inactivated
virus vaccine resist homologous strain challenge without detectable
levels of neutralizing antibody but with virus-specific cell-mediated
immune responses (17).
Accumulating evidence suggests that major histocompatibility complex
(MHC) class I-restricted virus-specific cytotoxic T lymphocytes (CTL)
may play an important role in the immune control of diseases caused by
HIV-1 and SIV infection (5, 26, 51). CTL appear to be
involved in both the clearance of the primary viremia in HIV-1
infection (26) and the prevention of disease progression to
AIDS (42). In EIAV infection, the appearance of activated CD8+ CTL (effectors) correlated with the control of the
initial viremic episodes (33). Although the CTL effectors
decline to low levels when plasma viremias become undetectable, a high
frequency of memory CTL (CTLm) has been detected in some carrier horses
(34), and these CTLm recognize either EIAV Env or Gag/Pr
proteins or both (15, 34). Both CD8+ and
CD4+ CTL activities have been detected in some
EIAV-infected horses (15), but their roles in disease
control are not known.
The epitopes recognized by CD8+ CTL are usually
peptides of 8 to 11 amino acids (aa) presented by MHC class I molecules
on the target cell surface. Identifying the CTL epitopes and the MHC class I molecules that restrict responses is necessary in order to
determine how CTL are involved in the control of disease and to
stimulate CTL by vaccination. However, the occurrence of escape mutants
which are no longer recognized by CTL is one of the major difficulties
for inducing effective CTL responses against different
variants (6). Gag protein epitopes recognized by CTL may
be of importance because Gag proteins are relatively conserved among
EIAV strains (21, 32, 40, 48). In this study, at least 12 peptides with CTL epitopes were recognized by stimulated peripheral
blood mononuclear cells (PBMC) from six long-term EIAV-infected horses
with different ELA-A alleles. These peptides were identified by using
retroviral vectors expressing individual Gag proteins and synthetic
overlapping peptides from recognized proteins. We identified two
nonamer peptides, one apparently restricted by ELA-A5.1, and another by
ELA-A9, molecules.
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MATERIALS AND METHODS |
Animals.
Six mixed-breed ponies (horses)
experimentally infected with EIAVWSU5 were used
as sources of PBMC. The derivation of EIAVWSU5 and the
infection of five of these horses were previously described (33,
34). A sixth horse, H513, was similarly infected with 108 50% tissue culture infective doses
(TCID50) of EIAVWSU5. At the time of this
study, H507 had been infected for 5 years, H521 had been infected for 4 years, H529, H532, and H540 had been infected for 3 years, and H513 had
been infected for 1.5 years.
Cell lines.
Equine kidney (EK) cells for CTL targets were
expanded from kidney biopsies of the six horses obtained before EIAV
infection (33). Other cell lines used in this study included
PA317 amphotropic retroviral packaging cells (ATCC CRL 9078) and PG13
packaging cells (ATCC CRL 10686). All cell lines were maintained in
Dulbecco modified Eagle medium (DMEM; Gibco BRL, Grand Island, N.Y.)
containing 5% fetal bovine serum (FBS).
ELA-A typing.
PBMC were typed for ELA-A molecules by
microcytotoxicity assays using described antibody reagents (2,
27). The ELA-A locus, the only well-defined MHC class I locus in
the horse, contains 17 internationally accepted alleles (A1 to A10,
A14, A15, A19, W16, W17, W18, and W20) (31).
Construction of retroviral vectors.
The EIAVWSU5
gag gene was amplified from pEIA5G (32) into five
DNA segments by PCR using specific primers (Table
1). These segments encoded p15, the
N-terminal half of p26 (p26a), the C-terminal half of p26 (p26b), p11,
and p9. The optimal primers for cloning resulted in overlaps of 12 bp
of the p26a with p26b segments and overlaps of 6 bp on both ends of the
p11 segment with the p26b and p9 gene segments. EcoRI and
XhoI restriction enzyme sites designed in the forward and
backward primers, respectively, were used for inserting the segments
into retroviral vector plasmid pLXSN (provided by A. Dusty Miller, Fred
Hutchinson Cancer Center, Seattle, Wash.), using previously described
procedures (30). pLXSN contains the neomycin
phosphoribosyltransferase gene under control of the simian virus 40 early gene promoter. Expression of the gag gene segments was
driven by the Moloney murine sarcoma virus long terminal repeat
promoter (35). The orientation of the inserts was determined
by restriction enzyme analysis, and the nucleotide sequences were
determined. Transfection of the retroviral packaging PA317 cells with
the vector plasmids with or without the gag gene segments
was accomplished by using LipofectAMINE (Gibco BRL). Forty-eight hours
after transfection, the supernatants were collected, filtered through a
0.45-µm-pore-size syringe filter, and used to transduce the PG13
packaging cell line, which incorporates gibbon ape leukemia virus Env
into vector virion membranes for cell entry with a wide host range
(35). The transduced PG13 cells were selected with medium
containing 700 µg of G418 sulfate (Calbiochem, La Jolla, Calif.) per
ml. Regular medium (DMEM with 10% FBS) was used on the resulting
G418-resistant PG13 cell line for collecting supernatant containing
vector virus. The supernatant was titrated on EK cells, and the titer
ranged from 1 × to 6 × 105 transducing
particles/ml.
Synthetic peptides.
Peptides 16 to 25 aa in length and
overlapping by 8 aa corresponding to p15 and p26 (Table
2) were synthesized by the Laboratory for
Biotechnology and Bioanalysis I, Washington State University, Pullman.
Additional peptides (9 to 12 aa) were synthesized for fine mapping of
two CTL epitopes. High-pressure liquid chromatography analysis
indicated that the 9- to 12-aa peptides were >95% pure. The peptides
were dissolved at 2 mg/ml in sterile phosphate-buffered saline with
10% dimethyl sulfoxide before dilution with medium and pulsing of EK
target cells.
Generation of CTLm.
PBMC were separated from the blood of
EIAV-infected horses by layering leukocyte-rich plasma over Histopaque
and centrifuging. To generate CTLm, 106 PBMC were cultured
with 106 irradiated, EIAV-infected or peptide-pulsed
autologous PBMC in each well of a 24-well plate with 1 ml of DMEM
containing 10% FBS and 20 mM HEPES. The stimulator PBMC were prepared
by irradiating 108 autologous PBMC with 3 krad of gamma
irradiation, mixing with 5 ml of 106 TCID50 of
EIAVWSU5 per ml or various peptides at 20 µg/ml and incubated at 37°C for 1 h. After 7 days, 5 × 105 viable cells were restimulated with 106
stimulator cells in 1 ml of medium/well containing 10 U of recombinant human interleukin-2 (Gibco BRL) per ml. After 4 days, the restimulated PBMC were split from one well to two wells with fresh medium and recombinant human interleukin-2. The cells were further expanded for
another 3 to 4 days before use as effector cells in CTL assays (34).
Cytotoxicity assay.
EK cells were transduced with retroviral
vectors with or without the gag gene segments in DMEM
containing 10% FBS for 24 h at a multiplicity of infection of 2 to 5. The transduced EK cells were then placed under selection in DMEM
containing 700 µg of G418 per ml for at least 1 week before the CTL
assay. EK target cells (3 × 104/well) were incubated
in collagen-coated wells of 96-well plates at 37°C with 5%
CO2 for 24 h before 51Cr labeling.
Peptide-sensitized EK target cells were prepared by pulsing the normal
EK cells with synthetic peptides at concentrations indicated in the
figure legends for 2 to 16 h in 96-well plates at 37°C in a 5%
CO2 and humidified atmosphere before 51Cr
labeling. Target cells were labeled for 2 h with 2.5 µCi of 51Cr per well in 50 µl of DMEM with 5% FBS. Different
effector-to-target cell ratios were incubated at 37°C with 5%
CO2 for 17 h (33), and then 100 µl of
supernatant was removed from each well to determine 51Cr
release. Percent specific lysis was calculated as [(E 
S)/(M S]) × 100, where E is the mean of six
test wells, S is the mean spontaneous release from six
wells without effector cells, and M is the mean maximal
release from six wells with 3% Triton X-100. The standard error (SE)
of percent specific lysis was calculated as described previously
(33). Only assays with a spontaneous lysis of <30% were used.
RT-PCR amplification of RNA from transduced EK cells.
Total
RNA was isolated from retroviral vector-transduced EK cells by using a
commercial kit (Qiagen Inc., Hilden, Germany). One microgram of the RNA
sample was treated with DNase I for 15 min at room temperature, and the
reverse transcription (RT) reactions were performed with 300 ng of
DNase-treated total RNA by using an RNA-PCR kit (Perkin-Elmer Cetus,
Norwalk, Conn.). Briefly, the cDNA was synthesized in a total volume of
20 µl, using backward primers specific to the sequences of the
gag segments (Table 1) and reaction conditions of 42°C for
15 min, 95°C for 10 min, and then 5°C for 5 min. Reactions without
RT were used as negative controls. Then PCR was performed by adding the
forward primers and DNA Taq polymerase to the cDNA followed
by 35 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 7 min. Ten-microliter aliquots of the PCR products were electrophoresed
on 1.5% agarose gels stained with ethidium bromide to visualize the
reaction products.
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RESULTS |
Retroviral vector transcription of EIAV gag genes in
transduced EK cells.
As an initial step to map CTL epitopes
from Gag proteins, retroviral vectors were constructed to express
segments of the gag gene encoding p15, the N-terminal half
of p26 (p26a), the C-terminal half of p26 (p26b), p11, and p9. To
verify transcription of the gene segments, total RNA was extracted from
each of the retroviral vector-transduced EK cells and used for RT-PCR.
The predicted size of cDNA was amplified from each RNA sample (data not
shown). No cDNA amplification was detected without RT or when RNA was extracted from EK cells transduced with the control retroviral vector,
vLXSN (data not shown). The DNA sequences of the RT-PCR products were
identical to the EIAVWSU5 gag gene
(32). These results indicated that the mRNAs for the five
gag gene segments were transcribed and had the correct RNA
sequence. The transduced EK cells were then used as targets in CTL assays.
Recognition of Gag proteins by CTLm from EIAV-infected
horses.
To determine which Gag proteins were recognized by
CTLm, EK cells transduced with the five retroviral vectors were used as CTL targets. PBMC from six long-term
EIAVWSU5-infected horses were used as sources of CTL
for mapping. These six horses were monitored by taking daily body
temperature and determining platelet counts, packed cell volumes, and
plasma viremia twice per week. Four horses (H507, H521, H529, and H540)
were considered inapparent carriers because they had no fever, anemia,
or thrombocytopenia and no detectable viremia during the 6-month period
when these experiments were done. H532 occasionally had
thrombocytopenia and low-level viremia detected by assay for
virus on EK cell cultures (38) but lacked fever. H513 had
two febrile episodes with associated viremia during the 6-month
period. EIAVWSU5-stimulated PBMC from all the horses
except H513 were previously reported to have CTLm activity against
target cells either infected with a recombinant vaccinia virus
expressing EIAV Gag/Pr (34) or transduced with a retroviral
vector expressing EIAV Gag/Pr (30). CTLm from four horses
(H507, H513, H521, and H529) recognized p15, CTLm from three horses (H513, H529, and H540) recognized p26a, and CTLm from four horses (H507, H521, H529, and H532) recognized p26b (Fig.
1). CTLm from H-513 were assayed with the
transduced target cells at an effector-to-target cell ratio of 20:1
(data not shown). None had CTLm recognizing p11 and p9, and none killed
transduced EK cells which were mismatched at the ELA-A locus and
expressing Gag protein segments. The results demonstrated that
ELA-A-restricted CTLm recognized epitopes in p15, p26a, and p26b
but not in p11 and p9. These CTL epitopes were then further mapped
by using overlapping peptides.
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FIG. 1.
Recognition of EIAV Gag proteins by CTLm from
EIAV-infected horses. Autologous and ELA-A-mismatched EK cells were
transduced with retroviral vectors expressing p15, p26a, p26b, p11, p9,
and the vector (vLXSN) expressing no EIAV proteins as a negative
control (NC). Effector-to-target cell ratios of 20:1, 10:1, and 5:1
were used in these assays. Vertical lines on columns are SEs, and
asterisks represent significant specific lysis  3 SE above the
negative control value.
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CTLm killing of EK target cells pulsed with synthetic peptides from
p15.
Ten peptides, 16 to 20 aa in length and overlapping by 8 aa,
were made from p15 and numbered 1 to 10 from N to C terminus (Table 2).
PBMC from the four horses with CTLm recognizing p15 (H513, H507, H521,
and H529) were stimulated with EIAVWSU5 and used in CTL
assays against EK targets pulsed with the p15 peptides (Fig.
2). The results indicated that at least
one p15 epitope was recognized by CTLm from H513 and H507. Because
the possibility of a single epitope in the overlapping region of
two positive adjacent peptides existed, at least two p15 epitopes
were recognized by CTLm from H521, and at least five epitopes were
recognized by CTLm from H529. Further, CTLm from H513, H507, and H529
recognized peptide 1, while CTLm from both H521 and H529 recognized
peptide 8.
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FIG. 2.
Recognition of overlapping p15 peptides by CTLm from
H507, H513, H521, and H529. Autologous EK target cells were pulsed with
p15 peptides 1 to 10 at a final concentration of 200 µg/ml, pulsed
with no peptide as a negative control (NC), or transduced with a
retroviral vector expressing p15. The effector-to-target cell ratio was
20:1. Vertical lines on columns are SEs, and asterisks represent
significant specific lysis 3 SE above the negative control value.
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CTLm killing of EK target cells pulsed with synthetic peptides from
p26a.
Nine peptides, 20 to 25 aa in length and overlapping by 8 aa, were made from p26a and numbered 11 to 19 from N to C terminus (Table 2). CTLm from H513, H529, and H540, which recognized retroviral vector-transduced targets expressing p26a, were used to identify p26a
peptides. CTLm from all three horses recognized targets pulsed with
peptide 18, while CTLm from H529 also recognized peptides 14, 16, and
17 (Fig. 3). Even though the killing of
target cells pulsed with peptides 14 and 16 was relatively low in the
assay presented in Fig. 3, in another assay, target cells pulsed with peptides 14, peptide 16, and no peptide had 29, 25, and 3% specific lysis, respectively. Differences in killing between assays may relate
to the effectiveness of the in vitro stimulation of the CTLm.
Therefore, at least one epitope in peptide 18 was recognized by
CTLm from H513, H529, and H540, and CTLm from H529 recognized at least
two additional epitopes.
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FIG. 3.
Recognition of overlapping p26a peptides by CTLm from
H513, H529, and H540. Autologous EK target cells were pulsed with p26a
peptides 11 to 19 at a final concentration of 200 µg/ml, pulsed with
no peptide as a negative control (NC), or transduced with retroviral
vector expressing p26a. The effector-to-target cell ratio was 20:1.
Vertical lines on columns are SEs, and asterisks represent significant
specific lysis 3 SE above the negative control value.
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CTLm killing of EK target cells pulsed with synthetic p26b
peptides.
Nine peptides, 20 to 22 aa in length and overlapping by
8 aa, were made from p26b and numbered 20 to 28 from N to C terminus (Table 2). CTLm from H507, H521, and H532, which recognized p26b, were
used against EK target cells pulsed with these peptides. CTLm from H532
recognized peptide 28, CTLm from H507 recognized peptides 24, 25, and
28, and CTLm from H521 recognized peptides 23 and 24 (Fig.
4). Because peptides 23 and 24 overlapped
by 8 aa, it was possible that CTLm from H521 recognized an epitope located in the overlapping region of peptides 23 and 24. To test this
possibility, a nonamer peptide (sequence VDRLLSOIK) that covered the
overlapping region was made and used in CTLm assays. However, CTLm from
H521 did not recognize this nonamer peptide (data not shown).
These results indicated that at least two different epitopes were
recognized by CTLm from H507 and H521. Finally, CTLm from both H507 and
H532 recognized peptide 28, and CTLm from both H507 and H521
recognized peptide 24.
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FIG. 4.
Recognition of overlapping p26b peptides by CTLm from
H507, H521, and H532. Autologous EK cells were pulsed with p26b
peptides 20 to 28 at a final concentration of 200 µg/ml, pulsed with
no peptide as a negative control (NC), or transduced with a retroviral
vector expressing p26b. The effector-to-target cell ratio was 20:1.
Vertical lines on columns are SEs, and asterisks represent significant
specific lysis 3 SE above the negative control value.
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Fine mapping of two CTLm epitopes located within
p26.
Because CTLm from H529 recognized both p26 peptides 17 and
18, a nonamer peptide (18a; sequence HPLPNAPLV) covering the
overlapping region was made. This nonamer peptide was recognized by
CTLm from H529 and by CTLm from H513 and H540, which also recognized
peptide 18 (Fig. 5). CTL assays using
PBMC from H513 stimulated with peptide 18a demonstrated that about 10 nM peptide 18a was required to sensitize EK cell targets for lysis.
Peptide 18a was recognized at 103- to
104-fold-lower concentrations than the 20-aa peptide 18 (Fig. 6A). To further determine if
peptide 18a was the optimal epitope, additional 8- to 10-aa
peptides with deletion or addition of residues to peptide 18a were made
(Fig. 6B) and compared in CTL assays with CTLm from H513. Deletion of
the first-position amino acid H from 8-, 9-, and 10-aa peptides
resulted in no killing of pulsed target cells (Fig. 6B). This
observation and the finding that there was no killing with CTLm from
H513 when the C-terminal V was missing from peptide 17 (Fig. 3)
demonstrated that the first-position H and the ninth-position V were
critical residues for this CTL epitope. Further, a 10-aa peptide
(HPLPNAPLVA) required a 100-fold increase in concentration to be
recognized by CTLm, further indicating that peptide 18a was the optimal
epitope.
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FIG. 5.
Recognition of p26a nonamer peptide 18a by CTLm from
H513, H529, and H540. Autologous EK cells were pulsed with p26a
peptides 18 and 18a at a final concentration of 200 µg/ml or with no
peptide as a negative control (NC). The effector-to-target cell ratio
was 20:1. Vertical lines on columns are SEs, and asterisks represent
significant specific lysis 3 SE above the negative control value.
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FIG. 6.
Optimal peptide epitope recognized by CTLm from
H513. Effectors were peptide 18a-stimulated CTLm from H513, and the
effector-to-target cell ratio was 20:1. (A) Efficiencies of recognition
of peptide 18a (HPLPNAPLV) and peptide 18 (HPLPNAPLVAPPQGPIPMTA); (B)
comparison of peptide 18a ( ) with 8-aa, 9-aa, and 10-aa versions of
the peptide. ( , HPLPNAPLVA; *, PLPNAPLVAP;  , PLPNAPLVA;
 , LPNAPLVAP;  , PLPNAPLV; +, LPNAPLVA).
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Fine mapping of the p26b peptide 28 epitope recognized by CTLm from
H532 was also performed. Peptides 28a and 28b but not 28c were
recognized (Table 3, experiment 1).
Peptides 28a and 28b overlapped by 8 aa, and the lysis of target cells
pulsed with peptide 28b was significantly higher than the lysis of
cells pulsed with peptide 28a (Table 3, experiment 1). Therefore, a
nonamer peptide epitope located in the N-terminal end of peptide
28b was made and designated 28b-1 (sequence GTTKQKMML).
Peptides 28b and 28b-1 were equally killed by CTLm from H532 (Table 3,
experiment 2). In dose-response experiments, 1 nM peptide 28b-1 was
required to be recognized by CTLm from H532, and peptide 28b-1 was
recognized at 103- to 104-fold-lower
concentrations than the 20-aa peptide 28 (Fig.
7A). Additional 8- to 10-aa peptides with
deletion and addition of peptide 28b-1 residues were compared (Fig.
7B). Peptides with a deletion of the first-position G or ninth-position
L were less efficiently recognized by CTLm from H532, indicating that
these two residues were needed in the epitope (Fig. 7B). Moreover,
adding an I to the N terminus or an L to the C terminus of peptide
28b-1 did not increase the efficiency of target cell lysis by CTLm
(Fig. 7B). These results further confirmed that the nonamer peptide (GTTKQKMML) was the optimal epitope recognized by CTLm from H532.
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FIG. 7.
Optimal peptide epitope recognized by CTLm
from H532. Effectors were peptide 28b-1-stimulated CTLm from H532, and
the effector-to-target cell ratio was 20:1. (A) Efficiencies of
recognition of peptides 28b-1 (GTTKQKMML), 28 (KMYACRDIGTTKQKMMLLAKAL), and 28b (GTTKQKMMLLA); (B) comparison of
peptide 28b-1 ( ) with 8-, 9-, and 10-aa versions of the peptide
(, IGTTKQKMML; *, GTTKQKMMLL; , TTKQKMMLL; , IGTTKQKMM;
, GTTKQKMM; +, TTKQKMML).
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ELA-A restriction of the CTL responses to EIAV Gag
epitopes.
The ELA-A type restricting H540 CTLm killing of
target cells pulsed with p26a peptide 18a was evaluated in assays using
EK target cells that were half-matched or mismatched at the ELA-A locus
and autologous EK target cells (Fig. 8).
Two EK target cells that shared (half-matched) ELA-A5 were lysed by
CTLm from H540, while one EK target cell that shared ELA-A5 was not
lysed. This unexpected observation suggested that while the ELA-A5
molecules from these four horses were recognized by the same typing
serum (44), one was not recognized by the T-cell receptor
(TCR) on H540 CTLm. Similar subtypes of human HLA-A2 and Cw3 alleles
have been reported for CTL from HIV-1- and influenza A virus-infected patients (4, 29). Because CTLm from H513 and H529 also
recognized peptide 18a, ELA-A restriction of epitope 18a from H513
and H529 was also evaluated, and the results were identical to those
for H540 (data not shown). Therefore, it is likely that
epitope HPLPNAPLV was restricted by a subtype of ELA-A5
molecules designated ELA-A5.1 (CTL1). CTLm from H532 were tested
against different EK target cells pulsed with p26b peptide 28 (Fig.
9). The results provided preliminary data
that the p26b peptide 28 epitope recognized by CTL from H532 was
restricted by ELA-A9 molecules.
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FIG. 8.
ELA-A5 restriction of CTLm recognizing p26a nonamer
peptide 18a. Autologous or heterologous EK target cells with
half-matched or mismatched ELA-A alleles were pulsed with p26a
peptides 18 (pep 18) and 18a (pep 18a) or with no peptide as a negative
control (NC). The effector-to-target cell ratio was 20:1. Vertical
lines on columns are SEs, and asterisks represent significant specific
lysis 3 SE above the negative control value.
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FIG. 9.
ELA-A9 restriction of CTLm recognizing p26b peptide 28. Autologous EK target cells or heterologous EK target cells with
half-matched or mismatched ELA-A alleles were pulsed with p26b peptide
28 (pep 28) or with no peptide as a negative control (NC). The
effector-to-target cell ratio was 20:1. Vertical lines on columns are
SEs, and asterisks represent significant specific lysis 3 SE above
the negative control value.
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DISCUSSION |
Identifying EIAV CTL epitopes provides the detailed
information necessary to evaluate the role of CTL responses in
controlling infection and perhaps to induce CTL in horses. In this
study, several ELA-A class I-restricted EIAV Gag epitopes were
identified with CTLm from six long-term EIAV-infected horses. The CTL
epitope mapping was facilitated by using target cells transduced
with retroviral vectors expressing gag gene segments.
Retroviral vectors have several advantages for use in CTL studies. (i)
Gene expression and thus epitope presentation are relatively stable
in the transduced target cells (46). (ii) Unlike the case
for other viral vectors, retroviral vector-transduced cells
express only the target gene and a neomycin phosphoribosyltransferase
gene (50). (iii) Retroviral vectors do not have a cytopathic
effect on transduced cells (19). (iv) Proteins expressed by
retroviral vectors undergo endogenous pathway processing and
presentation, thereby facilitating recognition by CTL (20).
Retroviral vectors have been used extensively for gene therapy
(19) but have had limited use for mapping CTL epitopes and for induction of CTL responses in vitro (47) and in vivo (16). These reports are extended by the results presented in this paper which demonstrated the utility of transduced EK target cells
expressing EIAVWSU5 gag segments for preliminary
mapping of CTL epitopes.
The Gag protein epitopes recognized by CTL from four EIAV-infected
carrier horses and two other long-term EIAV-infected horse were all
located on the p15 and p26 proteins and included at least 12 different
epitopes. These results are consistent with the location of the Gag
epitopes identified by CTL from HIV-1-infected patients, which are
almost exclusively located in HIV-1 p17 and p24 proteins (14, 18,
36, 37, 49). Moreover, the majority of HIV-1 patients have CTL
recognizing Gag epitopes. For instance, the Gag epitopes are
the protein epitopes most frequently recognized by CTL from
HIV-1-infected nonprogressors, with CTL from 16 of 25 (64%)
nonprogressors recognize HIV-1 Gag epitopes, while CTL from 11 of
25 (44%) recognize Env epitopes, CTL from 9 of 25 (36%) recognize
RT, and CTL from 1 of 25 (4%) recognize Tat (28). Our
results demonstrating at least one EIAV Gag epitope identified by
CTL from each of the six horses indicate that Gag epitopes may be
recognized by CTL from most EIAV-infected horses.
Stimulation of MHC class I-restricted CTL responses to viral
epitopes is determined by several molecular interactions in
addition to the requirement for recognition by TCR (11).
These other interactions include cleavage of the viral proteins by
proteases and transport of the viral peptides into the endoplasmic
reticulum by transport-associated proteins (11). Among these
interactions, presentation of epitopes by MHC class I molecules are
critical (45) because the epitopes have to bind to MHC
class I molecules to be recognized by TCR on CTL for both stimulation
and killing (11). It has been shown that the number of viral
CTL epitopes is restricted in virus-infected animals with limited
MHC class I polymorphism (7, 13). In contrast, in most
outbred populations, MHC class I genes are very polymorphic, leading to
the diversity of CTL responses in such populations. For example, more
than 266 different human MHC class I alleles have been identified by
nucleotide sequence analysis (39), and diverse CTL responses
have been found in HIV-1-infected patients (37). Our results
agree with the findings for HIV-1-infected patients in that diverse CTL
responses were generated from the six infected horses with different
HLA-A types.
The results also indicated that the Gag protein nonamer epitopes
were presented by products of ELA-A alleles. One nonamer epitope
recognized by CTLm from three horses was probably presented by the
product of the ELA-A5.1 allele. Another epitope recognized by CTLm
from a different horse was likely ELA-A9 restricted. This is because
the restriction of CTL responses detected in PBMC from EIAV-infected or
equine herpesvirus-infected horses with defined ELA-A alleles has so
far been associated with the ELA-A alleles (1, 15, 34).
Moreover, in this study, no target cells mismatched at both ELA-A
alleles were killed by CTL from the infected horses. Nevertheless, the
possibility of restriction by a second ELA class I locus (B locus)
(3) or another undefined locus exists.
In comparing the CTL responses in the slow and rapid progressors of the
HIV-1-infected patients, the responses cannot be distinguished by the
frequency of the CTL (22), and so it is assumed that the
quality of the CTL responses may differ in these patients (14). If the majority of CTL recognize conserved
epitopes, it may be harder for viruses to escape the CTL responses.
The CTL epitopes on the HIV-1 Gag proteins are reported to be
highly conserved among HIV-1 isolates (18, 49), and CTL
responses to HIV-1 Gag epitopes are associated with decreased risk
of progression to AIDS (43). In EIAV infection, most
infected horses eventually control the disease and become lifelong
carriers (9). This outcome may occur because the majority of
carrier horses have CTL recognizing highly conserved epitopes. The
predicted amino acid sequences of EIAV matrix protein (p15) and capsid
protein (p26) are identical among the five EIAV strains examined
(21, 32, 40, 48). However, these strains are all derived
from the Wyoming wild-type strain and do not reflect the possible
diversity of other horse-passaged strains. Data from five of the horses in this study suggested that despite possible diversity of Gag epitopes, CTL to Gag epitopes could still be an important
immune response for maintaining the carrier state of long-term
EIAV-infected horses.
In conclusion, the use of retroviral vectors expressing segments of the
gag gene greatly facilitated the mapping of CTL epitopes by reducing the number of synthetic peptides needed and the number of
peptides evaluated for each horse. Results from the six horses in this
study indicated that Gag epitopes are consistently recognized by
CTL from long-term EIAV-infected horses. Even though peptides with a
minimum of 12 different epitopes were identified, none were
recognized by CTLm from all horses. The CTLm were ELA-A restricted, and
different peptides were identified by CTLm from horses expressing different ELA-A molecules. The nonamer peptides which were likely restricted by ELA-A5.1 or ELA-A9 molecules could be used to induce CTL
in horses expressing these molecules. However, a successful EIAV
vaccine for horse populations with polymorphic MHC class I molecules
will need to include a large number of CTL epitopes in order to
induce CTL in all individuals. The use of whole viral proteins
expressed by retroviral or other vectors may present the diversity of
epitopes needed to stimulate CTL in horse populations. Gag proteins
should be included in the proteins expressed by such vectors.
This work was supported in part by Public Health Service NIAID
grant AI-24291 and U.S. Department of Agriculture NRICGP grant 96-35204-3427.
We thank Wendy C. Brown, William C. Davis, and William P. Cheevers for many valuable discussions, and we thank Steve Leib, Eldon Libstaff, and Emma Karel for technical assistance.
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Abstract
Introduction
Present address: Arthropod-Borne Animal Diseases
Research Laboratory, USDA-ARS, University Station,
Laramie, WY 82071-3965.
