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*
Scripps Research Institute, La Jolla, CA 92037;
Epimmune Corporation, San Diego, CA 92037;
Bioqual, Rockville, MD 20850; and
§
National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
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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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Experiments in HBV transgenic mice have enhanced our understanding of
the host-virus relationship during HBV infection. For example,
HBV-specific murine CTLs cause an acute necroinflammatory liver disease
when they are transferred into transgenic mice that express HBV Ags in
the liver (13, 14). In addition, Ag-specific CTLs can eliminate HBV
from the liver noncytopathically, by secreting IFN-
and TNF-
that
inhibit HBV gene expression and replication in the hepatocytes (15, 16). These results suggest that a strong intrahepatic immune response
to HBV can clear the infection both by killing infected hepatocytes and
by inactivating the virus in infected hepatocytes, i.e., curing them.
Alternatively, a weak immune response could contribute to viral
persistence by reducing the expression of viral Ags sufficiently for
infected cells to escape immune recognition.
Nonetheless, several important aspects of HBV immunobiology have not yet been defined, and the prospects of examining these remaining questions in infected patients or transgenic mice are quite remote. For example, the kinetics, quality, and vigor of the early immune response, especially the CTL response, soon after exposure to the virus are likely to determine the ultimate outcome of the infection. These parameters are not approachable in mice since they are not infectible; nor can they be studied in humans who acquire the infection several weeks or months before the onset of clinically apparent disease. For similar reasons, the extent to which viral escape from the CTL response contributes to the initiation of persistent infection cannot be studied in humans because the most effective selection events are likely to occur early in infection. For ethical reasons, the protective effect of vaccines designed to induce a CTL response in the absence of antiviral Ab, which is known to protect, cannot be examined in humans. Finally, the ability of therapeutic stimulation of the HBV-specific CTL response to terminate persistent HBV infection could be determined most effectively if a pertinent animal model were available.
The current study was undertaken to develop the technology needed to define the CTL response to HBV in acutely infected chimpanzees. Since HLA class I genes are conserved in higher primates (17) and since CTLs that recognize several HLA class I-restricted CTL epitopes have been identified in infected humans, we reasoned that HBV-infected chimpanzees might also respond to at least some of these epitopes. The current study demonstrates that the CTL repertoire overlaps in chimps and humans, and that the HLA-A2 and HLA-B7 supertypes extend to the chimpanzee. These results suggest that the CTL response to these and other predetermined HLA-restricted viral epitopes can be analyzed and manipulated in the chimpanzee.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thirteen healthy young adult chimpanzees were used in this
study. All animals were seronegative for all HBV, HCV, hepatitis 
virus, and HIV markers, and seropositive for IgG anti-hepatitis A
virus Abs. Two of these animals (ch. 1558 and ch. 1564) had been
recently inoculated i.v. with 0.5 ml of HBV DNA-positive serum from
transgenic mouse lineages 1.3.32 (
2 x 107 HBV
genomes) and 1.3.46 (7 x 107 HBV genomes) that
contain a terminally redundant copy of the complete HBV genome
(ayw subtype) and replicate the virus in their livers at
levels comparable with levels in infected livers of patients with
chronic hepatitis (18). The other 11 animals served as uninfected
controls. All chimpanzees were housed and analyzed at BioQual
Laboratories (Rockville, MD) under contract to National Institute of
Allergy and Infectious Diseases. All studies were approved by the
relevant Animal Care and Use Committees of National Institutes of
Health and of Bioqual Laboratories.
Serologic, biochemical, and histologic analysis
Blood was obtained from the two HBV-inoculated chimpanzees on a
weekly basis after inoculation. Serum was analyzed for HBsAg,
anti-HBs, anti-HBc, HBeAg, and anti-HBe by solid-phase RIA
(Ausria II for HBsAg, Ausab for anti-HBs, CorAb for anti-HBc,
and Abbott-HBe for HBeAg and anti-HBe; Abbott Laboratories, Abbott
Park, IL). HBsAg and anti-HBs levels were quantified by reference
to an internal standard provided by the manufacturer. Abs to hepatitis
A virus and hepatitis virus were assayed by solid-phase RIA (Havab
and anti-
, respectively; Abbott). Abs to HCV were assayed by
ELISA for anti-C100-3 Ag (Ortho Diagnostics, Raritan, NJ). Liver
function was evaluated by analysis of serum alanine aminotransferase
and isocitrate dehydrogenase activity, as previously described (19).
Serum DNase-resistant HBV DNA was measured by dot-blot analysis exactly
as described (18).
HBV peptides and rHBcAg
A panel of highly conserved HBV peptides (9–11 mers) that have been shown previously to be CTL epitopes restricted by HLA-A2, HLA-B7, and HLA-A3 supertype alleles in acutely infected patients (6) was either synthesized at Cytel (San Diego, CA), as previously described (20), or purchased from Chiron Mimotopes (Chiron, Clayton, Victoria, Australia) or from Research Genetics (Huntsville, AL). In addition, a group of longer peptides (10–27 mers) covering the entire HBV (ayw subtype) envelope (38 peptides) and nucleocapsid (16 peptides) proteins was purchased from Multiple Peptide Systems (La Jolla, CA). The nucleocapsid peptides have been previously described (2). The envelope peptides included the following residues, with the first amino acid of preS1 serving as residue 1 throughout: preS1–10, preS10–21, preS10–25, preS10–36, preS17–31, preS28–47, preS40–54, preS47–63, preS55–69, preS63–77, preS70–84, preS83–97, preS95–108, preS109–123, preS109–128, preS121–135, preS134–148, preS141–157, preS152–165, S164–183, S174–193, S184–203, S214–231, S224–243, S246–265, S258–272, S265–283, S289–308, S299–318, S308–327, S319–338, S329–348, S339–358, S349–368, S359–373, and S374–389. Lyophilized peptides were reconstituted at 20 mg/ml in DMSO (Malinckrodt, Paris, KY) and diluted to 1 mg/ml with RPMI 1640 medium (Life Technologies, Grand Island, NY). rHBcAg was obtained from bacterial extracts of Escherichia coli, as previously described (21). Peptides for these assays either were synthesized at Cytel according to standard t-BOC or F-MOC solid-phase synthesis methods (20) or were purchased from Chiron Mimotopes (San Diego, CA). The peptides synthesized at Cytel were reverse-phase HPLC purified to >95% homogeneity, and their composition was ascertained by amino acid analysis, sequencing, and/or mass-spectrometric analysis.
Recombinant vaccinia viruses
Recombinant vaccinia virus constructs that express the HBV major envelope protein (ayw subtype) were used to induce transient expression of endogenously processed HBV proteins in chimp EBV-B cell lines, as previously described (1). Wt-vaccinia virus was used as a control (22).
CTL induction protocol
Anticoagulated (ACD-A; Baxter Healthcare, Fenwal Division, Deerfield, IL) whole blood (30 ml) or leukopheresis products derived from 135 ml of blood from the infected animals and uninfected controls were transported from BioQual Laboratories to The Scripps Research Institute (La Jolla, CA) by overnight delivery. PBMC were separated on Ficoll-Histopaque density gradients (Sigma, St. Louis, MO), washed three times in PBS (Sigma), resuspended in RPMI 1640 (Life Technologies) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 µg/ml), and HEPES (10 mM) containing 10% heat-inactivated human AB serum (complete medium), and plated in a 24-well plate at 4 x 106 cells/well. Synthetic peptides, or pools of five to six peptides, were added at 10 µg/ml to each well, and rHBcAg (Biogen, Cambridge, MA) was added at 1 µg/ml to each well as a source of T cell help during the first week of stimulation. On days 3, 10, and 17, 1 ml of complete medium and IL-2 (Hoffman-LaRoche, Nutley, NJ) at 20 U/ml final concentration were added to each well. On days 7 and 14, the cultures were restimulated with peptide, rIL-2, and 106 irradiated (3000 rad) autologous feeder cells. The cultures were tested for cytotoxic activity on days 14 and 21. Duplicate assays yielding greater than 15% specific 51Cr release were considered positive.
EBV-transformed B cell lines
Before HBV infection, EBV-transformed B cell lines (BCL) were established from PBMCs and maintained in complete RPMI with 10% (v/v) heat-inactivated FCS (Life Technologies), as we have previously described for the establishment of human EBV BCLs (23).
Chromium release assay
The cytolytic activity of peptide-stimulated PBMCs was determined in a standard 6-h split-well 51Cr release assay using U-bottomed 96-well plates containing 5000 target cells/well at E:T ratios of 100:1, 50:1, and 25:1 on days 14 and 21, as previously described (6). Target cells (autologous or allogeneic BCLs) were incubated overnight with synthetic peptides at 10 µM and labeled with 200 µCi of 51Cr (ICN Biochemicals, Costa Mesa, CA) for 1 h, after which they were washed four times with HBSS (Life Technologies). Percentage of cytotoxicity was determined from the formula: 100 x [(experimental release - spontaneous release)/(maximum release - spontaneous release)]. Maximum release was determined by lysis of targets by detergent (2% Triton X-100; Sigma). Spontaneous release was <25% of maximum release in all experiments. In selected experiments, anti-human CD4 (Leu3a; clone SK3) and anti-human CD8 (Leu2a; clone SK1) mAbs (Becton Dickinson, San Jose, CA) were added to the effector cells for 1 h before and at the inception of the assay.
Cell-binding assay
Chimpanzee BCLs were maintained in RPMI 1640 media supplemented with glutamine (2 mM), penicillin-streptomycin (100 U/ml), geneticin (500 µg/ml), and 10% FCS. Cell-binding assays were performed essentially as previously described (24). Briefly, BCLs were washed twice with RPMI plus 5% FCS, then incubated overnight at 106 cells/ml at 26°C in RPMI plus 5% FCS in the presence of 3 µg/ml of human ß2-microglobulin. Following two washes in serum-free media, the cells were resuspended in serum-free medium at 107 cells/ml, and supplemental ß2-microglobulin (3 µg/ml) was added. Cells (2 x 106 in 200 µl) were used per data point. Cells were either plated in 96-well tissue culture plates (Falcon 3077) or distributed to 12 x 75 tubes (Falcon 2063), then incubated in the presence of 105 cpm of radiolabeled peptide (see below) and various concentrations of unlabeled competitor peptide at 20°C for 4 h. Following the incubation period, free and cell-bound peptides were separated by washing three times with serum-free media, then passed through a 175 µl FCS gradient in microcentrifuge tubes. Pelleted fractions were counted on a gamma scintillation counter.
In general, peptides were lyophilized, then dissolved in 100% DMSO at
4 to 20 mg/ml. Subsequent dilution of peptide stocks was done using
PBS. Peptides used as radiolabeled ligands were peptide 941.01
(HBc18–27 F6
Y; sequence FLPSDYFPSV) and peptide 1021.05
(B35CON2; sequence FPFKYAAAF). These peptides were radiolabeled with
125I, according to the chloramine T method (25).
Immunoprecipitation of MHC class I-peptide complexes
To assess whether radiolabeled peptides were specifically bound to MHC class I molecules, immunoprecipitation analysis was performed. Cell aliquots were incubated with radiolabeled peptides, as described above, and washed three times with PBS. Pelleted cells were lysed with 1 ml ice-cold PBS/1% Nonidet P-40 for 1 h at 4°C. Lysates were then centrifuged for 2 min at 10,000 rpm. Lysate aliquots were subsequently mixed with 50 µl of immunoabsorbent beads (protein A-Sepharose CL4B) coupled with specific mAbs, and then incubated for 1 h at 4°C. Beads were recovered by centrifugation and washed three times with PBS. The radioactivity contained in both the washed beads and the supernatants was then measured. The mAbs utilized (and their specificities) were: LB3.1 (anti-HLA-DR) (26), W6/32 (anti-HLA class I) (27), and B1.23.1 (anti-HLA-B, C) (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two HBV-seronegative chimpanzees (ch. 1558 and ch. 1564) were inoculated i.v. with serum derived from HBV transgenic mice that replicate the HBV genome (18). Both chimps developed typical cases of acute, self-limited HBV infection characterized by transient HBs and HBe antigenemia, biochemical and histologic evidence of viral hepatitis, Ab seroconversion, and clearance of viral Ags and HBV DNA. A detailed description of the virologic and pathologic characteristics of this infection, which proves that these transgenic mice actually produce infectious virus, will be reported separately (Guidotti et al., in preparation).
HBV-specific CTL responses in infected chimpanzees
Recent data indicate that different HLA types can be grouped into a relatively few functional supertypes, each defined on the basis of its main peptide-binding specificities (29). The overall frequency of a given supertype is conserved among very different ethnicities. Because of the close evolutionary relationship between humans and chimpanzees, we asked whether human HLA supertype cross-reactive peptides could bind to chimp class I molecules and be presented to chimp CTLs. Because CTL responses to these peptides are detected in acutely infected patients and not in uninfected controls, we began this study by analyzing the HBV peptide-specific CTL response in two acutely infected chimpanzees.
PBMCs from ch. 1558 and ch. 1564 were stimulated in vitro for 2 to 3 wk
with pools of synthetic HBV peptides (9–11 mers) known to be
recognized by CD8-positive CTLs from acutely infected patients in the
context of the HLA-A2, HLA-B7, and HLA-A3 supertype alleles (6).
Additionally, the PBMCs were also stimulated with pools of overlapping
15 to 20 mers covering the HBV envelope and nucleocapsid proteins. As
shown in Table I
, PBMCs obtained
from ch. 1558 displayed strong cytolytic activity (shown in bold)
against autologous BCLs pulsed with the HLA-A2 peptide pool, and lesser
responses to some of the core peptide pools. Similarly, PBMCs obtained
from ch. 1564 responded vigorously to the HLA-B7 peptide pool and to
the envelope peptide pool HBs-6. Lesser responses were also detected
against several other peptide pools as well.
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, PBMCs from ch. 1558
displayed CTL activity specific for Env 183–191 (FLLTRILTI), Env
335–343 (WLSLLVPFV), and Pol 575–583 (FLLSLGIHL), which have been
identified previously as HLA-A2 supertype-restricted CTL epitopes in
acutely infected patients (1, 3). In contrast, ch. 1564 responded to
Env 313–321 (IPIPSSWAF) and Pol 354–363 (TPARVTGGVF), which
have been identified previously as HLA-B7 supertype-restricted CTL
epitopes in acutely infected patients (1, 3, 6). In addition, ch. 1564
responded to Env 224–243 (SPTSCPPTCPGYRWMCLRRF), which contains six B7
supertype motifs (Env 229–238, 232–240, 228–235, 228–237, 228–238,
and 229–237), i.e., 8–11 mers bearing P in position 2 and hydrophobic
(L, I, V, M, or A) or aromatic residues (F, W, or Y) at the C terminus
(30, 31). By analyzing the ability of Env 224–243-specific CTL to kill
target cells pulsed with these peptides, we determined that Env
232–240 (CPGYRWMCL) is the minimal optimal epitope
recognized by these CTLs (Table I). These patterns of reactivity were
apparently stable, as they were observed for both chimps with PBMCs
obtained up to several months after resolution of infection and disease
(Table II).
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), was
chosen for further analysis. As shown in Figure 1B, the
cytolytic activity of this CTL line was blocked most efficiently by
anti-human CD8 mAbs, indicating that the cytolytic activity was
mediated principally by CD8-positive T cells. The relative affinity of
the Env 183–191-specific CTLs for their specific HLA-peptide complexes
was tested in a dose-response study using target cells that were pulsed
with varying amounts of the peptide. As shown in Figure 1A,
CTL lysis was not detectable below a peptide concentration of 0.1 µM,
suggesting moderate-to-low affinity. In keeping with this result, none
of the CTL lines established in this study were able to kill autologous
target cells infected with vaccinia viruses that expressed the
corresponding HBV proteins (not shown).
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Next, the capacity of ch. 1558 and ch. 1564 class I molecules to
bind human HLA-supermotif peptides was examined. To do so, we utilized
a live cell-binding assay, since we and others (24, 32, 33) have shown
that radiolabeled or biotinylated peptides can be utilized to detect
binding to peptide-receptive MHC molecules expressed by intact living
cells. In initial experiments, several HLA-A2, HLA-A3, or HLA-B7
supertype peptides (24, 30, 34) were tested for their capacity to bind
BCLs derived from ch. 1558 and ch. 1564. As shown in Figure 2A, prototype peptides 941.01
and 1021.05 that have been shown previously to bind the HLA-A2 and
HLA-B7 supertypes (20, 24, 30, 34) displayed significant binding to
BCLs derived from ch. 1558 and ch. 1564, respectively. The binding was
specific since it was inhibitable by excess unlabeled ligand, and it
displayed high affinity, with IC50 values in the 10 to 100
nM range (Fig. 2B). Immunoprecipitation experiments verified
that most of the counts were associated with class I molecules (Fig. 2C). Specifically, on average 47% of the 941.01 counts
bound to 1558, and 52% of the 1021.05 counts bound to 1564 were
immunoprecipitated by the anti-HLA class I Ab W6/32. For the sake
of comparison, it should be noted that in the case of binding of 941.01
to human A*0201 and A*0207 homozygous cell lines, 1000 to 2500 cpm were
bound, with IC50 values in the 1 to 25 nM range, and
50 to 90% of the bound radioactivity was immunoprecititated by
anti-class I Abs (24, 35). By contrast, only 17 and 10% of the
941.01 and 1021.05 counts, respectively, were immunoprecipitated by the
anti-HLA class II Ab LB3.1. Similarly, in the case of the control
.221 A2/Kb line, which lacks class II expression, 9% of the counts
could also be immunoprecipitated with the anti-class II Ab.
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To further define the specificity of the class I-peptide-binding
interactions, additional inhibition assays were performed. As shown in
Table III, binding of the prototype A2
supertype peptide 941.01 to ch. 1558 BCLs was inhibited by peptides
containing the A2 supermotif, but not by A3 and B7
supermotif-containing peptides. Conversely, binding of the B7 supertype
peptide 1021.05 to ch. 1564 BCLs was inhibited most efficiently by
peptides containing the B7 supermotif, although four of the A2
supertype peptides, two of which contain an internal B7 supermotif,
also inhibited the B7 supertype peptide-binding assay. Finally, none of
the seven control A3 supertype peptides tested inhibited the ch.
1564–1021.05-binding interaction (Table III). Table III also lists the
binding capacity of the same sets of peptides for the HLA molecules
A*0201 and B*0702, as measured in the molecular binding assay utilizing
purified class I molecules. These results suggest that a fundamental
similarity exists in the binding specificity of HLA-A2 and HLA-B7
supertype human class I molecules, and the class I molecules expressed
by ch. 1558 and ch. 1564, respectively.
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A, only substitutions at
positions 2 and the C terminus were able to reduce the binding of
peptide 941.01 to ch. 1558 BCLs more than 100-fold. In both positions,
only aliphatic or hydrophobic residues (L, V, T, and I in position 2,
and V, L, I, F, and T at the C terminus) were tolerated, exactly
mirroring the fine specificity of HLA-A*0201 molecules (20, 35, 36).
Similarly, as shown in Figure 3B, nonconservative
substitutions significantly reduced binding of the B7 supertype peptide
1021.05 to ch. 1564 BCLs only at position 2 and the C terminus. Further
analogue testing revealed that only V and P were allowed at position 2,
and hydrophobic or aromatic residues such as V, I, L, T, or F are
allowed at the C terminus of this peptide, similar to the reported fine
specificity of the HLA-B7 supertype (30, 31). These results illustrate
the close similarity of the peptide-binding specificities of the human
HLA-A2 and HLA-B7 supertypes and the presumed class I molecules
displayed by ch. 1558 and ch. 1564, respectively.
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The ability of the HLA-A2 and HLA-B7 supertype peptides to bind to
chimp class I molecules was examined in an additional 11 chimpanzees,
all of which were seronegative for current or previous exposure to HBV.
As shown in Figure 4A, the
HLA-A2 supertype peptide 941.01 was able to bind to BCLs derived from 7
of the uninfected animals (as well as ch. 1558 and ch. 1564), while
BCLs from 3 uninfected chimpanzees (as well as ch. 1564) were able to
bind the B7 supertype peptide 1021.05 (Fig. 4B). It is
noteworthy that peptide-binding specificity correlated with the genetic
relatedness of the chimpanzees. Specifically, ch. 1564, ch. 1573, and
ch. 1580 (group 1) shared the same sire and, except for 1573, the same
dam. Additionally, ch. 1530 and ch. 1581 (group 2) shared the same dam,
and ch. 1574 and ch. 1579 (group 3) shared the same sire. In all cases,
binding was inhibitable by excess unlabeled peptide. Thus, the class I
specificities responsible for the binding of these peptides are
relatively common in this group of chimpanzees.
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, a
relatively narrow range of avidity was measured, with all four assays
yielding IC50 values (for inhibition by the homologous
ligand) in the 50 to 150 nM range. Next, the ability of BCLs from all
13 chimpanzees to bind peptide 1021.05 was compared with their ability
to be lysed by Pol 354- and Env 232-specific CTLs from ch. 1564. As
shown in Table IV, high levels of killing were observed only in the
case of the autologous BCL from ch. 1564 and the three other BCLs
capable of binding the B7 supertype peptide. Low levels of killing were
observed with three BCLs (5852, 1574, and 1530) that displayed little
or no binding of the HLA-B7 supertype peptide 1021.05 (Fig. 4 and Table IV). Taken together, these data suggest that the class I specificities
of the lines binding 1021.05 are similar enough to allow binding of the
same B7 supertype peptides, and to allow for cross-reactive recognition
of specific peptide/MHC complexes. Furthermore, this broad specificity
appears to be relatively common in the chimpanzee population analyzed.
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Since BCLs from several of the uninfected chimps could present HBV
peptides Env 232 and Pol 354 to ch. 1564-derived CTLs, we asked
whether CTL responses to these and other HBV-derived peptides
recognized by the infected animals could be induced in vitro using
PBMCs derived from the uninfected animals. As shown in Table V, a primary in vitro CTL response to
env 232 was induced in 4 of the 10 uninfected animals tested
with that peptide, while CTL responses to Env 313 and Pol 354 were
induced in 2 and (perhaps) 1 of the 5 uninfected chimps tested,
respectively. Importantly, all of these animals were able to present
Env 232 and/or Pol 354 to ch. 1564 CTLs (Table IV) and, with the
exception of ch. 1574, BCLs from all of these animals were able to bind
the HLA-B7 supertype peptide 1021.05 (Fig. 4). Collectively, these
results suggest that the CTL responses to the B7 supertype peptides
detected in the HBV-infected ch. 1564 may have resulted at least in
part from in vitro priming of specific CTL precursors.
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),
despite the fact that BCLs from 7 of these animals were able to bind
the prototype HLA-A2 supertype peptide 941.01 (Table IV). One of the 11
uninfected chimps (ch. 1574), however, displayed a CTL response to a
single HLA-A2 supertype peptide (env 183). The basis for
this response is unclear at this time, especially since BCLs from this
animal did not bind the prototype HLA-A2 supertype peptide 941.01 (Fig. 4). The lack of any positive markers of HBV infection at the time of
analysis suggests that the response could have been due to in vitro
priming. Despite this single ambiguity, however, the current results
suggest that the HBV-specific CTL responses detected in the infected
ch. 1558 were probably primed in vivo. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The close similarity of the peptide-binding specificities of the human
HLA-A2 and HLA-B7 supertypes and the presumed class I molecules
displayed by ch. 1558 and ch. 1564, respectively, suggests that
orthologues of both of these HLA alleles are present in the chimpanzee.
This notion is compatible with the proposed close evolutionary
relationship between different human (HLA) and chimpanzee (Patr) class
I alleles (17). Genealogies of the A locus suggest that HLA and Patr
lineages predate speciation, and that Patr alleles are related to the
HLA-A1/A3/A11 family (37, 38, 39, 40, 41). In addition, at the structural level,
comparison of Patr and HLA residues believed to form the main
peptide-binding pockets (42, 43) suggests that several Patr and HLA
alleles may share overlapping peptide-binding repertoires. For example,
the B pocket motif shared by HLA alleles that bind peptides with
proline in position 2 (31) is present in at least three Patr alleles
(Patr-B*11–13). For this reason, and because some Patr-B alleles are
thought to be derived from an HLA-B*07-like ancestral gene (44), the
high frequency of B7-like binding specificities (Table IV, Fig. 4) and
CTL responses (Table V) in the current study was not completely
unexpected.
The results presented herein suggest that either a single B7 supertype
class I molecule is common in the chimpanzee population from which the
BCLs were derived, or that different molecules that share similar B7
supertype specificity may be expressed by these chimps, thereby
permitting promiscuous target recognition by peptide-specific CTL
lines. The single amino acid substitution analysis of HLA-B7 supertype
molecules (30) and ch. 1564’s class I molecules binding the B7
supertype peptide (Fig. 3) further demonstrate that binding motifs
associated with one or more of these functional HLA-B7 orthologues
expressed in the chimpanzee are virtually indistinguishable from those
associated with their human B7 counterparts.
In contrast, the HLA-A2-supermotif specificity of the CTL response in
ch. 1558 and one of the uninfected animals was unexpected, since no
Patr class I orthologue of the HLA-A2 family has been reported to date.
Nonetheless, the existence of an HLA-A2 orthologue is likely since
nonconservative substitutions at position 2 and the C terminus of an
HLA-A2 supertype peptide abrogated binding to ch. 1558 class I
molecules (Fig. 3), with a fine specificity pattern virtually
indistinguishable from various HLA molecules of the A2 supertype (35).
The current findings demonstrate the existence of and explain the functional basis for overlapping class I and CTL repertoires in humans and chimps (i.e., common HLA-binding specificities) previously reported by Kowalski et al. (45), who showed that chimp CTL lines expressing Patr-B*13 recognize a HCV epitope bearing the HLA-B7 supermotif, and who also identified a Patr-B*16-restricted CTL response to a previously defined HCV epitope that is restricted by HLA-B35 in chronically infected patients (46).
Additional studies are needed to identify the exact nature of the Patr
molecules associated with the A2- and B7-supertype-binding
specificities, and to examine whether any Patr class I molecules
associated with an A3 supertype binding specificity can also be
identified. Additional studies are also needed to determine whether the
CTL responses detected in the two infected chimps were induced in vivo
or in vitro, since 4 of 11 uninfected chimps responded to some of the
same peptides, especially those displaying the HLA-B7 supermotif (Table V). The apparent low affinity CTL recognition of these peptides by the
infected chimps (Fig. 1) could also reflect in vitro priming.
Nonetheless, the absence of a CTL response to Env 335, Pol 354, and Pol
575 in all of the uninfected chimps whose class I molecules were able
to bind these peptides argues that the CTL responses to these peptides
observed in the infected chimps were probably generated in vivo.
In conclusion, the current study suggests that shared A2 and B7 supertype specificities between humans and chimpanzees could be exploited to study many previously unapproachable aspects of the CTL response to viral infections, using CTL epitopes that have been defined previously in infected patients and are therefore relevant to man. For example, chimps can be selected for experimental viral infection according to the ability of their class I molecules to bind predetermined viral epitopes, permitting the precursor frequency of these specific CTLs to be correlated with the kinetics of viral spread, the emergence of escape mutants, and the outcome of the infection. Similarly, the protective and therapeutic effects of immunization strategies designed to elicit CTL responses to viral epitopes previously determined to be pertinent to human infection can be studied in this manner. The apparently high prevalence of HLA-A2-like and B7-like class I alleles in the chimp population included in the current study should greatly facilitate the selection of animals for these experiments.
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Acknowledgments |
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Footnotes |
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2 This is manuscript number 11416-MEM from The Scripps Research Institute.
3 Address correspondence and reprint requests to Dr. Francis V. Chisari, Department of Molecular and Experimental Medicine, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address:
4 Abbreviations used in this paper: HBV, hepatitis B virus; BCL, B cell line; HBc, hepatitis B core Ag; HBe, hepatitis Be Ag; HBs, hepatitis B surface Ag; HCV, hepatitis C virus.
Received for publication March 11, 1998. Accepted for publication June 19, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 K. J. Lavender and K. P. Kane Cross-Species Dependence of Ly49 Recognition on the Supertype Defining B-Pocket of a Class I MHC Molecule J. Immunol., December 15, 2006; 177(12): 8578 - 8586. [Abstract] [Full Text] [PDF]
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 S. Zhu, K. Udaka, J. Sidney, A. Sette, K. F. Aoki-Kinoshita, and H. Mamitsuka Improving MHC binding peptide prediction by incorporating binding data of auxiliary MHC molecules Bioinformatics, July 1, 2006; 22(13): 1648 - 1655. [Abstract] [Full Text] [PDF]
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J. T. Loffredo, J. Sidney, S. Piaskowski, A. Szymanski, J. Furlott, R. Rudersdorf, J. Reed, B. Peters, H. D. Hickman-Miller, W. Bardet, et al. The High Frequency Indian Rhesus Macaque MHC Class I Molecule, Mamu-B*01, Does Not Appear to Be Involved in CD8+ T Lymphocyte Responses to SIVmac239 J. Immunol., November 1, 2005; 175(9): 5986 - 5997. [Abstract] [Full Text] [PDF]
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 S. F. Wieland and F. V. Chisari Stealth and Cunning: Hepatitis B and Hepatitis C Viruses J. Virol., August 1, 2005; 79(15): 9369 - 9380. [Full Text] [PDF]
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 F. E. Tynan, N. A. Borg, J. J. Miles, T. Beddoe, D. El-Hassen, S. L. Silins, W. J. M. van Zuylen, A. W. Purcell, L. Kjer-Nielsen, J. McCluskey, et al. High Resolution Structures of Highly Bulged Viral Epitopes Bound to Major Histocompatibility Complex Class I: IMPLICATIONS FOR T-CELL RECEPTOR ENGAGEMENT AND T-CELL IMMUNODOMINANCE J. Biol. Chem., June 24, 2005; 280(25): 23900 - 23909. [Abstract] [Full Text] [PDF]
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K. J. Lavender, B. J. Ma, E. T. Silver, and K. P. Kane The Rat RT1-A1c MHC Molecule Is a Xenogeneic Ligand Recognized by the Mouse Activating Ly-49W and Inhibitory Ly-49G Receptors J. Immunol., March 15, 2004; 172(6): 3518 - 3526. [Abstract] [Full Text] [PDF]
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N. H. Shoukry, J. Sidney, A. Sette, and C. M. Walker Conserved Hierarchy of Helper T Cell Responses in a Chimpanzee during Primary and Secondary Hepatitis C Virus Infections J. Immunol., January 1, 2004; 172(1): 483 - 492. [Abstract] [Full Text] [PDF]
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J. Sidney, S. Southwood, V. Pasquetto, and A. Sette Simultaneous Prediction of Binding Capacity for Multiple Molecules of the HLA B44 Supertype J. Immunol., December 1, 2003; 171(11): 5964 - 5974. [Abstract] [Full Text] [PDF]
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D. Meyer-Olson, K. W. Brady, J. T. Blackard, T. M. Allen, S. Islam, N. H. Shoukry, K. Hartman, C. M. Walker, and S. A. Kalams Analysis of the TCR {beta} Variable Gene Repertoire in Chimpanzees: Identification of Functional Homologs to Human Pseudogenes J. Immunol., April 15, 2003; 170(8): 4161 - 4169. [Abstract] [Full Text] [PDF]
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R. Thimme, S. Wieland, C. Steiger, J. Ghrayeb, K. A. Reimann, R. H. Purcell, and F. V. Chisari CD8+ T Cells Mediate Viral Clearance and Disease Pathogenesis during Acute Hepatitis B Virus Infection J. Virol., December 6, 2002; 77(1): 68 - 76. [Abstract] [Full Text] [PDF]
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 P. L. Yang, A. Althage, J. Chung, and F. V. Chisari Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection PNAS, October 15, 2002; 99(21): 13825 - 13830. [Abstract] [Full Text] [PDF]
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N. G. de Groot, N. Otting, G. G. M. Doxiadis, S. S. Balla-Jhagjhoorsingh, J. L. Heeney, J. J. van Rood, P. Gagneux, and R. E. Bontrop Evidence for an ancient selective sweep in the MHC class I gene repertoire of chimpanzees PNAS, September 3, 2002; 99(18): 11748 - 11753. [Abstract] [Full Text] [PDF]
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P. Riedl, S. El Kholy, J. Reimann, and R. Schirmbeck Priming Biologically Active Antibody Responses Against an Isolated, Conformational Viral Epitope by DNA Vaccination J. Immunol., August 1, 2002; 169(3): 1251 - 1260. [Abstract] [Full Text] [PDF]
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E. Mizukoshi, M. Nascimbeni, J. B. Blaustein, K. Mihalik, C. M. Rice, T. J. Liang, S. M. Feinstone, and B. Rehermann Molecular and Immunological Significance of Chimpanzee Major Histocompatibility Complex Haplotypes for Hepatitis C Virus Immune Response and Vaccination Studies J. Virol., May 13, 2002; 76(12): 6093 - 6103. [Abstract] [Full Text] [PDF]
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J. L. Dzuris, J. Sidney, H. Horton, R. Correa, D. Carter, R. W. Chesnut, D. I. Watkins, and A. Sette Molecular Determinants of Peptide Binding to Two Common Rhesus Macaque Major Histocompatibility Complex Class II Molecules J. Virol., November 15, 2001; 75(22): 10958 - 10968. [Abstract] [Full Text] [PDF]
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D. M. McKinney, A. L. Erickson, C. M. Walker, R. Thimme, F. V. Chisari, J. Sidney, and A. Sette Identification of Five Different Patr Class I Molecules That Bind HLA Supertype Peptides and Definition of Their Peptide Binding Motifs J. Immunol., October 15, 2000; 165(8): 4414 - 4422. [Abstract] [Full Text] [PDF]
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 F. V. Chisari Viruses, Immunity, and Cancer: Lessons from Hepatitis B Am. J. Pathol., April 1, 2000; 156(4): 1117 - 1132. [Full Text] [PDF]
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S. Santra, P. N. Fultz, and N. L. Letvin Virus-Specific Cytotoxic T Lymphocytes in Human Immunodeficiency Virus Type 1-Infected Chimpanzees J. Virol., August 1, 1999; 73(8): 7065 - 7069. [Abstract] [Full Text]
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 L. G. Guidotti, R. Rochford, J. Chung, M. Shapiro, R. Purcell, and F. V. Chisari Viral Clearance Without Destruction of Infected Cells During Acute HBV Infection Science, April 30, 1999; 284(5415): 825 - 829. [Abstract] [Full Text]
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). In each case, binding was inhibitable by an
excess of unlabeled versions of the labeled peptide (
). Peptide
941.01 (sequence FLPSDYFPSV) was used as the radiolabled ligand with
ch. 1558 BCLs, and peptide 1021.05 (sequence FPFKYAAAF) for ch. 1564
BCLs. B, Inhibition of binding of radiolabeled ligands
to the ch. 1558 (
) and 1564 (
