Human Class I Supertypes and CTL
Repertoires Extend to Chimpanzees
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J Immunol 1998; 161:4447-4455; ;
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References
Roberto Bertoni, Alessandro Sette, John Sidney, Luca G.
Guidotti, Max Shapiro, Robert Purcell and Francis V. Chisari
Human Class I Supertypes and CTL Repertoires Extend to
Chimpanzees1,2
Roberto Bertoni,* Alessandro Sette,† John Sidney,† Luca G. Guidotti,* Max Shapiro,‡
Robert Purcell,§ and Francis V. Chisari3*
P
atients withcute viral hepatitis have been shown to develop
a strong, polyclonal, multispecific CTL response to the
hepatitis B virus (HBV)4 envelope (1), capsid (2), and
polymerase (3) proteins, and this response has been shown to be
restricted by multiple alleles in the HLA-A2 (A2.1-2.7, Aw68.2),
HLA-A3 (A3, A11, A31, A33, A68.1), and HLA-B7 (B7, B35,
B51, B53, B54) supertypes (1, 4 – 6). In contrast, the T cell response to HBV is usually weak or undetectable in chronically infected patients (7), apparently contributing both to viral persistence
and chronic hepatitis. Further analysis of the cellular immunobiology of HBV infection has been hampered by the absence of
conventional tissue culture or small animal models. Despite the
existence of related hepadnaviruses that infect woodchucks (8) and
peking ducks (9), the outbred nature of those species and the minimal definition of their immune systems have to date precluded
meaningful immunologic analysis beyond the humoral Ab response in those species. HBV itself is infectious for chimpanzees
(10 –12), but, because of the scarcity and cost of these animals and
their outbred nature, they have not been used to analyze the T cell
response to HBV.
Experiments in HBV transgenic mice have enhanced our understanding of the host-virus relationship during HBV infection. For
*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
Received for publication March 11, 1998. Accepted for publication June 19, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by National Institutes of Health Grants R01-AI20001,
R01-AI40696, and R37-CA40489, and with federal funds from National Institute for
Allergy and Infectious Diseases, National Institutes of Health, under Contracts N01AI-45241 and N01-AI-05069.
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: [email protected]
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.
Copyright © 1998 by The American Association of Immunologists
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-g and TNF-a 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
0022-1767/98/$02.00
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Using an in vitro peptide stimulation strategy, two chimpanzees that were acutely infected by the hepatitis B virus (HBV) produced
peripheral blood CTL responses to several HBV-encoded epitopes that are known to be recognized by class I-restricted CTL in
acutely infected humans. One animal responded to three HBV peptides that, in humans, are restricted by HLA-A2; the other
animal responded to three peptides that are restricted by HLA-B35 and HLA-B51, members of the HLA-B7 supertype in man.
The peptides recognized by each chimp corresponded with the ability of its class I molecules to bind peptides containing the
HLA-A2 and HLA-B7 supermotifs. Similar, apparently class I-restricted CTL responses to some of these peptides were also
detected in occasional HBV-uninfected chimps. These results demonstrate that the CTL repertoire overlaps in humans and chimps
and that the HLA-A2 and HLA-B7 supertypes extend to the chimpanzee. Based on these results, the immunogenicity and efficacy
of vaccines designed to induce CTL responses to human HLA-restricted viral epitopes may be testable in chimpanzees. The
Journal of Immunology, 1998, 161: 4447– 4455.
4448
IDENTICAL EPITOPES RECOGNIZED BY HUMAN AND CHIMPANZEE CTLs
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.
Materials and Methods
Chimpanzees and transgenic mice
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 d virus were assayed
by solid-phase RIA (Havab and anti-D, 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).
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 mg/ml), and
HEPES (10 mM) containing 10% heat-inactivated human AB serum (complete medium), and plated in a 24-well plate at 4 3 106 cells/well. Synthetic peptides, or pools of five to six peptides, were added at 10 mg/ml to
each well, and rHBcAg (Biogen, Cambridge, MA) was added at 1 mg/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 (HoffmanLaRoche, 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
mM and labeled with 200 mCi 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 3 [(experimental release 2 spontaneous release)/(maximum
release 2 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
mg/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 mg/ml of human b2-microglobulin.
Following two washes in serum-free media, the cells were resuspended in
serum-free medium at 107 cells/ml, and supplemental b2-microglobulin (3
mg/ml) was added. Cells (2 3 106 in 200 ml) were used per data point.
Cells were either plated in 96-well tissue culture plates (Falcon 3077) or
distributed to 12 3 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 ml 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
F63 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 ml of
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Thirteen healthy young adult chimpanzees were used in this study. All
animals were seronegative for all HBV, HCV, hepatitis d 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 3 107 HBV genomes) and 1.3.46 (;7 3 107 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.
CTL induction protocol
The Journal of Immunology
4449
Table I. Identification of CTL epitopes recognized by HBV-infected chimpanzees a
% Specific Lysis
Peptide Pool
No. of
peptides
Ch. 1558
Ch. 1564
Peptide Sequence
HLA A2
HLA A3
HLA A3/B7
6
6
5
52
19
8
9
9
24
Mixture
Mixture
Mixture
Core 1
Core 2
Core 3
Core 4
HBs 5
HBs 6
PreS 7
PreS 8
PreS 9
PreS 10
4
4
4
4
6
6
7
6
7
6
13
16
4
13
6
4
0
0
0
2
21
4
15
1
21
58
0
17
17
3
Mixture
Mixture
Mixture
Mixture
Mixture
Mixture
Mixture
Mixture
Mixture
Mixture
3
67
40
0
18
0
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
0
54
40
7
4
0
39
0
0
5
3
1
0
61
FLPSDFFPSV
FLLTRILTI
WLSLLVPFV
GLSRYVARL
FLLSLGIHL
ALMPLYACI
LPSDFFPSV
IPIPSSWAF
TPARVTGGVF
NVSIPWTHK
KVGNFTGLY
LVVDFSQFSR
SPTSCPPTCPGYRWMCLRRF
FLFILLLCLIFLLVLLDYQG
LVLIDYQGMLPVCPL
GMLPVCPLIPGSSTTSTGP
TPAQGTSMYPSCCCTKPSDG
SCCCTKPSDGNCTCIPIPS
PPTCPGYRWM
CPGYRWMCL
Origin
HLA A2 Pool
HBs 6 Pool
Env 224-243
Env 224-243
a
PBMCs were stimulated for 2 to 3 wk with the indicated peptides prior to a 4-h 51Cr release assay. E:T cell ratio 50 –100:1
in all assays except for the individual pool HBs 6 peptides, where it was 20:1. Peptides capable of stimulating CTL activity in
chimp PBMCs are shown in bold. NT, not tested.
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).
Results
HBV transgenic mouse serum causes acute viral hepatitis in
chimpanzees
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 crossreactive 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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HLA A3/B7 Pool
Residues
Core 18-27
Env 183-191
Env 335-343
Pol 455-463
Pol 575-583
Pol 642-650
Core 19-27
Env 313-320
Pol 354-363
Pol 47-55
Pol 55-63
Pol 377-386
Env 224-243
Env 246-265
Env 258-272
Env 265-283
Env 289-308
Env 299-317
ENV 229-238
ENV 232-240
4450
IDENTICAL EPITOPES RECOGNIZED BY HUMAN AND CHIMPANZEE CTLs
Table II. HBV-specific CTL response in acutely infected chimpanzees
% Specific
CTL Epitopes
Protein
Env
Env
Pol
Env
Env
Pol
a
b
51
Cr Releasea by Bleed Dateb
Ch. 1558
Ch. 1564
Position
Sequence
Supermotif
1
2
3
4
5
183
335
575
232
313
354
FLLTRILTI
WLSLLVPFV
FLLSLGIHL
CPGYRWMCL
IPPSSWAF
TPARVTGGVF
A2
A2
A2
B7
B7
B7
NT
NT
NT
67
40
7
NT
NT
NT
59
1
18
65
2
NT
1
2
3
4
5
31
NT
NT
NT
54
0
29
23
NT
33
1
40
61
NT
38
Numbers reflect the results of a 4-h 51Cr release assay using EBV B cell targets at an E:T ratio of 50 –100:1. NT, not tested.
Approximately monthly intervals after HBV infection.
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
Measurement of peptide binding to live chimp cells
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 HLAA2, 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
FIGURE 1. A, Cytolytic activity of PBMCs from ch. 1558 against autologous 51Cr-labeled, BCLs pulsed with varying concentrations of env 183
peptide. PBMCs, stimulated for 4 wk in vitro with the corresponding peptide, were analyzed at an E:T ratio of 100. B, Ch. 1558 CTLs were preincubated with mAbs to mouse CD4 or CD8, or with an isotype-specific
control mAb for 1 h before the addition of 51Cr-labeled, peptide-pulsed (10
mM) autologous BCLs at an E:T ratio of 100.
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PBMCs obtained at later time points from each chimp were
stimulated for 3 wk with the individual peptides from the peptide
pools to which they had previously responded. As shown in Table
I, 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).
A representative CTL line, derived from ch. 1558 and specific
for the Env 183–191 peptide (Fig. 1), 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 doseresponse 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 mM, 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).
The Journal of Immunology
4451
the counts could also be immunoprecipitated with the anti-class II
Ab.
Specificity of chimp BCL-peptide interactions
FIGURE 2. A, Radiolabeled HLA class I ligands bind chimp-derived
EBV-transformed BCLs (f). In each case, binding was inhibitable by an
excess of unlabeled versions of the labeled peptide (e). 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 (V)
and 1564 (f) BCLs by an excess of unlabeled peptide. C, Counts bound to
ch. 1558 and ch. 1564 BCLs were immunoprecipitated, as described in
Materials and Methods, using either HLA-class I-specific mAbW6/32 (f),
or the HLA-class II-specific mAb LB3.1 (M). An HLA-A2 transgenic line
(.221 A2/Kb), which binds peptide 941.01, was used as a control.
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
Correlation between binding capacity in the live cell assay and
target cell recognition by specific CTL lines
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.
In a separate experiment, the avidity of the peptide-MHC interaction was measured for the four BCLs (1564, 1573, 1578, and
Downloaded from http://www.jimmunol.org/ by guest on July 12, 2017
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.
To define the amino acid residues required for peptide binding
to the chimp BCLs, we tested two panels of single amino acid
analogues of peptides 941.01 and 1021.05 for their ability to bind
to ch. 1558 and ch. 1564 BCLs, respectively. As shown in Figure
3A, 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.
4452
IDENTICAL EPITOPES RECOGNIZED BY HUMAN AND CHIMPANZEE CTLs
Table III. Binding of peptides bearing HLA supermotifs to class I MHC molecules expressed by chimp-derived EBV lines
Binding Capacity (IC50, nM)
Ch. 1564b
A*0201a
B*0702b
7.8
24
126
380
459
495
2412
2883
191
163
1750
–c
–
–
–
2450
5
2.5
20
9.8
79
10
7.7
4.5
–
–
ND
ND
ND
ND
ND
–
HIV nef 84
HBV Env 313
HBV Pol 354
HBV core 19
B35 Con2
HBV Pol 429
HBV Pol 640
HBV Pol 530
ENV 232
4875
–
–
–
–
7429
–
–
–
13
15
30
41
49
51
54
202
420
–
ND
ND
–
–
ND
ND
ND
ND
16
29
17
1774
46
56
306
56
821
A3 Con1
HBV Pol 47
HBV Pol 377
HBV core 141
HBV Pol 654
HBV Pol 520
HBV Pol 149
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1429
ND
–
ND
ND
–
ND
ND
ND
Sequence
A2
FLPSDYFPSV
FLPSDFFPSV
YLVAYQATV
FLLTRILTI
GLSRYVARL
ALMPLYACI
FLLSLGIHL
WLSLLVPFV
HBV
HBV
HCV
HBV
HBV
HBV
HBV
HBV
B7
FPVRPQVPL
IPIPSSWAF
TPARVTGGVF
LPSDFFPSV
FPFKYAAAF
HPAAMPHLL
YPALMPLYA
FPHCLAFSYM
CPGYRWMCL
A3
KVFPYALINK
NVSIPWTHK
LVVDFSQFSR
STLPETTVVRR
QAFTFSPTYK
SAICSVVRR
HTLWKAGILYK
a
b
c
Source
18-27 F6.Y
core 18
lorf
Env 183
Pol 455
Pol 642
Pol 575
Env 335
Tested with the HLA-A2 supermotif peptide 941.01 (sequence FLPSDYFPSV) as the radiolabeled probe. ND, not done.
Tested with the HLA-B7 supermotif peptide 1021.05 (sequence FPFKYAAAF) as the radiolabeled probe. ND, not done.
A dash indicates IC50 $ 35,000 nM.
1580) that displayed significant binding capacity for the HLA-B7
supertype peptide 1021.05. As shown in Table IV, 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.
CTL responses in uninfected chimpanzees
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.
In contrast, two of the HLA-A2 supertype peptides (Env 335
and Pol 575) were not recognized by any of the uninfected chimps
tested (Table V), 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
The results presented herein indicate that human HLA class I
supertypes extend to the chimpanzee, and consequently, that a
significant overlap in the CTL repertoire of humans and chimpanzees also exists. In the course of this study, it was further
demonstrated that the inducibility of the CTL response in
chimps corresponds with the ability of their class I molecules to
bind the corresponding peptides; that the same peptide residues
determine its ability to bind to human and chimp class I molecules; and that the peptide-binding affinities of human and
chimp alleles appear comparable.
The close similarity of the peptide-binding specificities of the
human HLA-A2 and HLA-B7 supertypes and the presumed class
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Ch. 1558a
Supermotif
The Journal of Immunology
4453
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– 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 unex-
FIGURE 4. Direct binding to BCLs derived from uninfected chimpanzees. Binding of the radiolabeled HLA-A2 supertype degenerate binder
941.01 (A), or the HLA-B7 supertype degenerate binder 1021.05 (B), is
shown. Assays were performed with (M) and without (m) excess of unlabeled signal peptide. Binding of 1021.05 and 941.01 to BCLs from ch.
1558 and ch. 1564 is shown for comparison purposes.
pected, 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
Table IV. Correlation between binding in the live cell binding assay
and target cell recognition
CTLa
Chimp LCL
ID No.
Live Cell Binding
with 1021.05
Avidity
(IC50, nM)
Pol 354
Env 232
1573
1578
1580
1564
5852
1581
5835
5867
1574
1530
5829
1558
1579
1
1
1
1
6
2
2
2
2
2
2
2
2
148
104
110
49
NDb
ND
ND
ND
ND
ND
ND
ND
ND
57
47
40
40
0
0
11
0
3
0
10
0
0
57
59
56
48
15
ND
ND
ND
17
14
0
0
0
a
Numbers reflect results of a 4-h 51Cr release assay using peptide-pulsed autologous EBV B cell targets at an E:T ratio of 50-100:1.
b
ND, not done.
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FIGURE 3. A, Binding capacity of ch. 1558 BCLs for a panel of single
substitution analogues of peptide 941.01. Binding is expressed as relative
to the binding of 941.01 (7.8 nM). B, Binding capacity of ch. 1564 BCLs
for a panel of single substitution analogues of peptide 1021.05. Binding is
expressed as relative to the binding of 1021.05 (49 nM).
4454
IDENTICAL EPITOPES RECOGNIZED BY HUMAN AND CHIMPANZEE CTLs
Table V. CTL responsiveness in HBV infected and uninfected chimpanzees
Peptide Specificity
HLA A2 supertype
Chimp
1
1
2
2
2
2
2
2
2
2
2
2
2
Env 183
Env 335
380
2883
40
NT
0
0
51
0
3
0
0
0
0
0
0
40
NT
0
0
0
0
0
NT
NT
7
NT
NT
NT
Pol 575
Env 232
Env 313
Binding Affinity (IC50, nM)a
2412
420
15
% Specific Lysisb
18
NT
NT
53
NT
NT
0
47
0
25
0
2
0
0
NT
50
NT
2
0
1
NT
0
NT
45
NT
8
NT
54
NT
26
0
0
0
42
NT
NT
NT
NT
NT
Pol 354
Supertype
Peptide
Binding
30
NT
40
NT
14
0
0
7
1
NT
NT
NT
NT
NT
A2
A2 1 B7
A2
A2 1 B7
2
A2 1 B7
2
A2 1 B7
2
A2
A2
(B7)
A2
a
Binding affinity for HLA A2 supertype peptides is that measured in the ch. 1558/941.01 live cell assay, and for HLA B7
supertype peptides that measured in the ch. 1564/1021.05 assay.
b
PBMCs were stimulated for 2 to 3 wk in vitro with the indicated peptides. Results represent the percent specific 51Cr release
from peptide-pulsed autologous BCLs at an E:T cell ratio of 50-100 in a 4-h 51Cr release assay. Positive CTL responses are
shown in bold.
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.
Acknowledgments
We thank Ms. Patricia Fowler for technical assistance and Ms. Jennifer
Newmann for manuscript preparation.
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