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.

Patients 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, 5, 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, 11, 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 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-γ and TNF-α 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.

Thirteen healthy young adult chimpanzees were used in this study. All animals were seronegative for all HBV, HCV, hepatitis δ 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 × 107 HBV genomes) and 1.3.46 (∼7 × 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.

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 δ virus were assayed by solid-phase RIA (Havab and anti-Δ, 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).

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 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 μg/ml), and HEPES (10 mM) containing 10% heat-inactivated human AB serum (complete medium), and plated in a 24-well plate at 4 × 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.

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).

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 × [(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.

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 β2-microglobulin. Following two washes in serum-free media, the cells were resuspended in serum-free medium at 107 cells/ml, and supplemental β2-microglobulin (3 μg/ml) was added. Cells (2 × 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 × 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).

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).

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).

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.

Table I.

Identification of CTL epitopes recognized by HBV-infected chimpanzeesa

Peptide PoolNo. of peptides% Specific LysisPeptide Sequence
Ch. 1558Ch. 1564
HLA A2 52 Mixture 
HLA A3 19 Mixture 
HLA A3/B7 24 Mixture 
Core 1 13 21 Mixture 
Core 2 16 Mixture 
Core 3 15 Mixture 
Core 4 13 Mixture 
HBs 5 21 Mixture 
HBs 6 58 Mixture 
PreS 7 Mixture 
PreS 8 17 Mixture 
PreS 9 17 Mixture 
PreS 10 Mixture 
Origin Residues    
HLA A2 Pool Core 18-27 NT FLPSDFFPSV 
 Env 183-191 67 NT FLLTRILTI 
 Env 335-343 40 NT WLSLLVPFV 
 Pol 455-463 NT GLSRYVARL 
 Pol 575-583 18 NT FLLSLGIHL 
 Pol 642-650 NT ALMPLYACI 
HLA A3/B7 Pool Core 19-27 NT LPSDFFPSV 
 Env 313-320 NT 54 IPIPSSWAF 
 Pol 354-363 NT 40 TPARVTGGVF 
 Pol 47-55 NT NVSIPWTHK 
 Pol 55-63 NT KVGNFTGLY 
 Pol 377-386 NT LVVDFSQFSR 
HBs 6 Pool Env 224-243 NT 39 SPTSCPPTCPGYRWMCLRRF 
 Env 246-265 NT FLFILLLCLIFLLVLLDYQG 
 Env 258-272 NT LVLIDYQGMLPVCPL 
 Env 265-283 NT GMLPVCPLIPGSSTTSTGP 
 Env 289-308 NT TPAQGTSMYPSCCCTKPSDG 
 Env 299-317 NT SCCCTKPSDGNCTCIPIPS 
Env 224-243 ENV 229-238 NT PPTCPGYRWM 
Env 224-243 ENV 232-240 NT 61 CPGYRWMCL 
Peptide PoolNo. of peptides% Specific LysisPeptide Sequence
Ch. 1558Ch. 1564
HLA A2 52 Mixture 
HLA A3 19 Mixture 
HLA A3/B7 24 Mixture 
Core 1 13 21 Mixture 
Core 2 16 Mixture 
Core 3 15 Mixture 
Core 4 13 Mixture 
HBs 5 21 Mixture 
HBs 6 58 Mixture 
PreS 7 Mixture 
PreS 8 17 Mixture 
PreS 9 17 Mixture 
PreS 10 Mixture 
Origin Residues    
HLA A2 Pool Core 18-27 NT FLPSDFFPSV 
 Env 183-191 67 NT FLLTRILTI 
 Env 335-343 40 NT WLSLLVPFV 
 Pol 455-463 NT GLSRYVARL 
 Pol 575-583 18 NT FLLSLGIHL 
 Pol 642-650 NT ALMPLYACI 
HLA A3/B7 Pool Core 19-27 NT LPSDFFPSV 
 Env 313-320 NT 54 IPIPSSWAF 
 Pol 354-363 NT 40 TPARVTGGVF 
 Pol 47-55 NT NVSIPWTHK 
 Pol 55-63 NT KVGNFTGLY 
 Pol 377-386 NT LVVDFSQFSR 
HBs 6 Pool Env 224-243 NT 39 SPTSCPPTCPGYRWMCLRRF 
 Env 246-265 NT FLFILLLCLIFLLVLLDYQG 
 Env 258-272 NT LVLIDYQGMLPVCPL 
 Env 265-283 NT GMLPVCPLIPGSSTTSTGP 
 Env 289-308 NT TPAQGTSMYPSCCCTKPSDG 
 Env 299-317 NT SCCCTKPSDGNCTCIPIPS 
Env 224-243 ENV 229-238 NT PPTCPGYRWM 
Env 224-243 ENV 232-240 NT 61 CPGYRWMCL 
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.

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).

Table II.

HBV-specific CTL response in acutely infected chimpanzees

% Specific 51Cr Releasea by Bleed Dateb
CTL EpitopesCh. 1558Ch. 1564
ProteinPositionSequenceSupermotif1234512345
Env 183 FLLTRILTI A2 NT 67 NT 59 65      
Env 335 WLSLLVPFV A2 NT 40 NT      
Pol 575 FLLSLGIHL A2 NT NT 18 NT      
Env 232 CPGYRWMCL B7      31 NT 29 33 61 
Env 313 IPPSSWAF B7      NT 54 23 NT 
Pol 354 TPARVTGGVF B7      NT NT 40 38 
% Specific 51Cr Releasea by Bleed Dateb
CTL EpitopesCh. 1558Ch. 1564
ProteinPositionSequenceSupermotif1234512345
Env 183 FLLTRILTI A2 NT 67 NT 59 65      
Env 335 WLSLLVPFV A2 NT 40 NT      
Pol 575 FLLSLGIHL A2 NT NT 18 NT      
Env 232 CPGYRWMCL B7      31 NT 29 33 61 
Env 313 IPPSSWAF B7      NT 54 23 NT 
Pol 354 TPARVTGGVF B7      NT NT 40 38 
a

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.

b

Approximately monthly intervals after HBV infection.

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 1,B, 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 1 A, 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).

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 μM) autologous BCLs at an E:T ratio of 100.

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 μM) autologous BCLs at an E:T ratio of 100.

Close modal

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 2,A, 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. 2,B). Immunoprecipitation experiments verified that most of the counts were associated with class I molecules (Fig. 2 C). 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.

FIGURE 2.

A, Radiolabeled HLA class I ligands bind chimp-derived EBV-transformed BCLs (▪). 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 (▪) 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 (▪), or the HLA-class II-specific mAb LB3.1 (□). An HLA-A2 transgenic line (.221 A2/Kb), which binds peptide 941.01, was used as a control.

FIGURE 2.

A, Radiolabeled HLA class I ligands bind chimp-derived EBV-transformed BCLs (▪). 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 (▪) 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 (▪), or the HLA-class II-specific mAb LB3.1 (□). An HLA-A2 transgenic line (.221 A2/Kb), which binds peptide 941.01, was used as a control.

Close modal

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.

Table III.

Binding of peptides bearing HLA supermotifs to class I MHC molecules expressed by chimp-derived EBV lines

SupermotifSequenceSourceBinding Capacity (IC50, nM)
Ch. 1558aCh. 1564bA*0201aB*0702b
A2 FLPSDYFPSV HBV 18-27 F6>Y 7.8 191 – 
 FLPSDFFPSV HBV core 18 24 163 2.5 – 
 YLVAYQATV HCV lorf 126 1750 20 ND 
 FLLTRILTI HBV Env 183 380 c 9.8 ND 
 GLSRYVARL HBV Pol 455 459 – 79 ND 
 ALMPLYACI HBV Pol 642 495 – 10 ND 
 FLLSLGIHL HBV Pol 575 2412 – 7.7 ND 
 WLSLLVPFV HBV Env 335 2883 2450 4.5 – 
B7 FPVRPQVPL HIV nef 84 4875 13 – 16 
 IPIPSSWAF HBV Env 313 – 15 ND 29 
 TPARVTGGVF HBV Pol 354 – 30 ND 17 
 LPSDFFPSV HBV core 19 – 41 – 1774 
 FPFKYAAAF B35 Con2 – 49 – 46 
 HPAAMPHLL HBV Pol 429 7429 51 ND 56 
 YPALMPLYA HBV Pol 640 – 54 ND 306 
 FPHCLAFSYM HBV Pol 530 – 202 ND 56 
 CPGYRWMCL ENV 232 – 420 ND 821 
A3 KVFPYALINK A3 Con1 – – – – 
 NVSIPWTHK HBV Pol 47 – – – ND 
 LVVDFSQFSR HBV Pol 377 – – – ND 
 STLPETTVVRR HBV core 141 – – – – 
 QAFTFSPTYK HBV Pol 654 – – – ND 
 SAICSVVRR HBV Pol 520 – – 1429 ND 
 HTLWKAGILYK HBV Pol 149 – – ND ND 
SupermotifSequenceSourceBinding Capacity (IC50, nM)
Ch. 1558aCh. 1564bA*0201aB*0702b
A2 FLPSDYFPSV HBV 18-27 F6>Y 7.8 191 – 
 FLPSDFFPSV HBV core 18 24 163 2.5 – 
 YLVAYQATV HCV lorf 126 1750 20 ND 
 FLLTRILTI HBV Env 183 380 c 9.8 ND 
 GLSRYVARL HBV Pol 455 459 – 79 ND 
 ALMPLYACI HBV Pol 642 495 – 10 ND 
 FLLSLGIHL HBV Pol 575 2412 – 7.7 ND 
 WLSLLVPFV HBV Env 335 2883 2450 4.5 – 
B7 FPVRPQVPL HIV nef 84 4875 13 – 16 
 IPIPSSWAF HBV Env 313 – 15 ND 29 
 TPARVTGGVF HBV Pol 354 – 30 ND 17 
 LPSDFFPSV HBV core 19 – 41 – 1774 
 FPFKYAAAF B35 Con2 – 49 – 46 
 HPAAMPHLL HBV Pol 429 7429 51 ND 56 
 YPALMPLYA HBV Pol 640 – 54 ND 306 
 FPHCLAFSYM HBV Pol 530 – 202 ND 56 
 CPGYRWMCL ENV 232 – 420 ND 821 
A3 KVFPYALINK A3 Con1 – – – – 
 NVSIPWTHK HBV Pol 47 – – – ND 
 LVVDFSQFSR HBV Pol 377 – – – ND 
 STLPETTVVRR HBV core 141 – – – – 
 QAFTFSPTYK HBV Pol 654 – – – ND 
 SAICSVVRR HBV Pol 520 – – 1429 ND 
 HTLWKAGILYK HBV Pol 149 – – ND ND 
a

Tested with the HLA-A2 supermotif peptide 941.01 (sequence FLPSDYFPSV) as the radiolabeled probe. ND, not done.

b

Tested with the HLA-B7 supermotif peptide 1021.05 (sequence FPFKYAAAF) as the radiolabeled probe. ND, not done.

c

A dash indicates IC50 ≥ 35,000 nM.

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 3,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 3 B, 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.

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).

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).

Close modal

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 4,A, 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. 4 B). 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.

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 (□) and without (▪) 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.

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 (□) and without (▪) 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.

Close modal

In a separate experiment, the avidity of the peptide-MHC interaction was measured for the four BCLs (1564, 1573, 1578, and 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.

Table IV.

Correlation between binding in the live cell binding assay and target cell recognition

Chimp LCL ID No.Live Cell Binding with 1021.05Avidity (IC50, nM)CTLa
Pol 354Env 232
1573 148 57 57 
1578 104 47 59 
1580 110 40 56 
1564 49 40 48 
5852 ± NDb 15 
1581 − ND ND 
5835 − ND 11 ND 
5867 − ND ND 
1574 − ND 17 
1530 − ND 14 
5829 − ND 10 
1558 − ND 
1579 − ND 
Chimp LCL ID No.Live Cell Binding with 1021.05Avidity (IC50, nM)CTLa
Pol 354Env 232
1573 148 57 57 
1578 104 47 59 
1580 110 40 56 
1564 49 40 48 
5852 ± NDb 15 
1581 − ND ND 
5835 − ND 11 ND 
5867 − ND ND 
1574 − ND 17 
1530 − ND 14 
5829 − ND 10 
1558 − ND 
1579 − ND 
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.

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.

Table V.

CTL responsiveness in HBV infected and uninfected chimpanzees

ChimpHBVPeptide SpecificitySupertype Peptide Binding
HLA A2 supertypeHLA B7 supertype
Env 183Env 335Pol 575Env 232Env 313Pol 354
 Binding Affinity (IC50, nM)a        
  380 2883 2412 420 15 30  
 % Specific Lysisb        
1558 40 40 18 NT NT NT A2 
1564 NT NT NT 53 54 40 A2 + B7 
1530 − NT NT NT NT A2 
1573 − 47 26 14 A2 + B7 
1574 − 51 25 − 
1578 − A2 + B7 
1579 − − 
1580 − NT NT 50 42 A2 + B7 
1581 − NT NT NT NT − 
5829 − NT NT A2 
5835 − NT NT NT NT A2 
5852 − NT NT 45 NT NT (B7) 
5867 − NT NT NT NT A2 
ChimpHBVPeptide SpecificitySupertype Peptide Binding
HLA A2 supertypeHLA B7 supertype
Env 183Env 335Pol 575Env 232Env 313Pol 354
 Binding Affinity (IC50, nM)a        
  380 2883 2412 420 15 30  
 % Specific Lysisb        
1558 40 40 18 NT NT NT A2 
1564 NT NT NT 53 54 40 A2 + B7 
1530 − NT NT NT NT A2 
1573 − 47 26 14 A2 + B7 
1574 − 51 25 − 
1578 − A2 + B7 
1579 − − 
1580 − NT NT 50 42 A2 + B7 
1581 − NT NT NT NT − 
5829 − NT NT A2 
5835 − NT NT NT NT A2 
5852 − NT NT 45 NT NT (B7) 
5867 − NT NT NT NT 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.

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.

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 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.

We thank Ms. Patricia Fowler for technical assistance and Ms. Jennifer Newmann for manuscript preparation.

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 N01-AI-45241 and N01-AI-05069.

2

This is manuscript number 11416-MEM from The Scripps Research Institute.

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.

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