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Division of Immunology, New England Regional Primate Research Center, Harvard Medical School, Southborough, MA 01772;
Infectious Disease Unit and Partners AIDS Research Center, Massachusetts General Hospital, Charlestown, MA 02129;
Therion Biologics Corp., Cambridge, MA 02142; and
Divisions of Research Resources and Microbiology and Immunology, Yerkes Regional Primate Research Center, Emory University, Atlanta, GA 30322
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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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One of the remarkable features of SIV infection in naturally infected sooty mangabeys is the absence of immunodeficiency and the maintenance of a lifelong asymptomatic state in the face of high level SIV viremia. Plasma SIV viral RNA levels in naturally infected sooty mangabeys approximate those detected in macaques with end-stage AIDS and range between 105-107 copies/ml (6) (R. Grant et al., unpublished observations). Although SIV-specific humoral and proliferative responses and CD8+ noncytolytic suppressor activity have been described in vitro in naturally infected sooty mangabeys, their role in maintaining asymptomatic SIV infection is not known (7, 8, 9, 10, 11).
CD8+ CTL are an important component of the host immune response against many viral infections, and there is strong evidence for their role in the control of viral replication (12, 13, 14, 15, 16). We recently reported on SIV-specific CTL responses in asymptomatic SIV-infected sooty mangabeys (17). Depending on the type of SIV infection, two patterns of CTL activity and viral load are seen. In SIVmac239-infected sooty mangabeys, there is an inverse relationship between viral load and CTL activity during acute and chronic infection (17). The low plateau viremia levels of <1000 SIV RNA copies/ml plasma in SIVmac239-infected mangabeys are probably maintained by a combination of suboptimal SIV replication and strong CD8+ CTL activity (17). The significance of CTL in naturally infected sooty mangabeys is much less evident. Plasma SIV viremia ranges from 105–107 RNA copies/ml, and there is no correlation between viral load and bulk CTL activity (17) (A. Kaur et al., unpublished observations). Further, viral persistence in naturally infected mangabeys is not associated with CTL exhaustion, since memory CTL, although not effector CTL, are detected by chromium release assays (17) (A. Kaur et al., unpublished observations). Whether CTL have any role in suppressing viral replication in naturally infected highly viremic sooty mangabeys or whether they contribute to maintaining asymptomatic SIV infection is not known. The presence of CTL escape and whether it is a mechanism for persistent viremia in naturally infected sooty mangabeys also remain to be determined.
To better address what role CTL have during SIV infection in sooty mangabeys, we have conducted a detailed analysis of the fine specificity of the SIV-specific CTL response in sooty mangabeys. CTL clones were isolated from naturally infected and SIVmac239-infected sooty mangabeys, and their epitopes were characterized. Immunodominant CTL epitopes within Gag, Nef, and Env were identified in two naturally infected and three SIVmac239-infected sooty mangabeys. The identification of CTL epitopes in SIV-infected sooty mangabeys provides a tool to directly address the relationship between evolution of SIV and CTL responses in a nonhuman primate model of persistent asymptomatic lentivirus infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sooty mangabeys were housed at the Yerkes Regional Primate Research Center (Atlanta, GA) and maintained in accordance with federal guidelines (Guide for the Care and Use of Laboratory Animals, Department of Health and Human Services Publication (NIH) 85-23, revised 1985). Five sooty mangabeys naturally infected with SIVsmm and three sooty mangabeys experimentally inoculated with SIVmac239 were studied.
Blood for CTL assays was collected in heparinized CPT Vacutainer tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ), spun at 1500 x g for 20 min at room temperature to separate the PBMC from erythrocytes and granulocytes over a Ficoll gradient, and then shipped overnight at room temperature to the New England Regional Primate Research Center.
Recombinant vaccinia vectors
The following recombinant vaccinia viruses were used for the detection of bulk CTL activity: rVV-239, expressing SIVmac239 env (provided by Mark Mulligan, University of Alabama, Birmingham, AL); vAbT388, expressing SIVmac251 gag-pol and SIVmac239 env (Therion Biologics, Cambridge, MA); vAbT306, expressing SIVmac239 nef (Therion Biologics); vAbT252, expressing SIVmac251 gag-protease (Therion Biologics); and vAbT258, expressing SIVmac251 pol (Therion Biologics). Recombinant vaccinia viruses expressing SIVsmH4 gag-pol (vSmH4 gag-pol) or env (vSmH4 env) were provided by Philip R. Johnson, Ohio State University (Columbus, OH). SIVsmH4 is a molecular clone of SIV derived from a macaque that developed AIDS after being experimentally inoculated with SIV from an asymptomatic sooty mangabey (4).
In addition, recombinant vaccinia viruses expressing truncated Env of SIVmac239 were used to map Env-specific CTL clones. Sequences containing a portion of the SIVmac239 genome were obtained from Ronald Desrosiers (New England Regional Primate Research Center). The env gene was cloned and truncated using in vitro mutagenesis, restriction endonuclease digestion, or exonuclease III (Exo III) digestion. Recombinant vaccinia viruses were generated from a derivative of the New York City Board of Health vaccinia strain using a host range selection system (18, 19). Each env gene is under the control of the vaccinia early/late 40K promoter (20). Recombinant vaccinia virus vT60 contains a 2583 bp env gene that lacks the 5' signal sequence. Other recombinant vaccinia viruses contain env genes with intact 5' sequences and a series of truncations at the 3' end; stop codons were added to each truncated gene to terminate translation. The recombinant virus designation, the size of its truncated SIVmac239 env gene, and the enzyme used to generate the 3' truncation are as follows: vT51, 1470 bp (ClaI); vT53, 570 bp (PstI); vT54, 219 bp (HindIII); vT55, 2121 bp (Exo III); vT56, 888 bp (Exo III); vT58, 1281 bp (Exo III); and vT59, 1908 bp (Exo III).
Generation of B lymphoblastoid cell lines
Transformed B cell lines (B-LCL),3 for use as MHC-matched stimulator and target cells in CTL assays, were established for each animal. B cells were transformed by incubating PBMC at 37°C in a 5% CO2 incubator with herpesvirus papio derived from the supernatant of S594 cells (provided by Norman Letvin, Beth Israel Hospital, Boston, MA) and propagated in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 20% FBS (Sigma, St. Louis, MO), 10 mM HEPES (Life Technologies), 2 mM L-glutamine (Life Technologies), 50 IU of penicillin (Life Technologies)/ml, and 50 µg of streptomycin (Life Technologies)/ml.
Detection of bulk SIV-specific CTL activity
CTL activity was measured as previously described (17). Briefly, PBMC shipped overnight in CPT Vacutainer tubes were suspended at 2 x 106 cells/ml in RPMI 1640 medium supplemented with 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 50 IU of penicillin/ml, and 50 µg of streptomycin/ml (R-10 medium). Autologous B-LCL infected with recombinant vaccinia vectors vAbT388, vAbT306, vSmH4 gag-pol, or vSmH4 env were used as stimulator cells for Ag-specific stimulation. After 16–18 h of infection, virus was inactivated with long wave (400 nm) UV irradiation (Fisher model UV 350, Fisher Scientific, Pittsburgh, PA) in the presence of 10 µg/ml psoralen (furo(3,2-g)coumarin; P8399, Sigma). Cells were UV-irradiated at a distance of 3.5 cm from the light source, washed three times, and used as stimulators. The PBMC were mixed with stimulators in R-10 at a concentration of 2–3 x 106/ml at a responder to stimulator ratio of 10:1 and incubated at 37°C in a 5% CO2 incubator. Cells were half-fed with R-10 medium twice a week and recombinant human IL-2 (donated by M. Gately, Hoffmann-La Roche, Nutley, NJ; 10 IU/ml) was added to the feeding medium after 4–5 days. The CTL assays were performed after 12–14 days of Ag-specific stimulation.
Chromium release assay
Target cells consisted of autologous or allogeneic B-LCL infected with individual recombinant vaccinia viruses expressing SIV proteins. Recombinant vaccinia viruses used to infect target cells included the control vaccinia virus NYCBH, vAbT252, vAbT258, rVV-239, vSmH4gag-pol, vSmH4env, and vAbT306. Target cells were infected overnight at a multiplicity of infection of 5–10 PFU/cell and then labeled with 50 µCi 51Cr (DuPont-NEN, Wilmington, DE)/106 cells. Target cells (104 cells/well) were dispensed in duplicate for each E:T cell ratio into 96-well U-bottom plates (Costar, Cambridge, MA). Cold target inhibition was used in all assays to decrease background lysis. Cold targets consisted of unlabeled autologous B-LCL that had been infected with the control vaccinia virus NYCBH and were used at a cold/hot target ratio of 15:1. Chromium release was assayed after 5-h incubation at 37°C in a 5% CO2 incubator. Plates were spun at 1000 rpm for 10 min at 4°C, after which 30 µl of supernatant was harvested from each well onto wells of a LumaPlate-96 (Packard) and allowed to dry overnight. Emitted radioactivity was measured in a 1450 MicroBeta Plus Liquid Scintillation Counter (Wallac, Turku, Finland). Spontaneous release was measured from wells containing only target cells and medium. Maximum release was measured from wells containing target cells and 0.1% Triton X-100 (Sigma). The percent specific cytotoxicity was calculated as follows: ((test release - spontaneous release)/(maximum release - spontaneous release)) x 100%. Spontaneous release of target cells was <25% in all assays.
Generation of SIV-specific CTL clones
After 10–14 days of in vitro Ag-specific stimulation, bulk CTL were enriched for CD8+ T lymphocytes using magnetic beads coated with anti-CD4 Ab (CD4 Dynabeads, Dynal, Oslo, Norway) to remove CD4+ T lymphocytes. CD8+ T lymphocytes were plated in replicate wells at cell concentrations of 10, 3, and 1 cell/well into 96-well U-bottom plates in the presence of irradiated (100 Gy) autologous B-LCL (0.2 x 105 cells/well), irradiated (30 Gy) human PBMC (1 x 105 cells/well), Con A (5 µg/ml; Sigma), and IL-2 (50 IU/ml) and incubated at 37°C in a 5% CO2 incubator. The Con A was removed after 96 h and replaced with fresh R-10 medium and IL-2 (50 IU/ml), and subsequent media were exchanged twice a week. Wells with cell outgrowth were tested for CTL activity against autologous B-LCL infected with recombinant vaccinia SIV vectors. Wells with SIV-specific CTL activity were expanded into 48 or 24 wells in the presence of irradiated autologous B-LCL, irradiated human feeder PBMC, Con A, and IL-2 and maintained in culture by restimulation once every 2 wk.
Simian immunodeficiency virus peptides
SIV peptides were obtained from three sources. A panel of 201 screening peptides (25 aa long and overlapping by 8 aa) using the SIVmac251 sequence were provided by Norman Letvin (Harvard Medical School, Boston, MA). Additional overlapping peptides using the SIVmac239 sequence (gp120 and the first 60 aa of Nef) were synthesized at Quality Controlled Biochemicals (Hopkinton, MA). Peptides were synthesized by the method of Houghten et al. using t-butoxycarbonyl NH2-protected amino acids and 4-methylbenzhydramine resin (21). Peptides were cleaved from the resin with anhydrous hydrogen fluoride, washed with ether, extracted with 10% acetic acid, and evaluated for purity by reverse phase analytical HPLC. For fine mapping of CTL epitopes, short overlapping peptides, 8–15 aa long, were synthesized as free acids using F-moc-protected amino acids at Massachusetts General Hospital (Charlestown, MA) (22).
All peptides were reconstituted at 2 mg/ml in sterile distilled water with 10% DMSO (Sigma). A reducing agent (1 mM DTT) was added to peptides containing C, M, or W residues.
Peptide titration
Ten-fold dilutions of peptide with concentrations ranging between 100 µg/ml and 1 pg/ml were made in PBS with 2% FBS. Fifty microliters of peptide at each concentration was incubated in duplicate with 50 µl of labeled autologous B-LCL (2 x 105 cells/ml) for 1 h at 37°C in a 5% CO2 incubator. One hundred microliters of CTL clone was then added at an E:T cell ratio of 5 or 10:1, and CTL activity was measured in a 4-h chromium release assay.
Flow cytometric analysis
Three-color flow cytometry was used for immunophenotyping of CTL clones. The Ab and fluorochrome combinations used were anti-rhesus CD3 mAb conjugated to PE, anti-human CD4 FITC, and anti-human CD8 mAb conjugated to peridinin chlorophyl protein. Samples were analyzed on a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA). Antibodies other than anti-CD3 were obtained from Becton Dickinson Immunocytometry Systems and were mAbs of anti-human specificity that cross-react with rhesus Ags of the same specificity. Rhesus anti-CD3 (6G12) was provided by Johnson Wong, Massachusetts General Hospital (23).
Surface staining of PBMC was conducted using standard procedures. Briefly, 0.5–1 x 106 PBMC were washed with PBS containing 2% FBS, incubated with the conjugated Abs for 0.5 h at 4°C, and washed again. Stained cells were fixed in 2% paraformaldehyde and analyzed on a Becton Dickinson Immunocytometry Systems FACScan.
Isoelectric focusing gel electrophoresis
A modified one-dimensional isoelectric focusing technique (24, 25) was used for the characterization of class I MHC alleles in cultured B-LCL. Cultured cells (3 x 106) were incubated in methionine-free medium for 30 min and subsequently labeled with [35S]methionine for 6 h. Labeled cells were washed, lysed, and immunoprecipitated with monoclonal mouse anti-human MHC class I Ab W6/32 (Dako A/S, Copenhagen, Denmark) bound to protein A-Sepharose beads (Sigma). The labeled lysates were run on a 0.75-mm polyacrylamide gel (Sigma), containing 9 M urea, 1.6% (pH 5.0–7.0) ampholine, 0.4% (pH 3.5–10.0) ampholine, and 0.16% (pH 7.0–9.0) ampholine (Pharmacia Biotech, Piscataway, NJ). Fixed and dried gels were visualized by autoradiography.
Limiting dilution assays for determination of CTL precursor frequency assays
To quantitate CTL responses, we used a limiting dilution precursor frequency assay previously described for the detection of SIV-specific CTL (26). PBMC were seeded at 1,000–50,000 cells/well in 24 replicate wells of 96-well microtiter plates in the presence of 100 µl of R-10 medium supplemented with IL-2 to a final concentration of 100 IU/ml. To each well were added 5 x 104 irradiated (30 Gy) autologous PBMC, with 5 x 104 autologous B-LCL infected with the recombinant vaccinia vAbt388 expressing the SIV gag, pol, and env genes. Before use, vaccinia-infected B-LCL were inactivated using psoralen/UV light as described previously. On day 14, wells were split and tested for SIV-specific CTL activity using as targets autologous 51Cr-labeled B-LCL infected with recombinant vaccinia viruses expressing SIV genes as well as a control vaccinia virus. In addition, autologous B-LCL pulsed with peptide were used as target cells to measure the precursor frequency of epitope-specific CTL. The fraction of nonresponding wells was calculated for each dilution using a split-well analysis. Wells for which lysis was 7% above the background were scored as positive. Precursor cell frequency using split-well analysis was calculated by the maximum likelihood method using software developed by S. A. Kalams (Massachusetts General Hospital).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To facilitate characterization of CTL epitopes, we first
established T cell clones from sooty mangabeys experimentally infected
with SIVmac239 or naturally infected with SIVsmm. Clones were
established by limiting dilution cultures of CD8+
T lymphocytes fractionated from bulk CTL effectors generated by in
vitro SIV-specific stimulation and then screened for SIV-specific CTL
activity. In one naturally infected sooty mangabey (FDh), CTL clones
were isolated after peptide stimulation (Table I
). SIV-specific CTL clones could be
isolated from all three SIVmac239-infected sooty mangabeys but from
only two of the five sooty mangabeys naturally infected with SIVsmm
(Table I).
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). The low yield of
SIV-specific CTL clones in sooty mangabeys naturally infected with SIV
was partly a reflection of absent or low level SIV-specific CTL
activity, as bulk stimulated CTL assays in two animals (FBo and FWk)
were repeatedly negative (Table I and data not shown). Similar results
were obtained when vaccinia vectors expressing SIV proteins of
SIVmac239/251 or SIVsmH4 were used for stimulation or infection of
target cells (data not shown). However, in two other naturally infected
sooty mangabeys (FCp and FNg), a broad SIV-specific CTL response was
readily detected in bulk assays (Table I). The low yield of
SIV-specific CTL clones was not due to poor cloning efficiency, as
11–46% of seeded wells had sufficient cell growth to enable testing
in CTL assays (Table I). The negative wells either had no demonstrable
SIV-specific lytic activity even when recombinants expressing SIVsmH4
proteins (Gag, Pol, or Env) were used, or they showed high levels of
background lysis against control target cells infected with wild-type
vaccinia. Such clones were phenotypically CD3+
CD8+ and did not show classical NK activity, in
that they did not lyse K562 cells or allogeneic target cells (data not
shown). The specificity of these clones was not further
characterized.
In one naturally SIV-infected sooty mangabey, FDh, multiple clones
recognizing a single CTL epitope were isolated following
peptide-specific stimulation (Table I). During experiments testing for
lysis of HLA-mismatched target cells by CTL clones, it was found that
the Gag- and Env-specific CTL clones isolated from a SIVmac239-infected
mangabey, FLg, recognized cognate Gag or Env peptides presented on
target cell lines derived from FDh (Fig. 1a). This suggested the
presence of shared MHC class I alleles between FLg and FDh. When PBMC
from mangabey FDh were stimulated in vitro with cognate peptides
recognized by Gag- and Env-specific CTL clones of mangabey FLg, an
abundant yield of CTL clones recognizing the Env, but not the Gag,
epitope were obtained from FDh (Table I).
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). In
contrast, in the naturally infected sooty mangabeys and in one
SIVmac239-infected mangabey (FWl), clones specific for only a single
SIV protein were isolated (Table I). Seven CTL epitopes were
characterized in five SIV-infected sooty mangabeys (Table II). Three of these seven epitopes were
relatively immunodominant, accounting for 71–100% of all Env- or
Nef-specific clones isolated from a given animal (Table II). CTL
epitopes were not defined for Gag- and Pol-specific CTL clones isolated
from the SIVmac239-infected sooty mangabey FYg or the Nef- and
Pol-specific CTL clones from FLg (Table I).
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Previously we have shown that bulk SIV-specific CTL activity in
sooty mangabeys is MHC restricted and is mediated by
CD8+ T lymphocytes (17). Consistent
with this observation, SIV-specific CTL clones were
CD3+ CD8+ (data not shown).
The CTL activity mediated by CTL clones was MHC restricted, as shown by
lysis of autologous, but not most allogeneic, target cells loaded with
the cognate peptide (Fig. 1a). Both Env- and Gag-specific
CTL clones from mangabey FLg and Env-specific clones from mangabey FDh
were able to lyse each other’s target cells sensitized with the
cognate peptide (Fig. 1a and data not shown). By testing
against a panel of target cell lines, the MHC allele presenting the Env
and Gag epitope was detected in 2 of 12 (17%) unrelated sooty
mangabeys housed at the Yerkes Primate Research Center. Similarly,
Nef-specific CTL clones from FYg and FWl were able to recognize cognate
peptide presented on FNg and on each other’s target cells, but not on
cells from another mangabey FDh (data not shown). This suggested the
presence of shared MHC class I alleles between mangabeys FLg and FDh
and among mangabeys FYg, FNg, and FWl. Immunoprecipitation of MHC class
I alleles by the anti-MHC class I mAb W6/32 followed by IEF gel
electrophoresis revealed multiple common bands between these animals
(Fig. 1b), consistent with the presence of shared MHC class
I alleles.
Characterization of Gag-specific CTL clones in SIV-infected sooty mangabeys
The specificity of CTL clones directed against Gag or Nef was defined using target cells sensitized with screening peptides spanning the specific protein. After identification of a 20- to 25-aa region, further epitope mapping was performed using sequentially shorter peptides until a minimal 9- to 11-aa epitope was defined. Finally, peptide titration assays were performed to determine the optimal epitope.
Gag-specific CTL clones were isolated from two SIVmac239-infected
mangabeys (FYg and FLg; Table I). One CTL epitope in Gag accounted for
roughly 20% of Gag-specific CTL clones isolated from mangabey FLg
(Table II). The fine specificity of the remaining 80% of Gag-specific
clones from FLg and of all Gag-specific clones isolated from another
mangabey FYg could not be determined, since surviving clones did not
recognize the available SIV peptides. The sequence of SIV Gag peptides
was derived from SIVmac251, while the two mangabeys, FLg and FYg, were
infected with SIVmac239. Even though SIVmac251 has 98% amino acid
identity to SIVmac239 in Gag (27), SIVmac239 differs from
SIVmac251 in having 5 aa changes and 4 extra aa at the C-terminus
(28). Since a single amino acid change is sufficient to
abolish recognition (29), we may have failed to identify
epitopes located in regions with sequence diversity from the infecting
virus.
A single Gag-specific CTL epitope in p26 was identified in one
sooty mangabey, FLg, 6 wk following SIVmac239 infection (Table II). The
epitope mapping is shown in Fig. 2.
Despite testing a number of truncated peptides, the lowest sensitizing
dose of any peptide required for 50% maximal lysis
(SD50) was between 1–10 µg/ml (Fig. 2, a and b). The 10- or 11-aa epitope in p26,
HQAAMQIIRD(I), is highly conserved among SIV isolates from macaques and
sooty mangabeys (HQAAMQIIRDI in SIVmac239 and SIVmac251 and
HQAAMQIIREI in SIVsm isolates, H4, H9, and PBj; underline
refers to variant sequence) (28).
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In the instance of Env-specific CTL clones, epitope specificity
was first narrowed to roughly 100 aa using a panel of recombinant
vaccinia vectors expressing truncated Env proteins of SIVmac239. The
minimal 9- to 11-aa epitope was defined using overlapping peptides
spanning the identified region and the optimal epitope determined by
peptide titration assays (Fig. 3).
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). One of
ten Env-specific CTL clones isolated from the SIVmac239-infected sooty
mangabey FYg, recognized a 25-aa gp120 peptide located within and
outside the V3 cysteine loop (aa 339–363; Table II). The minimal
epitope for this clone was not identified, since it lost CTL activity
before further testing. This region is conserved between SIVmac239 and
SIVmac251, but differs among SIV isolates of sooty mangabeys
(28). The other CTL epitope recognized by the majority of
Env-specific CTL clones from two mangabeys, the SIVmac239-infected
mangabey FLg and the naturally infected mangabey FDh, was located just
beyond the V4 cysteine loop in gp120 (aa 428–437; Table II). This
region also is highly conserved among all SIV isolates and is in
alignment with the CD4 binding domain of HIV-1 (30, 31).
Even though mangabeys FLg and FDh shared MHC class I alleles and
recognized CTL epitopes in the same region of gp120, the optimal
epitope and fine specificity of the CTL clones were different in both
animals (Fig. 3, b–d). The CTL clones from FDh derived by
stimulation with the 12-aa Env peptide NYVPCHIRQIIN (peptide 121B),
which was well recognized by CTL clones from FLg, yielded Env-specific
CTL clones that, surprisingly, did not lyse target cells loaded with
the stimulating peptide 121B, but could lyse peptides 121H (NYVPCHIRQI)
and 121E (YVPCHIRQI; Fig. 3b). This suggested that the
C-terminal residues isoleucine and asparagine in the stimulating
peptide, although inhibiting target cell recognition, were still able
to stimulate CTL clones from FDh. The optimal epitope for FDh was
peptide 121H (NYVPCHIRQI) and peptide-sensitized target cells were
lysed at a SD50 of <0.1 µg/ml (Fig. 3d). On the other hand, the optimal epitope for the majority
of Env-specific CTL clones from the mangabey FLg, was peptide 121E
(YVPCHIRQI) and was recognized at a SD50 as low
as 0.001 µg/ml (Fig. 3c).
Although 29 of 29 CTL clones isolated from the naturally infected mangabey FDh recognized peptides 121E (YVPCHIRQI) and 121H (NYVPCHIRQI) to a comparable extent, they displayed a heterogeneous killing pattern for target cells sensitized with related peptides 121B (NYVPCHIRQIIN), 121F (VPCHIRQII), and 121G (PCHIRQIIN; data not shown). This indicated that even though CTL activity in this animal was directed toward a single dominant epitope, the clonal response was not monoclonal, but consisted of CTL clones with a diverse T cell repertoire.
Characterization of Nef-specific CTL clones in SIV-infected sooty mangabeys
Nef-specific CTL clones were isolated from three sooty mangabeys
(Table II), and remarkably, in all three animals, the cognate epitopes
mapped to the same region of SIV Nef (between aa 20–32). This epitope
accounted for the specificity of all or most of the Nef-specific
CTL clones isolated from two mangabeys, FNg and FWl (Table II). This
region of Nef is highly conserved among reported SIV isolates:
LRARGETYGR in SIVmac239 and SIVmac251 and
LQARGETYGR in SIVsm H4, H9, and PBj (28).
Nef-specific CTL clones from FYg and FWl were tested against a panel of
allogeneic target cells for their ability to induce lysis in a
MHC-restricted manner. The CTL clones from FYg were able to lyse
peptide-loaded target cells derived from FWl and FNg, while clones from
FWl recognized peptide-loaded target cells derived from FYg and FNg
(data not shown). This suggested that the Nef epitopes were presented
by the same class I MHC allele. Isoelectric focusing of cell lysates
from FNg and FYg immunoprecipitated with the anti-HLA class I Ab
W6/32 confirmed the presence of several shared MHC class I alleles
(Fig. 1b). The observation that the Nef-specific CTL
response in three sooty mangabeys with shared MHC class I alleles was
directed toward the same region of Nef suggests a high degree of
epitope focusing of the SIV CTL response in sooty mangabeys.
The optimal Nef epitope was defined by peptide titration assays in two
SIVmac239-infected mangabeys (FYg and FWl), and the sensitizing dose
for 50% maximal lysis ranged between 10–100 ng/ml (Table II and Fig. 4). Even though Nef-specific CTL clones
from the two SIVmac239-infected mangabeys mapped to the same region,
the pattern of recognition of related Nef peptides and the fine
specificity of clone recognition were different (Fig. 4), again
suggesting differences in the TCR repertoire of CTL clones generated in
the two mangabeys.
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To determine whether the characterized CTL epitopes were
immunodominant, bulk CTL generated by in vitro stimulation with
recombinant vaccinia expressing whole SIV proteins were tested in CTL
assays against target cells expressing whole SIV protein and target
cells pulsed with the optimal peptide (Fig. 5). These experiments showed that many of
the mapped CTL clones recognized epitopes that were immunodominant, in
that lysis of target cells pulsed with the peptide accounted for most
or all of the lysis of the intact protein. Surprisingly, even in the
instance of the naturally infected sooty mangabey FDh, despite the
selection bias during CTL cloning by peptide stimulation, the epitope
identified appears to be immunodominant (Fig. 5).
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). In all three mangabeys, the
identified CTL epitope appeared to be immunodominant, as
epitope-specific CTLp were greater or equal to CTLp specific for the
whole protein (Table III). Remarkably, the frequency of gp120-specific
CTLp in the SIVmac239-infected mangabey FLg and that in the naturally
infected mangabey FDh were comparable despite a 3-log difference in SIV
viral loads between these animals (Table III).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The detection of a limited repertoire of CTL epitopes that were similar
in SIV-infected mangabeys with shared MHC class I alleles was
surprising, given the differences in infecting SIV strain and viral
load between natural SIVsmm and experimental SIVmac239 infection.
Although the regions encoding Gag and Pol are relatively highly
conserved (
90% amino acid identity) between isolates of SIVsmm and
SIVmac, there is only a 77–81% amino acid identity seen in Env and
Nef (4). A high degree of focusing in epitope choice and a
remarkably conserved TCR usage is also seen in acute and persistent EBV
infection, so that only a limited number of allele-specific epitopes
elicit CTL responses (32, 33). This is in contrast to the
heterogeneous and broad gag-specific CTL response in HIV-1 infection,
where, using unstimulated PBMC, CTL clones targeting multiple epitopes
restricted by different MHC class I alleles were identified in Gag p24
in individual subjects (34). We isolated CTL clones only
after a period of in vitro stimulation, and this could have
artificially narrowed the detected CTL repertoire.
The extent to which CTL control in vivo SIV replication in naturally infected sooty mangabeys, albeit ineffectively, is not known. Viral persistence in naturally infected sooty mangabeys is not associated with CTL exhaustion, as memory CTL, although not effector CTL, are detected in functional assays (A. Kaur et al., unpublished observations). Interestingly, the frequency of CTL precursors in two MHC-matched sooty mangabeys, one naturally infected (FDh) and one SIVmac239 infected (FLg), targeting the same epitope in gp120, was comparable despite a 2- to 3-log difference in SIV viral load between the two animals. This paradoxical observation suggests that the differences in viral load between the low viremia (SIVmac239-infected) and high viremia (natural infection) models of SIV infection in sooty mangabeys are either due to qualitative differences in the rate at which CTL proliferate after encountering an infected cell (35) or to quantitative differences in the ratio of effector to memory CTL. A similar phenomenon has been observed in HTLV-1 infection, where symptomatic infection is associated with 1- to 2-log higher viral loads than asymptomatic infection, and yet there are no differences in the magnitude of CTL responses between asymptomatic and symptomatic individuals (36). Discordance between quantitative CTL responses and virus load have also been reported in viral hepatitis (37). It is possible that other factors, such as decreased TCR affinity due to antigenic variation of the virus in vivo or absence of TCR activation due to emergence of antagonistic variants, may be contributing to low CTL responsiveness in the naturally infected sooty mangabey.
Consistent with the observation of decreased CTL responses in natural SIVsmm vs experimental SIVmac239 infection (17) (A. Kaur, et al., unpublished observations), the yield of SIV-specific CTL clones in naturally infected mangabeys using nonpeptide methods of stimulation, was as low as 0–2.7% despite a good cloning efficiency. This may be due to two reasons. One possibility is that in naturally infected mangabeys, SIV-specific effector CTL are either absent or do not sufficiently expand from memory CTL after in vitro stimulation or are present but lack effector function. A second possibility is that a CTL response directed toward epitopes in SIV quasispecies may have been missed, since the recombinant vaccinia vectors and synthetic peptides used for screening CTL clones were derived from SIVmac239 or SIVmac251. This seems unlikely, since testing with another divergent SIV strain, SIVsmH4, did not increase the yield of SIV-specific CTL clones. SIVsmH4 is a molecular clone of SIV derived from a macaque experimentally inoculated with SIV from an asymptomatic sooty mangabey and is relatively divergent from both SIVmac239 and SIVmac251 (4).
The failure to detect SIV-specific CTL clones in three of five naturally infected sooty mangabeys and identification of only a single CTL epitope in the other two naturally infected mangabeys could also be due to the presence in vivo of multiple virus variants generated partly by CTL escape. Emergence of naturally occurring variants of CTL epitopes that are TCR antagonists is seen in HIV and HBV infection (38, 39) and may be yet another mechanism of immune escape relevant to viral persistence in naturally infected sooty mangabeys. Recently, Zajac et al. reported a novel mechanism of CTL escape whereby loss of recognition of a CTL epitope in persistent LCMV infection was associated with persistence of specific CD8+ T cells that had lost effector activity (40). Which, if any, of these mechanisms are operative in sooty mangabeys naturally infected with SIV is not yet known. The identification of specific CTL epitopes and their restricting MHC alleles in SIV-infected sooty mangabeys will enable the use of tetramers in future studies to more definitively examine the in vivo role of CTL in sooty mangabeys. Longitudinal studies on the frequency of nonsynonymous to synonymous mutations in the infecting virus strains and mutations resulting in loss of recognition of specific CTL epitopes will also help address whether CTL escape is operative or necessary for viral persistence in naturally SIV-infected sooty mangabeys.
The knowledge of specific CTL epitopes in SIV-infected sooty mangabeys, particularly in the setting of a high and a low viremia model of asymptomatic SIV infection, provides a unique opportunity to study the relationship among CTL, viral load, and pathogenicity in SIV infection. Even though asymptomatic infection in the face of high viral loads, as seen in naturally acquired SIV-infected sooty mangabeys, is not the paradigm for protection from AIDS in HIV infection, an understanding of mechanisms leading to such an equilibrium is likely to enhance our understanding of AIDS pathogenesis and provide alternate therapeutic approaches. In conclusion, the characterization of SIV-specific CTL epitopes in sooty mangabeys will help us to better understand and address issues of CTL escape, breadth of TCR usage, and how they relate to whether persistent SIV infection remains asymptomatic or results in AIDS.
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2 Address correspondence and reprint requests to Dr Amitinder Kaur, Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pinehill Drive, Southborough, MA 01772. E-mail address:
3 Abbreviations used in this paper: B-LCL, transformed B cell lines; CTLp, CTL precursor.
Received for publication July 8, 1999. Accepted for publication October 27, 1999.
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