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*
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and
Wisconsin Regional Primate Research Center and Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI 53715
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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A) replacement in the minimal epitope peptide bound by the MHC
class I molecule Mamu-A*01. SIVmac Gag-specific CTL lysed
Mamu-A*01+ target cells infected with recombinant vaccinia
virus expressing the wild-type but not the mutant Gag protein. In
addition, CTL recognized the mutant epitope peptide less efficiently
than the wild-type virus peptide. In studies to determine the mechanism
by which the mutant virus evaded CTL recognition, this peptide was
shown to bind Mamu-A*01 in a manner that was indistinguishable from the
wild-type peptide. However, experiments in which an increasing duration
of delay was introduced between peptide sensitization of target cells
and the assessment of these cells as targets in killing assays suggest
that the mutant peptide with a T A replacement had a higher
off-rate from Mamu-A*01 than the wild-type peptide did. Therefore,
these findings suggest that AIDS viruses can evade virus-specific CTL
responses through the accelerated dissociation of mutant peptide from
MHC class I. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SIV-infected rhesus monkey provides an ideal model system in which
to explore AIDS virus mutation to evade cell-mediated immune responses.
Monkeys can be selected for study on the basis of their MHC class I
alleles that will develop predictable, well-defined dominant SIV
epitope-specific CTL responses. Moreover, these animals can be
inoculated with a pathogenic virus isolate that has been molecularly
characterized, facilitating a precise definition of the virus that
initiates the infection. We previously addressed the possibility that
SIVmac might escape by mutation from a CTL response in chronically
infected rhesus monkeys. This study was done in monkeys expressing the
MHC class I gene Mamu-A*01 that develop an immunodominant
SIVmac Gag epitope-specific CTL response after simian immunodeficiency
virus of macaques
(SIVmac)4 infection
(19). In that study, mutant virus encoding a change from
threonine (T) to alanine (A) in the immunodominant Gag epitope
(residues 181–189 of the Gag protein) emerged in two of three
evaluated Mamu-A*01+ monkeys infected with SIVmac
(20). Experiments in which 12 amino acid peptides (p11C,
p11C/2A) containing the immunodominant epitope (20) were
used to sensitize target cells for lysis did not suggest that this T
A replacement would result in escape from the epitope-specific CTL
recognition (20). However, it still remained possible that
this mutant protein may not be properly processed in the intracellular
MHC class I pathway for recognition by the epitope-specific CTL or that
the mutant peptide may not form an optimally stable MHC class I peptide
complex. Therefore, we undertook further studies to assess the
possibility that SIVmac isolates with this T A replacement might
escape from the immunodominant CTL response in
Mamu-A*01+ rhesus monkeys.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rhesus monkeys (Macaca mulatta) were used in these studies. These animals were maintained in accordance with the guidelines of the Committee on Animals for Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (National Academy Press, 1996). All monkeys were inoculated i.v. with SIVmac 251 strain, as described previously (20). During the period of the studies, the monkeys were infected with SIVmac for 2–5 years but showed no evidence of an AIDS-like syndrome.
Site-directed mutagenesis and generation of recombinant vaccinia viruses
The plasmid pM40K containing the entire coding region of SIVmac
251 gag was engineered using site-directed mutagenesis to
encode the single T A replacement in the epitope-coding region
(21, 22). This was done following the instructions of the
QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
The sequence of oligonucleotides used to generate this change was 5'-
CAC TGT CAG AAG GTT GCG CCC CCT ATG ACA TTA ACT-3'. The
mutated base G and the other two bases, GCC,
encoded the change in amino acid from threonine to alanine. The
sequence of plasmid DNA containing the desired substitution was
confirmed by sequencing. A recombinant vaccinia virus expressing this T
A replacement of SIVmac was then created through homologous
recombination using a host range selection system and the plasmid pM40K
containing the sequence encoding the single T A replacement in the
epitope (23). The selected recombinant vaccinia viruses
were amplifed and titrated using RK13 cells. vGag/182A-2 and
vGag/182A-3 were viruses generated from two clones of the corresponding
mutated PK40 plasmids. As controls, recombinant vaccinia viruses were
also generated that expressed the wild-type SIVmac gag or
another SIVmac gag insert encoding a single amino acid
replacement 21 aa N-terminal to the epitope-coding sequence
(vGag/161V). These three recombinant vaccinia viruses were assessed for
the expression of SIVmac Gag after infection of B lymphoblastoid cell
line (B-LCL) expressing the Mamu-A*01 gene. In the
experiments using effector cells from monkeys 597 and 403, the target
B-LCL were infected overnight with the recombinant vaccinia viruses.
The infected B-LCL were then divided into two aliquots, one for CTL
assays and the other for 35S-translabeling and
SDS-PAGE characterization. The recombinant vaccinia virus-infected
B-LCL were assessed for viability by trypan blue exclusion before CTL
assays. Furthermore, immune fluorescence analysis showed that SIVmac
Gag protein was expressed in the B-LCL targets infected with the
recombinant viruses.
Cell lines
Rhesus monkey B-LCL were generated by incubating 105 Ficoll-diatrizoate-isolated PBL in 100 µl of culture medium with 100 µl of S594 supernatant. S594 is a cell line productively infected with the transforming baboon Herpesvirus papio (20). The B-LCL were transformed and maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 µg/ml), and 10% FCS (HyClone, Logan, UT).
Cytotoxicity assays
Rhesus monkey B-LCL immortalized with Herpesvirus papio served as target cells. The B-LCL were incubated at a cell concentration of 1 x 106/ml with recombinant vaccinia viruses carrying the SIVmac gag, with a control (equine herpesvirus gH) gene at 10 PFU, or with decreasing concentrations of synthetic peptides for 8 h at 37°C in a 5% CO2 humidified atmosphere (20). CTL derived from PBL of SIVmac-infected monkeys were used as effector cells in a standard 51Cr-release assay performed in U-bottom 96-well microtiter plates. 51Cr-labeled target cells were incubated for 5 h with effector cells at different E:T ratios. Spontaneous release varied from 10 to 20%. Specific release was calculated as [(experimental release - spontaneous release)/(100% release - spontaneous release)] x 100.
Live-cell binding assays
Peptide binding to a C1R cell line stably transfected with Mamu-A*01 was assessed as previously described (24). Cells were incubated overnight at 26°C in the presence of 3 µg/ml human ß2-microglobulin (ß2m). The next day cells were split into separate aliquots and incubated with 100,000 cpm of the iodinated reference peptide ATPYDINQM and different concentrations of the test peptides CTPYDINQM or CAPYDINQM at 20°C for 4 h. Cell pellets were then spun down, washed, and the incorporated 125I was measured by resuspension in Optiphase and counting in a gamma scintillation counter. Percent inhibition of binding was calculated as [1 - (incorporated cpm in the presence of competitor peptide)/(total incorporated cpm in the absence of competitor peptide) x 100].
In vitro folding of Mamu-A*01/ß2m with p11C,C-M or p11C, C-M/2A
Different concentrations (100 µM, 10 µM, and 1 µM) of the wild-type peptide p11C,C-M or the mutant peptide p11C,C-M/2A were added to fixed quantities of rhesus monkey Mamu-A*01 heavy chain and human ß2m as described previously (25). The same quantity of the 150-kDa protein alcohol dehydrogenase was added to each reaction as a control standard for determining the relative folding efficiency. Formation of the folded 43-kDa Mamu-A*01/peptide/ß2m complex was monitored by gel filtration on a TSK SWxl 3000 column (Tosohaas, Montgomeryville, PA)
CTL peptide-MHC class I stability assays
The kinetics of target cell-peptide sensitization for
CTL-mediated lysis was determined using a peptide-MHC class I stability
assay as described by Goulder et al. (10).
Mamu-A*01+ target cells were pulsed with 20 µM
of the 9 aa wild-type or mutant peptide and washed twice with FCS-free
RPMI 1640 medium. In the CTL experiments other than those shown in Fig. 6
, peptide sensitization and chromium labeling of target cells were
done simultaneously, with an 8-h incubation before the addition of
effector cells. In optimizing CTL peptide-MHC class I stability assays,
we found that the specific lysis of targets incubated for 8 h with
peptides was similar to the specific lysis of target cells incubated
for 2 h. Thus, the peptide-loaded target cells were incubated for
0, 2, 16, or 18 h before addition of CTL effector cells. The
impact of this period of incubation on CTL lysis was evaluated for the
mutant peptide in comparison with the wild-type peptide. An inverse
correlation between target cell lysis and the incubation time for a
given peptide indicates an unstable peptide-Mamu-A*01 complex.
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The circulating virions and proviruses in six
Mamu-A*01+ monkeys were assessed for the mutation
encoding the T A replacement in the epitope corresponding to
p11C,C-M. To this end, plasma SIV RNA was isolated and
reverse-transcribed to cDNA (20); proviral DNA was
extracted from PBL of infected monkeys as described previously
(20). The SIV cDNA and proviral DNA were then
characterized for mutations in the epitope-coding region using
PCR-based cloning and sequencing strategy (20). Up to 80
clones generated by PCR from each SIV cDNA or DNA sample were analyzed.
Sequence analyses showed that the mutation encoding the T A
replacement in the epitope could be identified both in the plasma SIV
RNA and proviral DNA in PBL of the infected monkeys (data not
shown).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A replacement in a dominant CTL
epitope were not lysed by Gag epitope-specific CTL
To determine whether virally expressed Gag containing the T A
mutation would be endogenously processed and recognized by
epitope-specific CTL, we generated a recombinant vaccinia-SIVmac
gag construct encoding this mutant Gag sequence. As
controls, recombinant vaccinia viruses were also generated that
expressed the wild-type SIVmac gag or a control mutant
SIVmac gag encoding a single amino acid replacement 21 aa
N-terminal to the epitope-coding sequence (vGag/161V). These three
recombinant vaccinia viruses expressed SIVmac Gag in infected
Mamu-A*01+ B-LCL (Fig. 1). B-LCL infected with these recombinant
vaccinia viruses were then assessed for recognition by Gag
epitope-specific CTL (Fig. 2).
Epitope-specific effector cells generated from
Mamu-A*01+ monkeys infected with SIVmac lysed
B-LCL infected with vaccinia virus expressing the wild-type and control
mutant SIVmac Gag but not the T A mutant Gag (Fig. 2). These results indicated that the
mutant Gag containing the T A replacement expressed in a
recombinant vaccinia virus failed to be recognized by the CTL and
suggested that processing or presentation of the mutant peptide was
impaired.
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We then sought to determine whether a peptide containing the T
A replacement could form a complex with Mamu-A*01 that would be
recognized by CTL. In experiments using 12 aa peptides containing the
epitope, differential CTL recognition of the mutant peptide with the T
A replacement and the wild-type peptide was not clear-cut. The
concentration of mutant peptide required for sensitizing a target cell
for CTL recognition appeared to be 10 times higher than the
concentration needed for sensitizing targets with wild-type peptide
(Refs. 20, 24 and data not shown). However, the ability
of the mutant peptide to evade CTL recognition was evident when two
9-aa peptides, which corresponded to the wild-type (p11C,C-M;
CTPYDINQM) and the mutant (p11C,C-M/2A; CAPYDINQM) viral sequences,
were used in CTL assays. Although target cells were sensitized for
lysis by the wild-type p11C,C-M at peptide concentrations as low as 1
ng/ml, 1000-fold higher concentrations of mutant p11C,C-M/2A were
required to sensitize target cells for comparable levels of
epitope-specific lysis (Fig. 3). Thus,
the results of experiments using optimal epitope peptides support the
observation made in the study of CTL recognition of the mutant Gag in
the vaccinia expression system. The mutant Gag containing a T A
replacement at the second position of the p11C,C-M epitope appears
capable of escaping from CTL recognition.
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We then sought to characterize the mechanism by which this mutant
viral epitope escapes CTL recognition. We first determined whether the
absence of CTL recognition of the T A mutant Gag could be
attributed to an inability of the viral peptide to bind to the
Mamu-A*01 molecule. Peptide-MHC class I binding studies were conducted
using the two 9-aa peptides, which corresponded to the wild-type and
the mutant sequences of the epitope (20). These two
peptides were assessed for their relative ability to compete with an
iodinated index peptide for binding to a
Mamu-A*01+ cell line. Interestingly, mutant
p11C,C-M/2A was still able to bind quite efficiently to Mamu-A*01
expressed on the surface of cells. Its binding capacity may at most
have been only slightly lower than that of the wild-type p11C,C-M
(Fig. 4).
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, the
mutant and wild-type peptides were equally efficient in inducing the
formation of a 43-kDa Mamu-A*01-peptide-ß2m
complex.
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A
replacement did not significantly interfere with peptide-Mamu-A*01
binding. Therefore, these observations suggest that the absence of CTL
recognition of the T A Gag mutant was not due to the inability of
the viral peptide to bind to Mamu-A*01 expressed on the cell
surface.
The mutant peptide with a T A replacement was unable to
stabilize the peptide-Mamu-A*01 complex for recognition by the
epitope-specific CTL
Despite the preserved ability of mutant p11C,C-M/2A to bind
Mamu-A*01, the T A replacement might allow the virus to escape CTL
recognition by altering the stability of the peptide-MHC complex,
increasing the off-rate of the bound peptide. To assess this
possibility, we employed a peptide-MHC class I stability assay (Fig. 6
). Mamu-A*01+
B-LCL were pulsed with 20 µM of wild-type p11C,C-M or mutant
p11C,C-M/2A and were washed with FCS-free medium. The peptide-loaded
cells were then incubated for 0, 2, 16, or 18 h before their use
as targets for CTL effector cells. When compared with the wild-type
peptide, the mutant peptide with the T A replacement exhibited a
decreased ability over time to maintain a stable Mamu-A*01-peptide
complex that was recognized by CTL. Epitope-specific CTL recognized
wild-type peptide- and mutant peptide-loaded target cells equivalently
when peptide-pulsed targets were incubated for 0 or 2 h before the
assay. In contrast, 16 and 18 h after peptide pulsing, the mutant
peptide-loaded targets were poorly recognized by the epitope-specific
CTL, whereas the wild-type peptide-loaded targets remained susceptible
to lysis. These results suggest that the T A replacement in the
epitope accelerates the dissociation of the peptide from Mamu-A*01
despite the preserved capacity of the peptide to bind Mamu-A*01.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A replacement in the p11C,C-M epitope confers upon SIVmac the
ability to escape from CTL recognition. The results of the experiments
employing peptide titration for sensitizing target cells using the
optimal 9-aa p11C,C-M/2A peptide contrasted with those seen in the
experiments using the 12-aa p11C and p11C/2A peptide-pulsed cells (Ref.
20 and Fig. 3). These differing results indicate that a
precise definition of the optimal CTL epitope is crucial for
characterizing escape mutants of AIDS viruses. On the other hand, viral
escape from CTL recognition by the T A replacement was most readily
appreciated when using target cells prepared by vaccinia virus
expression of the mutant Gag. The absence of CTL recognition of mutant
peptides generated in the cell through processing of vaccinia
virus-expressed Gag may mimic the naturally occurring events of CTL
escape because viral peptides must be processed from endogenously
synthesized proteins and presented by MHC class I molecules.
The present studies suggest that the absence of recognition of the
mutant epitope by CTL was a result of the rapid dissociation of the Gag
peptide from Mamu-A*01. The impact of the T A replacement on
peptide-MHC complex formation and viral escape from CTL recognition was
most evident when the peptide-loaded target cells was incubated for a
prolonged period of time before they were employed in the CTL assays.
Despite its inability to maintain a stable complex with Mamu-A*01,
mutant peptide exogenously maintained its ability to bind to Mamu-A*01.
This may explain the finding that CTL recognition of the mutant peptide
occurred when target cells were pulsed with high but not low
concentrations of this protein fragment. A high concentration of the
mutant peptide may maximize its association with MHC class I molecules,
overcoming the increased off-rate of MHC-peptide interaction. The
finding that the mutant peptide rapidly dissociates from Mamu-A*01 may
also explain the complete abrogation of CTL recognition of the target
cells infected with a recombinant vaccinia virus expressing the mutant
Gag. Mutant peptides derived from the endogenous processing pathway may
be particularly prone to dissociate from MHC class I molecules, given
that these endogenously generated peptides are produced in small
quantities in cells and take part in a number of biologic interactions
before expression on the surface of cells. The MHC-mutant peptide
complex may not be formed or stabilized on the surface of cells after
an endogenous processing and thus may not sensitize target cells for
recognition by CTL. Nevertheless, we cannot exclude the possibility
that the present results reflect a component of minor differences in
the on-rate of wild-type and mutant peptides because the peptide
binding assay did suggest a subtle, less favorable binding of the
mutant peptide to Mamu-A*01 (Figs. 3 and 4).
Mechanisms underlying viral escape from CTL recognition are likely to
be complex, involving peptide processing, binding of peptide to MHC
class I molecules, and peptide interactions with the TCR of CTL
(26). Although EBV and HSV as well as a mutant of murine
leukemia virus have been shown to inhibit peptide processing and
presentation for CTL recognition (27, 28, 29), precise
mechanisms by which HIV-1 mutants escape CTL recognition have not been
formally documented (9, 10, 11, 12, 13, 14, 15, 16, 17, 18). Some studies suggest that
HIV-1 mutants may evade CTL recognition through interfering with the
peptide binding to MHC class I molecules or through interactions with
the TCR of CTL (15, 16, 30). In fact, T cell recognition
depends on the functional density of the TCR ligand comprised of
MHC-peptide complex. It remains possible that the T A replacement
identified in the epitope corresponding to the P11C,C-M can result in a
decrease in the affinity threshold that is required for CTL lysis of
target cells. Additional studies are needed to address this
possibility.
The results of the present studies suggest that an amino acid
substitution in the epitope allows the virus to escape from CTL
recognition through an accelerated dissociation of the peptide from MHC
class I molecules. This in vitro observation implies that the single
mutation encoding the T A replacement in the CTL epitope may play a
role in the persistence of SIVmac infection in infected monkeys. The
rapid dissociation of the T-A mutant peptide from MHC class I can
certainly confer upon the virus a survival advantage through the escape
from CTL recognition. SIVmac may evade an antiviral T cell response
through the accumulation of mutations in CTL epitopes in infected
monkeys.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Zheng W. Chen, Division of Viral Pathogenesis, 330 Brookline Avenue, P.O. Box 15732, Re-113, Boston, MA 02215.
3 Current address: SmithKline Beecham Biologicals, Rue de l’Institut 89, 1330 Rixensart, Belgium.
4 Abbreviations used in this paper: SIVmac, simian immunodeficiency virus of macaques; B-LCL, B lymphoblastoid cell line; ß2m, ß2-microglobulin.
Received for publication August 31, 1999. Accepted for publication March 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R. Schirmbeck, D. Stober, S. El Kholy, P. Riedl, and J. Reimann The Immunodominant, Ld-Restricted T Cell Response to Hepatitis B Surface Antigen (HBsAg) Efficiently Suppresses T Cell Priming to Multiple Dd-, Kd-, and Kb-Restricted HBsAg Epitopes J. Immunol., June 15, 2002; 168(12): 6253 - 6262. [Abstract] [Full Text] [PDF]
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P. F. McKay, J. E. Schmitz, D. H. Barouch, M. J. Kuroda, M. A. Lifton, C. E. Nickerson, D. A. Gorgone, and N. L. Letvin Vaccine Protection Against Functional CTL Abnormalities in Simian Human Immunodeficiency Virus-Infected Rhesus Monkeys J. Immunol., January 1, 2002; 168(1): 332 - 337. [Abstract] [Full Text] [PDF]
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M. R. Betts, D. R. Ambrozak, D. C. Douek, S. Bonhoeffer, J. M. Brenchley, J. P. Casazza, R. A. Koup, and L. J. Picker Analysis of Total Human Immunodeficiency Virus (HIV)-Specific CD4+ and CD8+ T-Cell Responses: Relationship to Viral Load in Untreated HIV Infection J. Virol., December 15, 2001; 75(24): 11983 - 11991. [Abstract] [Full Text] [PDF]
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Z. Hel, J. Nacsa, B. Kelsall, W.-P. Tsai, N. Letvin, R. W. Parks, E. Tryniszewska, L. Picker, M. G. Lewis, Y. Edghill-Smith, et al. Impairment of Gag-Specific CD8+ T-Cell Function in Mucosal and Systemic Compartments of Simian Immunodeficiency Virus mac251- and Simian-Human Immunodeficiency Virus KU2-Infected Macaques J. Virol., December 1, 2001; 75(23): 11483 - 11495. [Abstract] [Full Text]
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T. U. Vogel, T. M. Allen, J. D. Altman, and D. I. Watkins Functional Impairment of Simian Immunodeficiency Virus-Specific CD8+ T Cells during the Chronic Phase of Infection J. Virol., March 1, 2001; 75(5): 2458 - 2461. [Abstract] [Full Text]
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) and mutant (
) 9-aa peptides, washed twice to remove
unloaded peptides, and then incubated at 37°C for increased numbers
of hours before the addition of CTL effector cells. The loss of CTL
recognition of target cells as the duration of time after peptide
pulsing increased indicated the lack of stability of the
peptide-Mamu-A*01 complex. Data were derived from up to three repeated
experiments, with error bars indicating the variability among the CTL
assays.
), mutant sequence (CAPYDINQM; 
), and control
sequence (p11B; 
) peptides were assessed for their ability to
compete with the iodinated reference peptide (ATPYDINQM) for binding to
Mamu-A*01-transfected C1R cells. The IC50 of binding for
CTPYDINQM was 5 nm and for CAPYDINQM was 30 nm.