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*
Department of Microbiology, University of Pennsylvania Medical School, Philadelphia, PA; and
Department of Crystallography, Birkbeck College, London, United Kingdom
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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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Falk et al. have described a series of motifs based on elution of peptides from class I molecules and peptide sequencing of bulk populations (5). These motifs, which we will call the Rammensee motifs, have been used to predict epitopes within Ags for many purposes, including the development of peptide vaccines (6, 7). In the mouse haplotype H-2d, for example, the motifs for all the loci have been identified, and, in particular, the Kd motif has been described to be a nonamer with residues tyrosine or phenylalanine in the second amino acid position and leucine, isoleucine, or valine at the carboxyl-terminal or ninth amino acid position as anchoring residues (8).
Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that enters the macrophage upon phagocytosis. It then escapes from the phagolysosome and lives in the cytoplasm of the cell (9). As a consequence, secreted L. monocytogenes Ags are processed and presented by both the class I and class II Ag presentation pathways. This results in strong cell-mediated immune responses, with the induction of both CD8+ CTL and CD4+ T cells (10, 11), thought to be of the Th1 phenotype (12, 13). It has recently been proposed that, in HIV infection, strong cell-mediated responses consisting of T helper cells of the type 1 phenotype and CTL may be protective (14, 15, 16).
We had previously described a genetic method to modify permanently the chromosome of L. monocytogenes so that HIV gene products can be expressed as secreted proteins under the control of a copy of the strong promoter of the hemolysin (hly)4 gene, which encodes listeriolysin-O (17). Furthermore, we demonstrated that mice immunized with one of these constructs, Lm-gag, mount a strong, specific, long-lasting CTL response against the HIV-1 gag protein and that the response is directed predominantly against an epitope present in the p24 portion of the protein.
In this article we have identified the immunodominant CTL epitope contained in the p24 portion of the protein and have shown that it is Kd restricted. Surprisingly, this peptide, gag 197–205 AMQMLKETI, does not contain the anchoring tyrosine residue in position two as predicted by the Rammensee motif (-Y------I/L) and displayed by all known Kd epitopes (8). Furthermore, other peptides in p24 that contain H-2d motifs do not appear to be selected for immune recognition, including some that contain tyrosine or phenylalanine in the second position, or to bind to Kd. In addition, competition assays suggest that AMQMLKETI binds to Kd with an affinity comparable to that of hly 91–99, a Kd-restricted L. monocytogenes epitope containing the Rammensee motif. In the absence of a crystal structure for Kd, we have used homology modeling to construct a model of this molecule and examined the contribution of individual residues to the stability of the Kd/AMQMLKETI complex using a molecular dynamics approach. By comparison with a similar analysis of the immunodominant hemolysin peptide GYKDGNEYI and the analogues AMAMLKETI and AYAMLKETI also bound to Kd, we show that the lack of an aromatic residue at position two in AMQMLKETI appears to be compensated by favorable interactions mediated by the glutamine residue at position three with pocket D in the Kd molecule. We have verified these predictions by showing that AMQMLKETI and AYAMLKETI effectively compete with hly 91–99 for recognition by hly-specific CTL whereas AMAMLKETI is a poor inhibitor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L. monocytogenes (Lm-wt) strain 10403S (11) was the wild-type organism used in these studies. It has an LD50 of approximately 3 x 104 when injected i.v. or i.p. into BALB/c mice (18). Lm-gag refers to either one of two recombinant strains of L. monocytogenes, each of which carries one copy of the HIV-1 strain HXB gag gene stably integrated into the listerial chromosome and both of which secret the gag gene product as determined by Western blotting of secreted proteins (Ref. 17; Z.-J. Yao and Y. Paterson, unpublished observations). All strains were grown in brain/heart infusion (BHI) medium (Difco Labs, Detroit, MI).
Cell lines
P815 mouse mastocytoma cells (H-2d) and tk- L cells (H-2k) transfected with H-2d class I molecules, L-Dd, L-Kd (kind gift from Dr. Eisenhlor, Thomas Jefferson University, Philadelphia, PA) (19), L-Ld, and the parental cell line L-tk- (kind gift from Dr. T. Hansen, Washington University, St. Louis, MO) (20) were grown in RPMI 1640 media supplemented with 10% FBS, 100 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine. Appropriate H-2d class I expression was verified by FACS analysis (data not shown).
Vaccinia constructs
The vaccinia viruses used in this paper were vVK1 (Vac-gag) (provided by B. Moss, National Institutes of Health, Bethesda, MD), NYCBH (Vac+), vAbT141 (p55), vAbT228 (p17), and vAbT286 (p24) (kindly provided by D. Panicali, Therion Biologics Corporation, Cambridge, MA) (21).
Viral stocks were grown in monolayer HeLa S3 cells and titrated from confluent monolayer cultures using a plaque assay with BSC-1 cells.
Generation and expansion of gag-specific CTLs
Six- to eight-week-old BALB/c female mice (H-2d) (Charles River Laboratories substrain AnNCrIBR, Charles River laboratories, Raleigh, NC) were immunized by i.p. inoculation with 106 or 107 live Lm-gag, or with 6 x 103 live Lm-wt (about 0.2 LD50), the wild-type strain. For some experiments, effectors were derived from mice boosted with a similar dose 2 to 4 wk later. Splenocytes, obtained from mice 1 to 2 wk after their last immunization with Lm-gag, were cultured after a third of the splenocytes were infected with five plaque-forming units per cell of Vac-gag (VVK1) or vAbT141 (p55). Splenocytes were cocultured for 7 days at 37°C in 40 ml of Iscove’s modified DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Alternatively splenocytes obtained from mice immunized with Lm-gag or Lm-wt were stimulated in vitro with 2 µM of peptides gag 197–205 or hly 91–99 (6), respectively, for 5 days in six-well plates at a concentration of 1 x 107 cells/ml (3 ml/well).
51Cr release assays
P815, L-Kd, L-Dd, L-Ld, and L-tk- target cells were washed twice with PBS, resuspended at a concentration of 107 cells/ml in Eagle’s minimal essential medium supplemented with 2.5% FBS and 100 mM HEPES, and infected with 16.5 plaque-forming units of vaccinia virus per cell. After adsorption for 2 h at 37°C, the infected cells were resuspended in RPMI 1640 medium supplemented with 10% FBS and antibiotics, and incubated at 37°C for 2 h. The cells were then pelleted, resuspended in 100 µl medium containing 50 µCi Na51CrO4 per 5 to 6 x 105 cells, and incubated at 37°C for 1 h.
In vitro-stimulated splenocytes were incubated at various ratios with 2 x 104 51Cr-labeled target cells suspended in RPMI 1640 medium (total volume of 200 µl/well) in round-bottom, 96-well plates for 4 h at 37°C. Released 51Cr was determined in 100 µl of supernatant, and the percentage of specific lysis was calculated as (experimental cpm-spontaneous cpm)/(total cpm-spontaneous cpm) x 100. All assays were performed in triplicate, and the mean and SD were calculated.
In assays where peptides were used as a source of Ag and as competitors, uninfected target cells were labeled with Na51CrO4, washed, and then incubated in 150 µl for 1 to 2 h at 37°C with peptides at various concentrations, before addition of splenocytes.
Overlapping peptides
Twenty-two overlapping 20-mer peptides with 10-amino acid overlaps, spanning residues 133 to 362 of the HIV-1SF2 gag p24 protein, were provided by the MRC AIDS Reagent Project, London, U.K. (Repository reference: ADP 788.1-22). HIV-1SF2 gag differs in amino acid sequence with the HXB strain gag, which was used to make the Lm-gag vaccine, only at two positions in the p24 fragment. One change is from I to L at position 138 and the other is an E to D change at position 132. Both of these substitutions would conserve the motifs associated with H-2d MHC class I presentation (6, 7).
Peptide synthesis
Peptides gag 193–201, gag 197–205A199, and gag 197–205Y198A199 were purchased as crude cleavage products from Biopolymer Synthesis Center, California Institute of Technology, Pasadena, CA. All other peptides were synthesized in our laboratory using DuPont’s RaMPS multiple peptide synthesis system procedure on Wang Resin (p-Alkoxy-benzyl alcohol resin) (DuPont Chemical Company, Wilmington, DE) using F-moc (9-fluorenylmethyloxycarbonyl) nitrogen terminal-protected amino acids (BACHEM Biotech Company, Philadelphia, PA). Couplings were conducted using DPC (N-N-diisopropylcarbodiimide), HOBT (1-hydroxybenzotriazole) in DMF (N,N-dimethylformamide) solvent and were monitored by ninhydrin reaction in 100°C water for 5 min. Simultaneous resin cleavage and side-chain deprotection were achieved by high concentration TFA (all chemical reagents purchased from Aldrich (Milwaukee, WI) and Sigma Chemical Company (St. Louis, MO)). All crude products were purified to 98% purity by reverse-phase HPLC (Perkin-Elmer, Norwalk, CT, Series 400) using a Vydac C18 column, and their m.w. was verified by matrix-assisted laser desorption ionization (MALDI)-mass spectroscopy.
Molecular modeling and calculations
All molecular modeling operations of the interaction of peptides
with Kd were performed using SYBYL Molecular Modeling
Software (Tripos Associates, St. Louis, MO) and a Silicon Graphics
display system (Silicon Graphics, San Diego, CA). Structures of
peptides bound to the 
1 and 2 domains of Kd were
built by homology modeling using a data set of known crystallographic
structures of peptide bound to human or mouse MHC class I molecules
deposited in the Brookhaven Protein Data Bank. Structures of peptides
of more than 9 residues or without a hydrophobic amino acid residue at
the carboxyl terminus were excluded, leaving 11 structures in the data
set, five of peptides bound to mouse H-2 and six of peptides bound to
human HLA. Lack of sequential homology between the
Kd-binding peptides and those in the homology data set was
treated by adopting for the test peptide the conserved backbone
coordinates between the first three and last two residues in the
peptide data set. The SYBYL LOOP closing algorithm was then used to
build the middle four residues. All structures were fully minimized
using the Powell algorithm, in which the coordinates of the structure
are modified in an iterative manner and the conformational energy is
calculated. Further changes in the coordinates are based on reducing
the energy by a path of steepest descent. This method, as is the case
with all minimization algorithms, is limited to relaxing the structure
into the local energy minimum.
Conformational energies were calculated, for minimization and molecular dynamics calculations, using an AMBER force field (22, 23) as the sum of torsional, electrostatic, van der Waals, and hydrogen bonding interaction energies between nonbonded atoms separated by at least three bonds. Because bonded atoms move, the impact of bond angle bending and bond stretching on the total energy are also included as specific energy terms in AMBER. Molecular dynamics analyses of the stability of peptide binding to Kd were performed on hydrated (approximately 2,500 water molecules) and minimized structures at constant temperature. Water is treated explicitly in these calculations; therefore, a dielectric constant of 1.0 was used for electrostatic interactions. Only MHC atoms within 6.5 Å of the peptide residues were allowed to move during the simulations. Preliminary molecular dynamics simulations, which were conducted for 12 ps for AMQMLKETI/Kd showed that dissociation of the peptide from Kd occurred within 6 ps. Molecular dynamic simulations were also performed on the VSV-8/Kb and SEV-9/Kd complexes (24) in addition to AMQMLKETI/Kd to compare the behavior of the modeled structure of the peptide/H-2 Kd complex with the x-ray crystallographically determined complexes. The behavior of the complexes during this period was essentially similar, and dissociation occurred for all three molecules within 6 ps. Subsequent simulations were thus restricted to this time frame.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the MHC restriction of the Ag-specific response to
gag delivered by Lm-gag, a series of L cell lines transfected with each
of the class I molecules, Dd, Kd, and
Ld, were used as target cells in 4-h 51Cr
release assays (Fig. 1
). Only L cells
transfected with Kd infected with vaccinia virus expressing
gag were lysed, suggesting that the epitope was Kd
restricted. In our previous studies (17), we had shown that the
immunodominant region of gag was contained within the p24 protein
fragment. To confirm that the Kd-restricted epitope was
indeed contained within p24, we compared the CTL activity against
L-Kd cells and P815 cells when infected with VVp55, VVp24,
and VVp17 (Fig. 2). The CTL activity
against both cells lines was comparable, and lysis was observed only
when the cells were infected with p55 or p24, indicating that the
epitope contained within p24 is Kd restricted.
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In previous studies (17), two 20-mer peptide sequences within p24
were observed to induce lysis of P815 target cells by Lm-gag-induced
splenocytes whereas a number of peptides of appropriate length
containing the Ld, Dd, or Kd motifs
did not target P815 cells for lysis (data not reported). Long peptides
have been shown to be effective in CTL assays because they either
contain truncated contaminants of appropriate length from the peptide
synthesis procedure (25, 26) or because they are cleaved to shorter
peptides by extracellular proteases such as angiotensin converting
enzyme (ACE) found in FBS in culture media (27, 28). It seemed likely,
therefore that these sequences contained the Kd-restricted
CTL epitopes of appropriate size. However, while the 20-mer peptides,
gag 193–212 and 233–252, did contain a number of potential
H-2d carboxyl-terminal anchor residues, none was positioned
at an appropriate distance from other required dominant anchor residues
usually associated with H-2d binding, as described by Falk
et al. (5) (Table I). Therefore, we
tested four nonamer peptides contained within gag 193–212 and 233–252
that contain the appropriate carboxyl-terminal residue, either leucine
or isoleucine, and three other peptides contained within p24 that had
the other anchoring residue, a tyrosine or a phenylalanine at the
second position from the amino-terminal, for their ability to induce
lysis of target cells by Lm-gag-induced splenocytes (Fig. 3, A and B). Two
nonamers, gag 197–205 (AMQMLKETI) contained within gag 193–212 and
gag 239–247 (TTSTLQEQI) contained within gag 233–252, induced
significant lysis of the target cells, suggesting that these are the
epitopes recognized in the H-2 Kd context.
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). In both cases, the shorter peptides
show higher activity than their longer counterparts, suggesting that
the nonamers are of a more appropriate size to bind to Kd
and that the longer peptides are not the ideal length for recognition
by gag-specific CTL. In the case of gag 193–212, while activity is
lost at about one nM for the longer peptide, the shorter peptide
targets cells for CTL activity at concentrations as low as 10 pM. On
the other hand, gag 233–252 is recognized only at the highest
concentration (10 µM) while the shorter peptide gag 239–247 shows
activity similar to that of gag 193–212, the other 20-mer, and loses
its ability to target cells for lysis at one nM. From these data, we
conclude that gag 197–205 is the immunodominant epitope in the immune
response to HIV-gag delivered by L. monocytogenes in
BALB/c mice.
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Since the immunodominant epitope identified did not contain the
full Rammensee motif, we compared the ability of this peptide to bind
Kd to that of other antigenic epitopes that do contain the
motifs by comparing the ability of these peptides to inhibit
Kd-restricted CTL recognition of another epitope in the
presence of competing peptide. This assay has been shown to be a more
sensitive measure of MHC class I binding than other widely used assays,
such as the RMA-S assay, which measures peptide-induced stability of
cell surface MHC class I molecules (29). We were particularly
interested in hly 91–99 (Table I
), the Kd-restricted
immunodominant epitope in the L. monocytogenes-secreted
virulence factor hemolysin, for a number of reasons. First, Pamer and
associates successfully identified this as an immunodominant epitope
for the L. monocytogenes response by synthesizing the eleven
nonamer peptides contained in the hly sequence that fitted the
Kd-restricted Rammensee motifs and then testing their
ability to target P815 cells for lysis by a CTL line derived from
BALB/c mice immunized with L. monocytogenes (6). Further
studies, using peptide-binding competition assays, showed that hly
91–99 along with two other hly peptides had the highest binding
affinity for H-2 Kd of all 11 hly peptides (30). In
addition, immunization with Lm-gag results in strong CTL responses to
hly 91–99, as well as to gag 197–205, eliminating possible
variability due to different ways of presenting the Ags to the immune
system. Finally, as an immunodominant epitope of L.
monocytogenes, hly 91–99 is a potential competitor in vivo for
gag 197–205 when delivered by Lm-gag since hemolysin is produced
copiously under the same conditions as gag when delivered by Lm-gag.
To compare the ability of hly 91–99 or gag 197–205 to inhibit
lysis by the other peptide, effector cells specific for either peptide
were prepared from splenocytes from mice immunized with Lm-gag and
expanded in vitro with either gag 197–205 or hly 91–99. CTL assays
were performed in which the same peptide with which splenocytes had
been expanded was used as the antigenic peptide at varying
concentrations in the presence of the competitor peptide at 2 µM.
Both peptides were equally able to compete for the Kd
molecules, as shown by a decrease in lysis (a 2.5 log shift of the
curve) in the presence of the competitor peptide (Fig. 5, A and B).
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). In contrast to
Kd-binding peptides hly 91–99 or
NP148–157(R-156) (31), none of the tested peptides were
able to compete with gag 197–205, even at concentrations of 10 µM,
suggesting that these peptides do not bind to Kd or bind
with an affinity lower than can be detected by this assay.
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Currently, there are no crystal structures of the mouse H-2
Kd molecule. Therefore, to determine how gag 197–205,
AMQMLKETI, binds to Kd in the absence of an
aromatic residue at position two, we constructed the Kd
molecule by homology modeling and compared the binding of AMQMLKETI to
that of hly 91–99, GYKDGNEYI (Fig. 7,
a and c). Although a methionine at position two
in the gag peptide could fit into the B pocket of the Kd,
it did not fill the pocket as well as the tyrosine in that position in
GYKDGNEYI. Whereas only one MHC residue (Gln63) was within
3.5 Å of the side chain of Met198 of the gag peptide,
additional residues (Tyr7, Ala24,
Gln63, Tyr92, Phe99,
Tyr159) were within 3.5 Å of Tyr92 of the hly
peptide. This suggested that the stability of the
AMQMLKETI/Kd complex must be derived from compensating
interactions by other residues. To determine the source of this
stability, we performed molecular dynamics analyses of both
peptide/Kd complexes. The atoms in the hydrated complexes
were subjected to Newtonian motion for 6 ps, maintaining the
temperature of the system at 293°K and constant volume. Quite early
in these dynamics runs (at about 2 ps), the peptides began to
disassociate from the peptide binding groove at the carboxyl terminus,
followed by the charged amino terminus, remaining anchored at the end
of 6 ps by only the tyrosine at position two in the case of GYKDGNEYI,
or glutamine at position three in the case of AMQMLKETI (Fig. 7, b and d). It is of interest that the dissociation
of AMQMLKETI from the Kd peptide binding groove is no more
extensive than for GYKDGNEYI (Fig. 7, b and d),
consistent with our experimental data that these peptides have a
comparable affinity for Kd.
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We next established that the glutamine at position three was, indeed,
acting as the major anchor residue for AMQMLKETI. We changed this
residue to an alanine and, after minimization, explored its
dissociation from Kd by molecular dynamics for 6 ps. This
change resulted in the complete dissociation of the peptide from the
Kd molecule (see Fig. 7e), confirming that the
interaction of AMQMLKETI with Kd is mainly stabilized by
this residue binding to pocket D. Finally, to determine whether the
glutamine at P3 was as efficient an anchor as Tyr at P2, we examined
the stability of the AYAMLKETI/Kd complex (Fig. 7f). At the end of a 6-ps molecular dynamics simulation,
this peptide remained anchored by the Tyr at P2. This change appeared
to restore stability to AMAMLKETI in the absence of Gln at P3 (Fig. 7, e and f) but remained less closely associated
with Kd than AMQMLKETI (Fig. 7, f and
d).
Gag 197–205Q199 and 197–205Y198 are better competitors than 197–205A199 for hly 91–99 CTL recognition, as theoretically predicted
The results of the molecular dynamics simulations described above
predict that AMQMLKETI and AYAMLKETI have an equivalent affinity for
Kd that is higher than AMAMLKETI. To verify this, we used
synthetic analogues with these sequences as competitors for the
recognition of effector CTL specific for hly 91–99. The assays were
performed essentially as described for Figure 5, using hly 91–99 as
the antigenic peptide at varying concentrations in the presence of the
competitor peptide at 2 µM. Both AMQMLKETI and AYAMLKETI were equally
able to compete for the Kd molecules, as shown by a
decrease in lysis (a 2.0 log shift of the curve) compared with the
titration curve in the absence of inhibitor. In contrast, AMAMLKETI did
not compete with hly 91–99 in this assay (Fig. 8), as anticipated from our molecular
dynamics findings.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2) and have further defined the epitopes by
identifying two nine-amino acid sequences within the 20-mers (Fig. 3)
that are recognized at concentrations lower than the 20-mer peptides
(Fig. 4). One of the peptides, gag 197–205, could target cells for
lysis at picomolar concentrations and was 105 times more
potent than the other short peptide, suggesting that it is the
immunodominant epitope for this response.
The motifs described for peptides binding class I molecules not only
specify the number of residues but also describe anchoring positions
where specific residues are located. In this case, for Kd,
the motif contains tyrosine or phenylalanine in position two and
isoleucine, leucine, or valine in position nine (5). It is of interest
then that, although we have identified two epitopes that fit the motif
in size, both fail to meet the requirements for the anchoring position
at position two since the immunodominant epitope, gag 197–205,
contains a methionine at this position whereas a threonine is present
in the case of gag 239–247. It is important to emphasize, at this
time, that these are the first identified epitopes that do not meet the
Kd motif criteria (reviewed in 8 although there have
been reports describing epitopes for other class I molecules, including
HLA-A (33), Kb (34), and Kk (35), that do not
conform to their corresponding described motifs. Two questions then
arise from this finding. The first is why, in a sequence containing
several classical Rammensee Ld and Dd motif
peptides, in addition to other partial Kd motifs (Table I)
that do contain the appropriate anchoring amino acid at position two,
the emerging epitopes do not meet these criteria. This selection could
take place at two potential levels: the processing pathway, including
MHC binding, or the T cell recognition of the peptide in the context of
Kd. There are no peptides with complete Kd
Rammensee motifs in the gag p24 sequence, and those partial
Kd motif peptides with a tyrosine at position two do not
bind to MHC as determined by competition assays (Fig. 6). Therefore,
control may occur at MHC binding in the case of the generation of
Kd-restricted epitopes. However, this explanation is not
sufficient to account for the lack of recognition of Ld-
and Dd-restricted epitopes in this response.
There are several checkpoints at which epitope selection may occur in
the MHC class I-restricted pathway. Peptide generation, which takes
place in the cytoplasm and appears to be the most critical step to
determine immunogenicity (36), can be regulated by latent membrane
protein (LMP)-2 and LMP-7, two proteins that modulate proteasome
specificity (37, 38, 39, 40). The generated peptides then must be transported
to the endoplasmic reticulum by TAP (41, 42), which could play a role
in peptide selectivity, although to a lesser degree (36, 43, 44, 45).
Finally, the peptide is required to bind to the MHC molecule with an
affinity high enough to stabilize the class I molecule and remain bound
on the surface of the cell (46, 47). All these steps have been the
result of evolutionary changes for a highly regulated mechanism of
defense. During this process, sequences flanking the epitope
particularly at the carboxyl terminal (48, 49, 50) appear to play an
important role in epitope selection. Thus the peptides we have
identified may have been generated in preference to all the other
sequences to be presented by H-2d MHC molecules by simply
being the only epitopes to emerge from all of these checkpoints for
recognition by CD8+ T cells. In addition, the
immunodominant epitope, 197–205, is capable of inducing lysis of P815
cells at concentrations as low as 1 to 10 pM. Therefore, although gag
197–205 lacks the predictable tyrosine or phenylalanine at position 2,
it nevertheless appears to bind Kd with an affinity similar
to that of other conventional Kd-binding peptides such as
hly 91–99 (Fig. 5, A and B), a peptide that was
identified as an immunodominant epitope by searching the hemolysin
sequence for peptides with the Rammensee motif (6).
A second question then arises as to the source of this stability in the
absence of an aromatic residue at position 2. Molecular dynamics is a
powerful tool for examining receptor/ligand interactions and
particularly for thermodynamic properties and is particularly suited to
study time-dependent phenomena (51). However, this technique has not
been widely exploited for studying MHC/peptide interactions, possibly
because early attempts to use this approach (52) before the structure
of MHC-bound peptides had been solved by x-ray crystallography,
incorrectly predicted an -helical conformation for an MHC class I
bound peptide. Better and more reliable predictions have been made
since the known x-ray structures of MHC-bound peptides have been used
as starting points for these analyses (53, 54, 55). Indeed, the stability
of binding of a set of HLA-B27 peptide analogues, as determined by the
buried surface areas of the peptide residues after molecular dynamics
perturbation, could be qualitatively correlated with the ability of
each analogue to catalyze the assembly of HLA-B27 in vitro (55).
We thus performed a theoretical analysis of the stability of the
interaction of gag 197–205 with Kd, using molecular
dynamics. This simulation of the dissociation of this peptide from the
MHC class I molecule indicated that, within a 6-ps simulation, it
remained more firmly anchored to the Kd molecule than other
peptide/MHC complexes, such as hly 91–99 to Kd (Fig. 7, A-D) and VSV8 or SEV9 to Kb (data not
shown). In addition, the binding of the glutamine residue at position
three to pocket D of Kd appeared to convey stability to the
gag peptide/Kd interaction (Fig. 7, C and
D). Indeed, changing this residue to an alanine resulted in
total dissociation of the peptide during the molecular dynamics
simulation (Fig. 7E), indicating that the interaction of
glutamine with pocket D was crucial to the stability of the complex and
compensated for the absence of an anchor residue at position two in the
peptide. Restoration of binding of this analogue (AMAMLKETI) to
Kd could be restored by providing an anchor residue at
position two, by there substituting a tyrosine for methionine (Fig. 7F). The confirmation of these findings in competition
assays using these analogues as competitors for hly 91–99 recognition
is an important experimental verification of the molecular dynamics
approach.
Although secondary anchor residues have been reported for other MHC class I molecules, including HLA-A2 (56) and Kb (57), position three has not previously been shown to be important in stabilizing peptide binding to Kd. Indeed, in a recent compilation of 26 Kd-binding peptides, all contained a tyrosine at position two except one which had a phenylalanine at this position whereas the residues found at position three were most variable, and glutamine was found in only four peptides (8).
In this study we have clearly shown that a peptide that does not contain the Rammensee motif is recognized as the major epitope in an immune response to gag delivered by Listeria in H-2d mice. Several groups have been using peptide-binding motifs to identify possible epitopes in states of disease to develop peptide vaccines and therapies (6), often with the help of computer algorithms (7). The data here presented suggest that these theoretical methods can fail to identify dominant epitopes that do not meet the criteria described by these motifs and indicate that caution should be used in their application. Even a predictive algorithm that is more broadly based on empirical measurements of the contribution of individual residues to the half time of dissociation of a peptide binding to MHC class I (58) failed to identify AMQMLKETI as the immunodominant epitope within the gag p24 sequence. In contrast, molecular dynamics analyses of putative peptide epitopes associated with MHC class I appears to be a useful tool in designing peptide analogues with differing affinities for MHC molecules.
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Acknowledgments |
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Footnotes |
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2 Current address: DuPont Industries, Wilmington, DE.
3 Address correspondence and reprint requests to Dr. Yvonne Paterson, Department of Microbiology, 323 Johnson Pavilion, 3610 Hamilton Walk, University of Pennsylvania, Philadelphia, PA 19104.
4 Abbreviations used in this paper: hly, hemolysin;, Lm-gag, L. monocytogenes strain expressing HIV-gag; Lm-wt, L. monocytogenes wild-type strain.
Received for publication January 23, 1998. Accepted for publication May 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß-TCR transgenic T cells is macrophage dependent. Int. Immunol. 5:371.
Th2 switch is a critical step in the etiology of HIV infection. Immunol. Today 14:107.[Medline]
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 Z. Li, M. Zhang, C. Zhou, X. Zhao, N. Iijima, and F. R. Frankel Novel Vaccination Protocol with Two Live Mucosal Vectors Elicits Strong Cell-Mediated Immunity in the Vagina and Protects against Vaginal Virus Challenge J. Immunol., February 15, 2008; 180(4): 2504 - 2513. [Abstract] [Full Text] [PDF]
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 J. Lin, Y. Zhi, L. Mays, and J. M. Wilson Vaccines Based on Novel Adeno-Associated Virus Vectors Elicit Aberrant CD8+ T-Cell Responses in Mice J. Virol., November 1, 2007; 81(21): 11840 - 11849. [Abstract] [Full Text] [PDF]
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 E. A. Reap, S. A. Dryga, J. Morris, B. Rivers, P. K. Norberg, R. A. Olmsted, and J. D. Chulay Cellular and Humoral Immune Responses to Alphavirus Replicon Vaccines Expressing Cytomegalovirus pp65, IE1, and gB Proteins Clin. Vaccine Immunol., June 1, 2007; 14(6): 748 - 755. [Abstract] [Full Text] [PDF]
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C. Ranasinghe, S. J. Turner, C. McArthur, D. B. Sutherland, J.-H. Kim, P. C. Doherty, and I. A. Ramshaw Mucosal HIV-1 Pox Virus Prime-Boost Immunization Induces High-Avidity CD8+ T Cells with Regime-Dependent Cytokine/Granzyme B Profiles J. Immunol., February 15, 2007; 178(4): 2370 - 2379. [Abstract] [Full Text] [PDF]
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J. N. Kochenderfer, C. D. Chien, J. L. Simpson, and R. E. Gress Synergism between CpG-Containing Oligodeoxynucleotides and IL-2 Causes Dramatic Enhancement of Vaccine-Elicited CD8+ T Cell Responses J. Immunol., December 15, 2006; 177(12): 8860 - 8873. [Abstract] [Full Text] [PDF]
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 L.-M. Chen, G. Briones, R. O. Donis, and J. E. Galan Optimization of the Delivery of Heterologous Proteins by the Salmonella enterica Serovar Typhimurium Type III Secretion System for Vaccine Development. Infect. Immun., October 1, 2006; 74(10): 5826 - 5833. [Abstract] [Full Text] [PDF]
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X. Zhao, M. Zhang, Z. Li, and F. R. Frankel Vaginal Protection and Immunity after Oral Immunization of Mice with a Novel Vaccine Strain of Listeria monocytogenes Expressing Human Immunodeficiency Virus Type 1 gag. J. Virol., September 1, 2006; 80(18): 8880 - 8890. [Abstract] [Full Text] [PDF]
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L. B. Arruda, D. Sim, P. R. Chikhlikar, M. Maciel Jr, K. Akasaki, J. T. August, and E. T. A. Marques Dendritic Cell-Lysosomal-Associated Membrane Protein (LAMP) and LAMP-1-HIV-1 Gag Chimeras Have Distinct Cellular Trafficking Pathways and Prime T and B Cell Responses to a Diverse Repertoire of Epitopes J. Immunol., August 15, 2006; 177(4): 2265 - 2275. [Abstract] [Full Text] [PDF]
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 C. Trumpfheller, J. S. Finke, C. B. Lopez, T. M. Moran, B. Moltedo, H. Soares, Y. Huang, S. J. Schlesinger, C. G. Park, M. C. Nussenzweig, et al. Intensified and protective CD4+ T cell immunity in mice with anti-dendritic cell HIV gag fusion antibody vaccine J. Exp. Med., March 20, 2006; 203(3): 607 - 617. [Abstract] [Full Text] [PDF]
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M. A. Kutzler, T. M. Robinson, M. A. Chattergoon, D. K. Choo, A. Y. Choo, P. Y. Choe, M. P. Ramanathan, R. Parkinson, S. Kudchodkar, Y. Tamura, et al. Coimmunization with an Optimized IL-15 Plasmid Results in Enhanced Function and Longevity of CD8 T Cells That Are Partially Independent of CD4 T Cell Help J. Immunol., July 1, 2005; 175(1): 112 - 123. [Abstract] [Full Text] [PDF]
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 G. L. Zhang, K. N. Srinivasan, A. Veeramani, J. T. August, and V. Brusic PREDBALB/c: a system for the prediction of peptide binding to H2d molecules, a haplotype of the BALB/c mouse Nucleic Acids Res., July 1, 2005; 33(suppl_2): W180 - W183. [Abstract] [Full Text] [PDF]
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X. Chen, M. T. Rock, J. Hammonds, J. Tartaglia, A. Shintani, J. Currier, B. Slike, J. E. Crowe Jr., M. Marovich, and P. Spearman Pseudovirion Particle Production by Live Poxvirus Human Immunodeficiency Virus Vaccine Vector Enhances Humoral and Cellular Immune Responses J. Virol., May 1, 2005; 79(9): 5537 - 5547. [Abstract] [Full Text] [PDF]
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S. B. J. Wong, C. B. Buck, X. Shen, and R. F. Siliciano An Evaluation of Enforced Rapid Proteasomal Degradation as a Means of Enhancing Vaccine-Induced CTL Responses J. Immunol., September 1, 2004; 173(5): 3073 - 3083. [Abstract] [Full Text] [PDF]
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C. Peters, X. Peng, D. Douven, Z.-K. Pan, and Y. Paterson The Induction of HIV Gag-Specific CD8+ T Cells in the Spleen and Gut-Associated Lymphoid Tissue by Parenteral or Mucosal Immunization with Recombinant Listeria monocytogenes HIV Gag J. Immunol., May 15, 2003; 170(10): 5176 - 5187. [Abstract] [Full Text] [PDF]
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K. Haglund, I. Leiner, K. Kerksiek, L. Buonocore, E. Pamer, and J. K. Rose Robust Recall and Long-Term Memory T-Cell Responses Induced by Prime-Boost Regimens with Heterologous Live Viral Vectors Expressing Human Immunodeficiency Virus Type 1 Gag and Env Proteins J. Virol., June 27, 2002; 76(15): 7506 - 7517. [Abstract] [Full Text] [PDF]
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M. Lomas, E. Hanon, Y. Tanaka, C. R. M. Bangham, and K. G. Gould Presentation of a new H-2Dk-restricted epitope in the Tax protein of human T-lymphotropic virus type I is enhanced by the proteasome inhibitor lactacystin J. Gen. Virol., March 1, 2002; 83(3): 641 - 650. [Abstract] [Full Text] [PDF]
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M. Rayevskaya, N. Kushnir, and F. R. Frankel Safety and Immunogenicity in Neonatal Mice of a Hyperattenuated Listeria Vaccine Directed against Human Immunodeficiency Virus J. Virol., January 15, 2002; 76(2): 918 - 922. [Abstract] [Full Text] [PDF]
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V. Molinier-Frenkel, R. Lengagne, F. Gaden, S.-S. Hong, J. Choppin, H. Gahery-Segard, P. Boulanger, and J.-G. Guillet Adenovirus Hexon Protein Is a Potent Adjuvant for Activation of a Cellular Immune Response J. Virol., January 1, 2002; 76(1): 127 - 135. [Abstract] [Full Text] [PDF]
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G. R. Gunn, A. Zubair, C. Peters, Z.-K. Pan, T.-C. Wu, and Y. Paterson Two Listeria monocytogenes Vaccine Vectors That Express Different Molecular Forms of Human Papilloma Virus-16 (HPV-16) E7 Induce Qualitatively Different T Cell Immunity That Correlates with Their Ability to Induce Regression of Established Tumors Immortalized by HPV-16 J. Immunol., December 1, 2001; 167(11): 6471 - 6479. [Abstract] [Full Text] [PDF]
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M. V. Rayevskaya and F. R. Frankel Systemic Immunity and Mucosal Immunity Are Induced against Human Immunodeficiency Virus Gag Protein in Mice by a New Hyperattenuated Strain of Listeria monocytogenes J. Virol., March 15, 2001; 75(6): 2786 - 2791. [Abstract] [Full Text]
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S. Zucchelli, S. Capone, E. Fattori, A. Folgori, A. Di Marco, D. Casimiro, A. J. Simon, R. Laufer, N. La Monica, R. Cortese, et al. Enhancing B- and T-Cell Immune Response to a Hepatitis C Virus E2 DNA Vaccine by Intramuscular Electrical Gene Transfer J. Virol., December 15, 2000; 74(24): 11598 - 11607. [Abstract] [Full Text]
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M. Mata and Y. Paterson Th1 T Cell Responses to HIV-1 Gag Protein Delivered by a Listeria monocytogenes Vaccine Are Similar to Those Induced by Endogenous Listerial Antigens J. Immunol., August 1, 1999; 163(3): 1449 - 1456. [Abstract] [Full Text] [PDF]
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K. R. Prilliman, K. W. Jackson, M. Lindsey, J. Wang, D. Crawford, and W. H. Hildebrand HLA-B15 Peptide Ligands Are Preferentially Anchored at Their C Termini J. Immunol., June 15, 1999; 162(12): 7277 - 7284. [Abstract] [Full Text] [PDF]
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