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J. Biol. Chem., Vol. 277, Issue 1, 161-168, January 4, 2002
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From the Department of Biochemistry, Tulane University Health
Sciences Center, New Orleans, Louisiana 70112-2699
Received for publication, March 13, 2001, and in revised form, October 9, 2001
Antigen three-dimensional structure potentially
limits the access of endoproteolytic processing enzymes to cleavage
sites and of class II major histocompatibility antigen-presenting
proteins to helper T-cell epitopes. Helper T-cell epitopes in
bacteriophage T4 Hsp10 have been mapped by restimulation of splenocytes
from CBA/J and C57BL/6J mice immunized in conjunction with mutant
(R192G) heat-labile enterotoxin from Escherichia coli.
Promiscuously immunogenic sequences were associated with unstable loops
in the three-dimensional structure of T4 Hsp10. The immunodominant
sequence lies on the N-terminal flank of the 22-residue mobile loop,
which is sensitive to proteolysis in divergent Hsp10s. Several mobile
loop deletions that inhibited proteolysis in vitro caused
global changes in the helper T-cell epitope map. A mobile loop deletion
that strongly stabilized the protein dramatically reduced the
immunogenicity of the flanking immunodominant helper T-cell epitope,
although the protein retained good overall immunogenicity. Antisera
against the mobile loop deletion variants exhibited increased
cross-reactivity, most especially the antisera against the strongly
stabilized variant. The results support the hypothesis that unstable
loops promote the presentation of flanking epitopes and suggest that
loop deletion could be a general strategy to increase the breadth and
strength of an immune response.
The structure of protein antigens controls the immunogenicity of
helper T-cell epitopes at the level of antigen processing as well as
binding of peptides to class II
MHC1 proteins. Analyses of
published structural data and epitope maps for several model antigens
suggested that local structural instability increases immunogenicity in
the adjacent sequences (1, 2). In this work, we test this hypothesis by
analyzing the immune response to bacteriophage T4 Hsp10 (T4Hsp10).
As in all Hsp10s examined thus far, T4Hsp10 has a flexibly disordered
Hsp60-binding mobile loop (3) that we suspected would favor
presentation of flanking sequences to helper T-cells. T4Hsp10 is
homologous to bacterial and mammalian Hsp10s by comparison of the
respective three-dimensional structures, although it exhibits less than
20% sequence identity with any of these Hsp10s (4). Earlier studies
have mapped helper T-cell epitopes to sequences flanking the mobile
loop of Mycobacterium leprae Hsp10 in infected humans and
immunized mice (5-7), but the possibility that the mobile loop should
be responsible for the immunodominance of these epitopes has only been
hypothesized (1).
Preferred proteolytic cleavage in Hsp10 mobile loops could enhance
presentation of sequences flanking the mobile loop. Evidence indicating
the proteolytic sensitivity of Hsp10 mobile loops is presented in a
companion paper (8). The proteolytic sensitivity of the mobile
loop in divergent Hsp10s was verified by mapping peptides generated by
limited proteolysis with several proteases. Moreover, proteolytic
sensitivity of T4Hsp10 is reduced when the mobile loop is made smaller
in recombinant variants of T4Hsp10. Low resolution structural analysis
of the mobile loop deletion variants reveals little difference in
three-dimensional structure from wild-type T4Hsp10, suggesting that the
changes were confined to the mobile loop.
We examined the propensity for three-dimensional structure to influence
processing and presentation of T4 Hsp10 by mapping T-cell epitopes in
mice. In two strains of mice, immunogenic sequences were found adjacent
to each of three disordered loops of T4Hsp10, with the single
immunodominant sequence located on the N-terminal flank of the mobile
loop. The immunogenicity of the dominant sequence was almost eliminated
when a deletion in the mobile loop substantially reduced proteolytic
sensitivity. Surprisingly, the immunogenicity of distal epitopes was
increased by mobile loop deletions that only modestly affected
proteolytic sensitivity. The deletions also affected the antibody
response. Antibody titers induced by all three deletion variants were
greater than those induced by T4Hsp10. Furthermore, antibodies raised
against the deletion variants exhibited increased cross-reactivity,
indicating that antibody epitope immunodominance shifted from epitopes
within the loop to epitopes outside of the mobile loop.
Proteins and Peptides--
Preparation of T4Hsp10 was as
previously described (9), except that the gel-filtration step was
substituted with hydroxyapatite chromatography employing a 20-300
mM gradient of sodium phosphate, pH 6.8. Construction,
expression, and characterization of mobile loop deletion variants have
been described in a companion paper (8). Overlapping peptides spanning
the sequence of T4Hsp10 were synthesized by Core Laboratory, Louisiana
State University Health Sciences Center (New Orleans, LA). All cysteine
residues were incorporated as the acetamide derivative.
Immunization and Necropsy Procedure--
Pathogen-free CBA/J
(H-2k) or C57BL/6J (H-2b) mice, female, 8-12
weeks of age (Jackson), were intranasally administered T4Hsp10, T4Hsp10dLIG, T4Hsp10d8, or T4Hsp10d8C in combination with mutant R192G heat-labile toxin (mLT, kindly provided by John Clements, Tulane
University Health Sciences Center) as adjuvant (10). Each mouse
received 10 µl of protein (2 mg/ml) and 10 µl of mLT (0.5 mg/ml) in PBS. Control mice received the same amount of mLT only. Mice
were boosted twice using the same protocol at intervals of 7 days. One
week after the second boosting, mice were euthanized with drops of
Metofane into their noses. The abdominal cavity of each mouse was
opened aseptically, and the spleen was removed for lymphocyte isolation
by gently teasing against a stainless steel mesh. The cells were
treated with red blood cell lysis buffer (Sigma) for 3 min at
room temperature and washed three times in RPMI 1640 medium. Viability
was determined by trypan blue exclusion, and the number of cells was
adjusted to the desired concentration in RPMI 1640 medium supplemented
with 10% heat-inactivated fetal bovine serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 2 mM
L-glutamine (working medium).
Lymphocyte Blastogenesis--
Splenocytes were distributed into
96-well flat-bottom tissue culture plates at 4 × 105
cells/well in RPMI 1640 working medium. Triplicate (CBA/J) or duplicate
(C57BL/6J) cultures were stimulated with peptide or protein at a final
concentration of 15 µg/ml. In an initial experiment, we found that
cultures stimulated with peptide at 50 µg/ml gave only slightly
higher levels of proliferation. Thus, the level of stimulation probably
reflects the population of epitope-specific helper T-cells, which was
primed during the immunization. In no case was the proliferation
reduced at the higher peptide concentration, indicating that
differences in stimulation are not caused by an inhibitory activity in
any of the peptides. The cultures were incubated for 3 days at 37 °C
in a 5% CO2 atmosphere, labeled with 1.0 µCi of
tritiated thymidine/well for another 18 h, and harvested onto
glass wool fiber filters using a cell harvester. Tritiated thymidine
incorporation into cellular DNA was measured in a liquid scintillation
counter. Outliers in the triplicate assays were eliminated from the
analysis if the standard deviation exceeded the average cpm. Using this
criterion, 60 of 1500 assays were eliminated. The result for each
peptide was expressed as the stimulation index (SI), which is the
quotient of the average cpm for stimulated wells divided by the average
cpm for medium-only control wells.
Biotinylation of gp31 Peptides--
Peptides bind poorly to
enzyme-linked immunoassay/radioimmunoassay plates and therefore
were tethered to avidin-coated plates by covalently attached biotin.
Immediately prior to use, sulfo-NHS-LC-biotin (Pierce) was dissolved in
distilled water and mixed with peptides in PBS, pH 7.4, at a molar
ratio of 1.5:1. The mixture was placed at room temperature for 30 min.
Tris-HCl, pH 8.0, was added to a final biotin:Tris ratio of 1:20 to
quench unreacted biotin. After 30 min at room temperature, sodium azide
was added to achieve a final concentration of 0.1%. The resulting
solutions of biotinylated peptides were stored at 4 °C.
Serum IgG Measurement--
Sera from immunized mice were
assessed for IgG antibodies against T4Hsp10, T4Hsp10dLIG, T4Hsp10d8,
T4Hsp10d8C, and T4Hsp10 peptides by ELISA as described previously
(11-13) with modification. All samples were assayed in duplicate. For
detection of IgG reacting with proteins, enzyme-linked
immunoassay/radioimmunoassay plates (EIA/RIA, Corning Inc.) were coated
with 100 µl of 4 µg/ml T4Hsp10, T4Hsp10dLIG, T4Hsp10d8, or
T4Hsp10d8C. For the detection of IgG reacting with T4Hsp10 peptides,
Reacti-Bind NeutrAvidin-coated plates (Pierce) were incubated with 100 µl of 2 µg/ml individual biotinylated T4Hsp10 peptides. Plates
incubated with PBS, pH 7.4, alone were used as a background control.
After 1 h at room temperature, the plates were washed five times
with 0.1% Triton-PBS, pH 7.4, using a plate washer (Dynatech
Laboratories) and then blocked with 4% whey in 0.5% Tween, PBS, pH
7.4, for 1 h at room temperature. Blocking buffer was removed, and
100-µl aliquots of serum dilutions were incubated in plate wells at
room temperature for 1 h. For some assays, the 1:100-diluted serum
had been preincubated (blocked) for 1 h at room temperature with
PBS or with one of the proteins (T4Hsp10, T4Hsp10dLIG, T4Hsp10d8, or
T4Hsp1-d8C) at a concentration of 0.4 mg/ml in PBS. After washing
thoroughly as before, the plate wells were incubated with 100-µl
aliquots of 1:2000 diluted peroxidase-labeled goat anti-mouse IgG ( Statistical Analysis--
Pearson correlation coefficients were
calculated using the facility implemented in Excel (Microsoft) or
InStat (GraphPad). Significance tests for correlations were by analysis
of variance. B-factors for amide nitrogen atoms were obtained from the
Protein Data Bank (code 1G31, subunit A).
T-helper Cell Immunogenicity in Sequences Adjacent to Disordered
Loops
Splenocyte proliferative responses to T4Hsp10 were mapped for five
CBA/J and five C57BL/6J mice. Immunization was by three intranasal
infusions of soluble antigen into one nostril and mLT into the
other nostril at 1-week intervals. This protocol most likely preserves
the native antigen structure prior to processing by antigen presenting
cells. Control mice received only mLT. The panel of 19 15-mer peptides
and one 16-mer peptide used for restimulation spanned T4Hsp10 residues
1-15, 6-21, etc. Splenocytes from all immunized mice proliferated
strongly in response to several peptides as well as the native T4Hsp10
(Fig. 1). Proliferative responses were
expressed as the SI, defined as the quotient of cpm in stimulated wells
divided by cpm in medium-only control wells (1.9 × 103 cpm average for CBA/J mice and 0.6 × 103 cpm average for C57BL/6J mice). Mice in which
splenocytes were stimulated with SI > 4 were regarded as having a
positive response. By this criterion, each mouse responded to an
average of 3 ± 1 (CBA/J) or 6 ± 2 (C57BL/6J) peptides. Only
a minor fraction of the 20 peptides was stimulatory. Six peptides
stimulated splenocytes from at least one immunized CBA/J mouse, and
none of these peptides stimulated control CBA/J mice. Eight peptides
stimulated splenocytes from at least one immunized C57BL/6J mouse. Two
of these peptides (6 and 19) also stimulated splenocytes from one
control mouse. For peptide 6, the average SI in the immunized group was
much higher than the average in the control group (10 versus
3.0); and thus, peptide 6 was considered stimulatory in C57BL/6J mice. For peptide 19, the average SI values were comparable in immunized and
control mice (1.9 versus 2.0); thus, peptide 19 was not
considered stimulatory in C57BL/6J mice.
Sequences corresponding to stimulatory peptides are presumed to contain
at least one epitope and thus are identified as "immunogenic sequences" (Fig. 2). Some sequences
were immunogenic in both strains of mice, whereas others were unique to
one strain. The sequence corresponding to peptide 4 was immunodominant
in both strains of mice. Peptide 4 stimulated proliferation with the
highest average SI for five mice (24 in CBA/J and 28 in C57BL/6J mice),
and it stimulated the largest number of mice with SI > 4, four
out five of both strains. Peptides 5 and 12 stimulated at least one
mouse of both strains. Other peptides stimulated one or more mice of only one strain, including peptides 8, 19, and 20 in CBA/J mice and
peptides 6, 7, 17, and 18 in C57BL/6J mice.
Structural Basis for Helper T-cell and Antibody Epitope
Immunodominance in Bacteriophage T4 Hsp10
ROLE OF DISORDERED LOOPS*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
)
(Kirkegaard & Perry Laboratories) at room temperature for 1 h.
Finally, the plates were washed and developed by the addition of
100-µl aliquots of substrate (0.1 M sodium acetate, pH
6.0, 0.2 mg/ml 3,3',5,5'-tetramethylbenzidine, 0.01% H2O2). After 3 min at room temperature, the
reactions were stopped by adding 100-µl aliquots of 1 M
phosphoric acid. The plates were read immediately at a wavelength of
450 nm by an ELISA reader (Dynatech MR5000).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 1.
Proliferative responses to T4Hsp10 peptides
of splenocytes from T4Hsp10-primed CBA/J and C57BL/6J mice. Each
bar represents the average response of splenocytes from a
single mouse assayed in triplicate (CBA/J) or duplicate
(C57BL/6J).
View larger version (21K):
[in a new window]
Fig. 2.
Correlation of helper T-cell epitope
immunogenicity with adjacent peaks of structural disorder in the
T4Hsp10 crystal structure. A and B,
peptide-specific immunogenicity reported as the efficacy of the recall
response to the particular peptide by splenocytes from T4Hsp10-primed
mice of the indicated strain (total of five mice of each strain).
C and D, residue-by-residue epitope frequency
scored by occurrence of the corresponding immunogenic sequence in the
indicated strain. E, profiles of summed epitope frequency
(CBA/J + C57BL/6J) and crystallographic B-factors for backbone amide
nitrogens, indicating a peak of epitope frequency associated with each
peak in B-factors. The horizontal line indicates the average
B-factor value. F, plot indicating correlations between
combined epitope frequency and B-factor (squares) and
between epitope frequency in CBA/J mice and in C57BL/6J mice
(triangles) at various offsets. The maximum correlation of
combined epitope frequency with B-factors at an offset of +2 indicates
that epitopes occur near and on the N-terminal side of peaks in
B-factor. In hen lysozyme and HIV gp120, maxima were obtained with
offsets of 
6 and 8, respectively. The difference in T4Hsp10 may be
due to the exceptionally strong immunodominance of a sequence (peptide
4) on the N-terminal flank of the mobile loop. The strong correlation
of epitope frequencies from the two mouse strains indicates that the
epitopes tend to occur in the same sequences despite the fact
that the two strains have different alleles of MHC class II antigen
presenting protein. This may be a consequence of the strong influence
of T4Hsp10 structure on helper T-cell epitope immunodominance.
The relationship of immunogenicity to structural disorder, and presumably also to proteolytic sensitivity, was investigated by correlating epitope frequency with crystallographic B-factors in T4Hsp10. Other work showed that the mobile loop contains the most proteolytically sensitive sites in Hsp10s (8), and the mobile loop exhibits the highest B-factors in the protein (4). Epitope frequencies were assigned to each residue in the sequence according to the frequency that the peptide stimulated either CBA/J or C57BL/6J mice. Because the peptides overlap, it is possible for a residue to have an epitope frequency greater than the number of mice in the experiment. The summation of epitope frequencies from both strains of mice emphasizes the influence of T4Hsp10 structure on epitope selection and de-emphasizes the influence of peptide affinity for the MHC class II protein.
Pearson correlation coefficients for epitope frequencies with B-factors
were sampled for a series of offsets ranging from 8 to +8 residues to
relate immunogenicity to disorder in adjacent sequences (Fig. 2F,
squares). In previous studies, epitope frequencies in hen egg
lysozyme (2) and the outer domain of HIV gp120 (14) were found to
correlate best with B-factors for residues located six and eight
residues N-terminal (6 and 8), respectively. Thus, epitopes tended
to be on the C-terminal flanks of disordered loops in those antigens.
However, in T4Hsp10, the correlation was at a maximum
(rmax = 0.60) when the offset was +2. In the
profiles of epitope frequency and B-factors, epitopes occur on both
sides of loops. One peak lies on the N-terminal side of the mobile
loop, one straddles the roof 
-hairpin, and one lies on the
C-terminal flank of the peripheral loop (Fig. 2E).
Immunogenic sequences of T4Hsp10 that were identified in the different mouse strains tend to coincide with each other despite potential sequence discrimination by different MHC alleles. Aside from sequences noted above that were immunogenic in both strains, additional immunogenic sequences were located adjacent to each other. The sequence corresponding to peptide 8 was immunogenic in CBA/J, and the sequence corresponding to peptides 6-7 was immunogenic in C57BL/6J. The sequences corresponding to peptides 19-20 were immunogenic in CBA/J, and the sequence corresponding to peptides 17-18 was immunogenic in C57BL/6J. A strong correlation (rmax = 0.79) between epitope frequencies in CBA/J and C57BL/6J mice was obtained with offset equal to 0, and thus epitopes in the two strains were coincident or narrowly distributed to the N- and C-terminal sides of each other (Fig. 2F, triangles).
Modulation of T-helper Epitope Immunogenicity by Mobile Loop Deletions
The location of the immunodominant sequence adjacent to the mobile loop in T4Hsp10 could be due to proteolytic sensitivity in the mobile loop, which may favor presentation of the adjacent sequence to T cells. We hypothesized that a reduction in mobile loop proteolytic sensitivity would reduce immunogenicity of the adjacent sequence. Three variant T4Hsp10 proteins, each with a different deletion in the mobile loop, were constructed with the expectation that the remaining loop segments would not so easily conform to the binding site of a protease. The reduced proteolytic sensitivity would result in reduced immunogenicity of the flanking epitope.
The mobile loop deletions were designed to shrink the loop size and, at
the same time, preserve a nascent -hairpin structure in the mobile
loop (Fig. 3). Three residues were
deleted to create variant the T4Hsp10dLIG. The resulting sequence
places an isoleucine opposite a threonine on the other strand in the
nascent hairpin, which is predicted to stabilize the hairpin on the
basis of amino acid preferences for -sheet secondary structure (15).
T4Hsp10d8 and T4Hsp10d8C were constructed from T4Hsp10dLIG by deleting
an additional eight residues. For T4Hsp10d8, four residues were removed symmetrically from each side of the nascent hairpin; and for
T4Hsp10d8C, eight residues were removed from the C-terminal flank of
the mobile loop, including the entire segment that forms an 
-helix
in the T4Hsp10 crystal structure.
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Each mobile loop deletion variant was expressed in E. coli and purified in a manner similar to that for T4Hsp10 (8). The biochemical and biophysical properties, including behavior on ion-exchange chromatography, circular dichroism spectroscopy, and chemical cross-linking were comparable with those of T4Hsp10. Each protein behaved as a well folded protein with native-like secondary and quaternary structure. Moreover, as expected for the removal of a disordered segment, each protein was more thermostable than T4Hsp10, exhibiting midpoints of thermal denaturation ranging from 3 to 7° higher than T4Hsp10.
Reductions in proteolytic sensitivity ranged from slight for T4Hsp10dLIG to dramatic for T4Hsp10d8C (8). Sensitivity to each of the four proteases, protease K, trypsin, protease V8, and cathepsin S, followed the progression: T4Hsp10>T4Hsp10dLIG> T4Hsp10d8>T4Hsp10d8C. Depending on the protease, sensitivity decreases were as follows: T4Hsp10dLIG, 0-30%; T4Hsp10d8, 10% to 6-fold; and T4Hsp10d8, 4-36-fold.
Each protein elicited an immune response in all five CBA/J mice
as indicated by the production of IgG antibody (see below). T-helper
epitope mapping results obtained for T4Hsp10 were similar to those
obtained in the first mapping experiment (Fig.
4). A majority of mice responded to
peptide 4, and at least one mouse responded to previously identified
peptides 5, 8, 12, and 20. Peptides 9 and 11 were newly stimulatory,
but they share sequence, and possibly all or part of the epitope, with
peptides 8 and 12, respectively.
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T-helper epitope mapping of the deletion variants utilized the T4Hsp10 peptides and therefore was restricted to epitopes shared with T4Hsp10. Nevertheless, these partial maps of the deletion variants deviate from that for T4Hsp10. The map for T4Hsp10dLIG retains peptides 4, 9, and 12 but acquires several newly stimulatory peptides: 2, 10, 13-15, and 18. The map for T4Hsp10d8 retains peptides 11, 12, and 19 (which was stimulatory in the first experiment) but acquires peptides 3, 13, 16, and 17. The map for T4Hsp10d8C is least similar to that for T4Hsp10. It retains only peptide 4, albeit at low frequency, and acquires peptides 1-3, 13, and14. As many as three mice responded to peptides corresponding to sequences altered by the deletions. Although these stimulatory peptides share partial sequences with the mobile loop deletion variants, it is not clear that the stimulation arises from an epitope that was present in T4Hsp10. Because epitopes unique to one deletion variant cannot be compared with epitopes in the other immunogens, responses to the altered peptide ligands were disregarded.
Variance in the intensity of proliferative responses from mouse to mouse is a well established phenomenon, even in inbred strains of mice (16). Because variance in presentation efficiency of the immunodominant epitope could be a source of variance in the response to the whole protein, dominance of a given epitope should be evident in a correlation of SI for the peptide with SI for the intact protein. SI values for peptides 4, 12, and 19 were tested for correlation with SI for the corresponding intact immunogen in the five mice of each group. SI for peptide 4 correlated well with SI for intact T4Hsp10 (r = 0.88, p = 0.05), less well with SI for intact T4Hsp10dLIG (r = 0.83, p = 0.08), and poorly with SI for intact T4Hsp10d8C. SI for peptide 12 correlated with SI for only intact T4Hsp10d8 (r = 0.97, p = 0.01). SI for peptide 19 marginally correlated with SI for intact T4Hsp10d8C (r = 0.86, p = 0.06).
Antibody Immunodominance in the Mobile Loop
IgG Titer--
Reactivity was compared at a serum dilution of
1:100, at which level the reaction was strong but not saturated
(Fig. 5A). The reactions of
sera raised against T4Hsp10 were consistently low, but none was less
than 2-fold greater than the reaction without antigen
(A450 = 0.05). The average titers for
sera raised against T4Hsp10dLIG and T4Hspd8C were more than 10-fold
higher than for sera raised against T4Hsp10, and the average titer for
T4Hspd8 was 6-fold higher than for T4Hsp10 (Fig. 5B).
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Cross-reactivity-- Cross-reactivity of the various antisera was analyzed by ELISA. Unexpectedly, the five sera raised against T4Hsp10 cross-reacted more strongly with T4Hsp10d8 and T4Hsp10d8C than with the immunogen (Fig. 5C), whereas sera raised against the deletion variants cross-reacted with T4Hsp10 only as well or less than they reacted with the immunogen.
Cross-blocking--
The unexpectedly strong cross-reaction of
anti-T4Hsp10 with the mobile loop deletion variants suggested that
T4Hsp10 epitopes were disrupted or sequestered upon binding of the
protein to the microtiter plate. Because ELISA may not accurately
reflect cross-reactivity in solution, the cross-blocking of sera from
two mice of each group was analyzed by incubating the serum with
a 4-fold excess of antigen (relative to the typical IgG concentration)
prior to its reaction with the immobilized immunogen (Fig.
6). Reactions for sera blocked with
antigens were normalized to the reaction of the same serum incubated
without blocking antigen. As expected, the immunogen blocked each serum
with equal or greater effectiveness than any other antigen. However,
there were clear differences in the extent to which the sera
cross-reacted. Whereas T4Hsp10 blocked more than 90% of the antibody
raised against T4Hsp10, the deletion variants blocked an average of
only 50%. In contrast, greater than 95% of the antibody raised
against T4Hsp10d8C was blocked by each protein including T4Hsp10. Sera
against T4Hsp10dLIG and T4Hsp10d8 exhibited intermediate levels of
blocking by cross-reacting antigens.
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Continuous Antibody Epitope Mapping--
The stronger
cross-reaction by sera raised against deletion variants suggested that
the mobile loop contained one or more immunodominant epitopes that were
eliminated by the deletions. Because the mobile loop is essentially
unstructured, it should present continuous epitopes that can be
recognized by antibody in the context of synthetic peptides. Sera from
three mice immunized with T4Hsp10 were screened with peptides 3-20
corresponding to T4Hsp10. Only six peptides reacted with any of the
sera, and each of the reactive peptides overlapped one of the three
disordered loops (Fig. 7). Peptides 6 and
7 overlap the mobile loop, peptide 11 overlaps the roof -hairpin,
and peptides 15-17 overlap the peripheral loop.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Helper T-cell Epitope Immunodominance-- T4Hsp10 was used to study the relationship of antigen structure to helper T-cell epitope immunodominance because the flexibly disordered mobile loop provides a natural target for an initial endoproteolytic processing event (1). Immunodominant epitopes had already been identified on the flanks of the mobile loop in mycobacterial Hsp10s, but the possibility of a mechanistic connection between the loop and the immunodominant epitopes was not discussed (5-7). Although the amino acid sequences of heat shock proteins from various sources typically are very similar, the bacteriophage T4Hsp10 has very little sequence identity to other Hsp10s (17 and 19% with Mycobacterium tuberculosis and human Hsp10s, respectively). Thus, any similarity in immunology is likely to be related to similarity in three-dimensional structure. In addition, immune tolerance of the mouse Hsp10 probably does not limit the repertoire of T-cells specific for T4Hsp10 sequences. Peculiar immune responses to other heat shock proteins have been attributed, in part, to their peptide-binding function (17). However, this is not likely to be an issue for Hsp10, which is thought to bind only to the Hsp60 chaperonin and only in the presence of ATP (18).
For this work, we have identified flexible regions of Hsp10 using crystallographic B-factors for backbone amide nitrogen atoms. B-factors weight the contribution of atoms in the back-calculation of electron density from the model structure (19). High B-factors can result from time-averaged structural fluctuations or static inhomogeneity. Thus, B-factors should provide a measure of the ease with which a protein segment can be molded to fit into a protease active site, and B-factor maxima in protein loops predict protease nick sites quite well (20).
Helper T-cell epitopes in T4Hsp10 were located in three regions
associated with flexible loops (Fig. 8). On the N-terminal flank of the
mobile loop, peptide 4 stimulated the most consistent and intense
responses in both CBA/J and C57BL/6J mice. The immunodominance of the
peptide 4-5 sequence also was evident in a correlation between the SI
for peptide 4 and the SI for the protein. A similar correlation was
reported for the immunodominant epitopes of diphtheria toxoid and
tetanus toxoid in a collection of human subjects (21). These
correlations suggest that a large portion of the variance in individual
immune responses is due to variance in the processing of the
immunodominant epitope(s). Peptides 8 and 12 were the second most
consistently immunogenic in CBA/J mice. Peptide 8 lies on the
C-terminal flank of the mobile loop, and peptide 12 overlaps the roof
-hairpin. Although the roof -hairpin is much smaller than the
mobile loop, five residues in the roof -hairpin have higher than
average crystallographic B-factors (Fig. 2), and the corresponding loop
in human Hsp10 is highly flexible (22). The roof -hairpin also was
able to present a continuous epitope to antibodies. To date, we have
not identified products of cleavage in the roof -hairpin after
limited proteolysis in vitro. Nevertheless, a low level of
proteolytic processing may be sufficient for priming in
vivo. Other immunogenic sequences also were associated with disordered loops. Peptides 6, 7, and 9 overlap the mobile loop, peptide
11 overlaps the roof -hairpin, and peptides 17-20 overlap the
peripheral loop. The peripheral loop is nearly as large as the mobile
loop, although it is much less flexible, which may account for the weak
immunogenicity of these sequences.
The relationship of disordered loops to helper T-cell epitope immunodominance was confirmed in a residue-by-residue correlation of epitope frequency with crystallographic B-factors in adjacent sequences. We summed epitope frequencies in two strains of mice to emphasize the MHC protein-independent contribution to immunodominance, in effect focusing on the "promiscuous" epitopes (23). The strong correlation of epitope frequencies in CBA/J and C57BL/6J mice justifies this procedure. The coefficient of determination (rmax2) indicates that structural disorder predicts epitopes two residues N-terminal to the extent of 36%. For comparison, rmax2 was 26% for lysozyme and 40% for the outer domain of HIV gp120. These values indicate the minimum possible influence of antigen structure on immunodominance. This analysis attempts to correlate epitopes with a single optimum distance from peaks in B-factor, but this obviously does not take into account the fine tuning in agretope selection by MHC protein or the possibility that exopeptidases eliminate agretopes prior to MHC protein binding. Nevertheless, these correlations are highly significant. The probability that epitope frequencies are not correlated with B-factors in T4Hsp10 is very low (p < 0.00001). In a similar qualitative analysis, Diethelm-Okita et al. (21) found that sequences with high relative B-factors were included in and/or flanked one or both ends of all of the "universal" epitopes in diphtheria toxoid and tetanus toxoid.
In lysozyme and gp120, T-helper epitopes tended to be located six to eight residues C-terminal from the center of disordered loops, as opposed to two residues N-terminal for T4Hsp10. In T4Hsp10, the most immunogenic sequence is located 10 residues N-terminal from the center of the mobile loop. Thus, the statistical correlation probably was weakened by the N-terminal position of the single most immunogenic epitope. However, this is a limitation of the analysis and not the model. We have proposed that preferential cleavage in disordered sites relieves conformational restraints and promotes presentation of the flanking sequences. It is not clear why the C-terminal flank should be presented more frequently, although one can imagine mechanisms that would create such a bias, such as a high ratio of carboxy- to amino-peptidase activity or a bias against C termini in the MHC peptide-binding site. Immunodominance of the N-terminal flank of the mobile loop may simply indicate the presence of compensating factors that overcome this bias and lead to selection of the N-terminal flank instead of the C-terminal flank.
The strongly immunogenic T-helper regions of Hsp10s are conserved along
with the three-dimensional structure. The three promiscuously immunogenic regions in T4Hsp10 correspond to the stimulatory peptides observed in BALB/C mice upon immunization with the M. leprae
or M. tuberculosis Hsp10s (Fig.
8). T4Hsp10 shares less than 20% sequence identity with either of the mycobacterial sequences. Thus, the
immunodominance pattern transcends mouse strains and diverse antigen
sequences. In addition, peptide 8, which was strongly immunogenic in
CBA/J mice, corresponds to a peptide that contains the immunodominant
epitope in humans exposed to M. leprae (5, 24). The
observations suggest that immunodominance caused by antigen
three-dimensional structure can supersede peptide selectivity by the
MHC protein.
|
Deletions in the mobile loop caused shifts in epitope immunodominance, and the magnitude of the shifts correlated with reduction in proteolytic sensitivity. We considered only the peptide sequences that are shared by deletion variants and native T4Hsp10. The proteolytic sensitivity of T4Hsp10dLIG was almost unaffected by the three-residue deletion (8), and sequences flanking the mobile loop (peptides 4 and 9) retained strong immunogenicity in this protein. As in T4Hsp10, SI for the immunodominant peptide 4, but not peptide 12 or 19, correlated with SI for intact T4Hsp10dLIG. In contrast, removal of an additional eight residues from the mobile loops in T4Hsp10d8 and T4Hsp10d8C rendered these proteins much more resistant to proteolysis (8). In T4Hsp10d8, both flanks of the mobile loop were changed by the deletion, and thus their immunogenicity cannot be compared. Nevertheless, the sequence corresponding to peptides 11-13 became more immunogenic, and peptide 12 became immunodominant, as indicated by a strong correlation of its SI with the SI for intact T4Hsp10d8. In T4Hsp10d8C, the sequence corresponding to peptide 4 became much less immunogenic, and the SI for peptide 19, but not 4 or 12, correlated with SI for intact T4Hsp10d8C. Thus, sequences beyond the mobile loop became immunodominant in the protease-resistant variants, and, in T4Hsp10d8C, the previously immunodominant sequence was encrypted.
We propose that the reduced immunogenicity of peptide 4 in T4Hsp10d8C results from reduced proteolytic cleavage in the adjacent sequence. However, other explanations cannot be excluded. The creation of a new epitope spanning the rejoined sequence in the mobile loop could block presentation of the original epitope by determinant capture (25). Alternatively, two epitopes flanking the now shorter mobile loop could simultaneously engage MHC proteins, and the failure to proteolytically separate them interferes with their transport to the cell surface. Such a circumstance recently was described for two epitopes of hen egg lysozyme (26). On a different level, B-cells acting as antigen-presenting cells could change the T-helper epitope map because mobile loop deletions affect the B-cell epitope immunodominance pattern (see below). Antigen-specific B-cells could diversify (27-30) or restrict (28) presentation. Further work will be necessary to distinguish these possibilities for any particular epitope. Nevertheless, the overall patterns of immunodominance in this and other antigens suggest that local variation in proteolytic sensitivity is an important influence on epitope presentation.
Antibody Epitope Immunodominance-- Mobile loop deletions produced large increases in antibody titer. A trivial explanation is that the proteins contained different levels of a contaminating mitogen such as lipopolysaccharide. However, all four proteins stimulated similar levels of splenocyte proliferation, and the highest level of proliferation for a given antigen was obtained when splenocytes had been primed against that antigen (data not shown). Thus, the level of any immune-activating contaminants must be comparable in the four preparations. Another possible explanation is that the deletion variants bypassed immune tolerance of the structurally similar (but sequence dissimilar) mouse Hsp10. If T4Hsp10 were challenging tolerance, then we expected to see cross-priming of T-helper responses against the endogenous mouse Hsp10. Cross-priming of self-reactive responses has been associated with exposure to well conserved heat shock proteins (31). As part of a study on cross-priming, we have observed that E. coli Hsp10 and a hybrid T4-mouse Hsp10 each cross-prime T-helper responses specific for mouse Hsp10; but we have not observed cross-priming of mouse-Hsp10 responses by native T4Hsp10 (data not shown). Thus, we do not think that tolerance affected the antibody titers observed here. Our favored explanation for the increased antibody titers for the deletion variants is that they resulted from increased breadth in the specificity of both B- and T-cell responses.
The location of the immunodominant antibody epitopes can be inferred from the patterns of cross-blocking. Antisera raised against T4Hsp10 were only partially blocked by the mobile loop deletion variants, indicating that a large fraction of these antibodies recognized epitopes that are absent from the deletion variants. Likewise, antisera raised against T4Hsp10dLIG and T4Hsp10d8 were only partially blocked by the other three antigens. Because each immunogen most effectively blocked the corresponding serum, T4Hsp10, T4Hsp10dLIG, and T4Hsp10d8 have uniquely dominant epitopes, most likely within the mobile loop. ELISA using immobilized peptides confirmed the presence of antibodies against mobile loop epitopes in T4Hsp10. Antisera raised against T4Hsp10d8C were distinctive in that they were completely blocked by each of the other antigens, indicating that these antibodies recognized shared epitopes. The exceptional cross-reactivity of T4Hsp10d8C antisera indicates a shift of immunodominance from the mobile loop to other epitopes that are shared by all four proteins.
Structural changes local to the mobile loop could be responsible
for the shift of antibody immunodominance in T4Hsp10d8C. The remaining
mobile loop segment in T4Hsp10d8C is both smaller and more structured
(8). Although the remaining loop segment in T4Hsp10d8 is the same size,
T4Hsp10d8 is more sensitive to proteases and heat. According to the
circular dichroism spectrum, T4Hsp10d8C had more -sheet; and,
because all other structural features appear to be native-like, the new
-sheet must be in the remaining mobile loop segment. The new
structural feature appears not to be very immunogenic, as the
T4Hsp10d8C antisera were completely blocked by the other three proteins.
Implications-- Strong helper T-cell epitope immunodominance creates a mechanism for pathogens to evade immune responses. Recent studies indicate that certain HCV and HIV "escape" variants contain alterations in the sequence of immunodominant helper T-cell epitopes, resulting in a failure to stimulate immune responses (32, 33). During the evolution of these chronically infecting viruses, pressure exerted by immune surveillance could have selected features in antigen three-dimensional structure that increase epitope immunodominance and therefore increase the probability that a mutation would substantially reduce the overall immune response to the virus. The elaboration of weakly structured polypeptide inserts in the antigens of parasitic protozoans may represent an extreme investment in this opportunity (34).
The changes in immunogenicity of the mobile loop deletion variants should encourage the use of loop deletions in subunit vaccines. Loop deletion has already been applied to HIV gp120 with mixed results (35). As observed for T4Hsp10, the loop-deleted "core" gp120 was more immunogenic than native gp120, a property that Lu et al. (35) attributed to the structural modifications. Core gp120 also acquired new antibody epitopes. The fact that antibodies against core gp120 could not neutralize HIV was attributed to the immunodominance of epitopes in the subunit interface of the gp120 trimer, which become accessible only after gp120 is shed from the virus. Our results with T4Hsp10 suggest that subunit vaccines composed of a trimeric protease-resistant core will induce the most broadly neutralizing antibodies.
The immunologic mechanism that produced the shift in antibody epitope
immunodominance remains obscure. Because the mobile loop is a large
flexible segment, which becomes smaller and more ordered in T4Hsp10d8C,
a shift of immunodominance out of the mobile loop may not be
surprising. Flexible segments could be intrinsically more immunogenic.
However, structural analyses of antigens and antigen-antibody complexes
have been inconclusive on this issue (36). The question of whether
flexibility is important or even common in antibody epitopes remains
open. Our findings suggest that helper T-cell epitope immunodominance
could be a determining factor in antibody immunodominance. Weak helper
T-cell epitope immunodominance in T4Hsp10d8C may support a broader
spectrum of antibody specificities.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Ken Bost, John Clements, and James Robinson and their co-workers for technical assistance and critical discussion, and we thank John Clements for mLT.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grant R01-AI42350.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Tulane University Health Sciences Center, 1430 Tulane Ave., New
Orleans, LA 70112-2699. Tel.: 504-586-3990; Fax: 504-584-2739; E-mail: landry@tulane.edu.
Published, JBC Papers in Press, October 15, 2001, DOI 10.1074/jbc.M102259200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: MHC, major histocompatibility complex; Hsp, heat shock protein; T4Hsp10, bacteriophage T4 Hsp10; mLT, mutant R192G heat-labile toxin; PBS, phosphate-buffered saline; SI, stimulation index; ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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